各位战友，你们好。小分子RNA是新近发现的非常重要的一类非编码调节性RNA分子，已经发现它们在多个生物学（生长，增值，分化，调亡等）过程中起非常重要的作用。是目前国际上研究的热点问题，被认为是基因表达调控的革命性发现。因为，以前公认的基因表达调节主要集中在蛋白分子（蛋白信号、转录因子等）。但本版好像没多少这方面的内容。就我本人从事的干细胞领域而言，已经发现microRNA（miRNA）在造血干细胞分化方面起重要作用（Science杂志2004年），同时在胚胎干细胞、神经干细胞中也起重要作用。最近另外一种小分子RNA：piRNA,被发现与生殖系干细胞的发育有密切的关系。关于小分子RNA与干细胞发育的关系，目前国际上也刚刚起步，预示着干细胞研究的未来方向之一。这也给了国内从事干细胞研究的人员一个机会：敢英超美。从我们国家863、973、自然基金支持的力度也可看出国家看到了小分子RNA的重要性。本人特在此发贴，希望广大战友热烈讨论，以期共同进步。同时，如果版主觉得这个话题有价值，请置顶。
我对这个领域非常有兴趣!
miRNA最早发现于线虫,研究的领域主要集中在发育学.miRNA是一类约22nt的小分子非编码单链RNA，通过与靶mRNA的3'非翻译区（3'-UTR）不完全互补配对，抑制mRNA的翻译或影响mRNA的稳定性，减少蛋白的表达。miRNAs具有以下特点：① miRNAs在物种进化中相当保守；②只在特定的组织和发育阶段表达，可作为某些特定细胞的分子标志；③在不同发育阶段、分化进程中出现特定的miRNA，能够决定细胞的分化方向以及分化时相
干细胞被认为有分化能力,因此在分化过程中必然伴随着miRNA的变化.现有的研究已证实:
1 人类胚胎干细胞中存在某些独特的miRNA，可以作为未分化的标志(Human embryonic stem cells express a unique set of microRNAs. Dev Biol, 2004, 270(2): 488-498)
2 miR-181控制哺乳动物造血细胞分化为B细胞［MicroRNAs modulate hematopoietic lineage differentiation. Science, 2004, 303(5654): 83-86］
3miR-143在脂肪细胞分化起作用［ MicroRNA-143 regulates adipocyte differentiation. J Biol Chem, 2004, 279(50): 52361-5.］
4miR-124a 和miR-9在胚胎干细胞向神经元和胶质细胞分化过程中发挥重要的调节作用［Specific microRNAs modulate embryonic stem cell-derived neurogenesis. Stem Cells,2006 ,24(4): 857-864］
等等,我认为miRNA将成为干细胞研究的热点之一
kaize兄讲的非常好。miRNA是目前研究的较为广泛而深入的小分子RNA。国际上已经出现不少的miRNA数据库（包括已发现的miRNA数据库、miRNA靶标数据库以及不少预测miRNA及其靶标的软件等）供研究人员使用。下面我为从事生殖系干细胞研究的朋友们推荐三篇高质量研究paper，其中2篇涉及最新发现的小分子RNA：piRNA。
1. The miRNA pathway intrinsically controls self-renewal of drosophila germline stem cells. Curr Biol. 2007,17:533-538.
2. A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature, 2006, 442:199-202.
3. A novel class of small RNAs bind to MILI protein in mouse testes. Nature, 2006,442: 203-207.
这个主题非常好，是目前国际干细胞研究方兴未艾而迅速发展的领域，希望同志们跟上国际步伐。下面是造血干细胞领域的一篇好文章。
约翰霍普金斯大学Kimmel癌症中心的Curt Civin博士小组发现了一套能够使成体造血干细胞处于原始状态的“主控开关”。这些开关密码的破译可能使研究人员在将来能够培养用于癌症和其它骨髓疾病患者移植的新血细胞。研究人员不是在基因水平上定位出这种控制开关，而是在最近新发现的核酸水平的蛋白质生产形式上，即RNA水平。MicroRNA分子曾经被认为是细胞的垃圾，但是现在已经知道它是关闭较大的RNA链的蛋白质装配。Civin博士解释说，干细胞能够制造对分化成血细胞至关重要的蛋白质，但是microRNA将它们封在了原地。microRNA-155已经被证实能够终止干细胞发育成红血球和白血球。和期望的一样，没有microRNA-155的干细胞成熟了，而携带microRNA-155的干细胞则很少能成熟为红血球和白细胞。目前，Civin和他的研究组已经申请了这种microRNA技术专利。相关论文如下：
CD34+ hematopoietic stem-progenitor cell microRNA expression and function: a circuit diagram of differentiation control. Proc Natl Acad Sci U S A. 2007, 104: 2750-2755.
我2003年写的一篇综述！
RNAi在干细胞研究中的应用及其进展
jmtang
（中南大学基础医学院生理系实验血液学研究室 湖南长沙 410078）
[摘要]在各类生物中，双链RNA（dsRNA）诱导产生特异性基因沉默的机制，被称为RNA干扰（RNAi）。dsRNA干扰技术可通过降解靶基因的mRNA进行基因干涉，是研究多种生物基因功能的有效手段。本文从RNAi相关的Dicer、Agronaute基因家族等来认识其基因沉默必需基因与干细胞功能特性的关系，并综述利用RNAi干扰技术在干细胞研究与应用的进展。
[关键词] RNAi 干细胞 增殖 分化 Dicer Agronaute
中图分类号 Q2 文献标识码A 文章编号
几十年来，一直认为RNA仅仅从DNA获取遗传信息，并将信息转换成蛋白质。但最近研究发现，小RNA(small RNA)发挥着基因调控的作用。小RNA能关闭基因的表达，或改变基因表达的水平。在发育过程中通过关闭或开放基因的表达，小RNA可能指导着细胞的定向分化并决定细胞的命运[1]。
九十年代初期研究发现21到28个核苷酸的小RNA能抑制植物的基因表达，随后在动物细胞中也发现了这一现象。但直到1998年2月华盛顿卡耐基研究院的Fire和马萨诸塞大学癌症中心的Mello首次将双链RNA（dsRNA）注入线虫，结果发现诱导了比单独注射正义链或者反义链都要强的基因靶向专一性的基因表达沉默（gene silencing）。他们将这种现象称为RNA干扰（RNA interference ,简称RNAi）。随后许多研究者先后采用不同长度的dsRNA使线虫、果蝇、植物、动物卵细胞和哺乳类细胞等的靶向基因表达明显降低或沉默，因此人们把这种利用dsRNA使目的基因敲低或使目的基因沉默，从而研究目的基因的功能即为RNA干扰技术（RNAi技术）[2]。RNA干扰技术由于其独特优点在干细胞研究中备受关注和应用。本文主要综述RNA干扰技术在干细胞研究中的应用及其进展。
一、 RNAi的机制及其必需基因
1、1 RNAi的机制
实验表明：RNAi反应中，加入的dsRNA被切割为21-23nt小片段RNA即siRNA，后者会使目的mRNA被切割为21-23nt的siRNA。Hammond等人部分纯化了一种RNase Ⅲ型酶（如Dicer等），该核酸酶具有序列特异性，它仅降解与引起RNAi的dsRNA具有同源序列的mRNA。当dsRNA导入细胞后，被一种dsRNA特异的RNase Ⅲ型酶Dicer识别,切割成21-23 nt的siRNA，这些片段可与该核酸酶的dsRNA结合结构域结合，并且作为模板识别目的mRNA；识别之后，mRNA与dsRNA的有义链发生链互换，原先dsRNA中的有义链被mRNA代替，从酶-dsRNA复合物中释放出来，而mRNA则处于原先的有义链的位置。核酸酶在同样位置对mRNA进行切割，这样又产生了21-23nt的siRNA,与Dicer形成复合物即RNA诱导基因表达沉默复合物（RISC），继而RISC特异性地对目的mRNA进行切割，从而使目的基因沉默，产生RNAi现象[2-4]。就目前的研究知道在各种生物均存在通过不同长度的dsRNA引起基因表达沉默的RNAi现象。如哺乳动物细胞无论RNA干扰活动,它们都有降解长片段dsRNA产生21-22 nt的siRNA的能力。Tuschl研究则进一步说明甚至在几种更长片段dsRNA不能产生RNAi的哺乳动物细胞中，siRNA依然能够引起基因表达沉默[5]。而在哺乳动物细胞长片段dsRNA不能产生RNAi的原因可能是长于30nt的dsRNA掩敝RNAi的IFN反应的非特异性激活。但Billy研究提示在鼠未分化的EC细胞和ES细胞通过长片段dsRNA诱导RNAi引起特异基因表达沉默[6]。同时Yang的研究显示鼠ES细胞通过siRNA诱导RNAi引起相应目的基因沉默。但通过长片段dsRNA和siRNA引起基因沉默的程度目前仍不清楚[7]。
1、2RNAi的必需基因
通过遗传分析的方法，目前已从线虫中分离到RDE-2，RDE-3和Mut-7等、果蝇中的ago2等、拟南芥中的sgs-2、sgs-3、argonaute1(ago1)、sde1、sde3、caf、ddm-1、met-1等RNAi相关的基因。研究发现这些基因多数属于多基因家族，有很大的保守性。进一步研究发现，许多基因沉默必需蛋白与一些基因表达调控因子结构相似,如拟南芥的DDMI蛋白与染色体再构因子SW12/SNF2相似；拟南芥的MET1是一种维持DNA甲基化转移酶；线虫的RDE-1与翻译因子Eif2C结构相似[3、4]。这可能表明，基因沉默机制在生物的进化中有很大的保守性和多样性。
二、RNAi在细胞分裂或分化中的作用
通过改变染色体的形状（更紧或更松），能够决定某一个基因表达。近来研究揭示参与RNAi的siRNA在染色体形状的改变时起着非常重要的作用。这样RNAi能永久的关闭基因的表达，而不仅仅是短期的抑制它。而我们知道染色体中心粒周围的异染色质控制着细胞的分裂。在比较研究缺乏RNAi的酵母及正常酵母时，发现当酵母分裂时染色质聚集并移位到细胞的对侧。而在缺乏siRNA的酵母里，染色体中心粒周围不能正常形成异染色质，细胞分裂也不能正常进行。因此研究者推测siRNA能促进异染色质聚集到正确的部位并促进细胞分裂的发生。同时在具有将遗传给子代的DNA储存在一个核里，而将表达的DNA储存在另一个核里特性的四膜虫（Tetrahymena）的研究中发现，当四膜虫分裂时，siRNA促进一些基因的删除或重组。RNAi似乎是作用在四膜虫中类似于异染色质的某种结构上，删除或将DNA移到另一个位置。但机制目前仍不清楚。在酵母和四膜虫中，siRNA的作用集中在基因组的中心粒周围，这个区域包含有转座子造成的基因重复序列。有假说认为siRNA在进化的很早阶段就出现了，以防止转座子造成的基因组不稳定。当然，研究发现RNAi在发育和疾病中也具有作用。研究证实RNAi能定向诱导植物干细胞（分生组织）的分化，因而研究者认为RNAi也可能参与人的干细胞的分化。如果在人的细胞分裂中RNAi的作用也像它在酵母和四膜虫中一样，那么对RNAi微小的干扰就可能导致细胞命运的转变[1]。
2、1RNAi在植物干细胞（分生组织）增殖分化中的作用
在芽尖分生组织发现包含类似动物干细胞一样的干细胞。如果它们处于未分化的状态，将为组织再生提供一干细胞池。研究提示ago1和zwille对于芽生组织中的干细胞具有重要的调控作用[8]。Zwille在芽生组织从胚胎发育到组织形成的过程中，为维持芽生组织中未分化的干细胞起关键作用。在芽尖分化时，Zwille像ago1一样诱导STM(shoot meristemless)基因的表达。而STM基因在整个发育过程中涉及茎端分生组织的结构建成,STM基因是芽生组织功能维持和启动分化所必需的[9]。在Zwille突变的芽尖分生组织，尽管能正确的启动分化，但不能不对称分裂,而牙生组织中心区里的干细胞失去了干细胞的特性，并终末分化[9、10]。
2、2 RNAi在哺乳动物干细胞增殖分化中的作用
线虫sting缺失突变会导致雄性不育。线虫AGO2是基因沉默所必需的，它和Dicer蛋白以及siRNA组成RISC复合体进行特异性降解mRNA,根据最近的研究结果，基因沉默可能通过控制内源基因表达来调控生长发育，而果蝇和线虫利用基因沉默必需元件Dicer加工生成的siRNA来调控发育的时序性[11、12]。而Dicer行使切割功能需要RDE-1或其同源基因ALG1/ALG2的协同[13、14]。
果蝇Agronaute家族成员Piwi是干细胞保持自我更新、不对称分裂等所必需的，piwi过表达影响干细胞的特性，引起干细胞增殖、分裂；而两个与Piwi同源的鼠Agronaute家族成员Miwi和Mili。尽管它们与Piwi有所不同，但它们涉及精子发生和原始生殖细胞的调控，而且目前认为Miwi是精子发生的主要调控开关[15-18]。
在鼠未分化的EC细胞和ES细胞通过长片段dsRNA诱导RNAi引起特异基因沉默的研究中细胞分离和免疫定位发现Dicer位于细胞质。但Dicer在EC细胞的高水平是否与stRNA合成有关，以及是否是胚胎细胞和/或未分化细胞的共同特性，目前仍不很清楚[6]。
同时，在人类干细胞的研究发现人Agronaute家族成员Hiwi在原始造血干细胞（CD34+骨髓干细胞）表达，而在已分化的血细胞不表达[19]。可能Hiwi表达缺失与干细胞分化有关。
2、3RNAi参与调控干细胞增殖分化的机制
在调控各种生物细胞命运以及发育模式的研究中发现参与干细胞功能特性调控的Agronaute家族蛋白也参与了Wingless/Wnt和Hedgehog信号转导途径。譬如：果蝇Agronaute1（dAgo1）是Wingless信号转导途径的调节子。dAgo1过表达挽救了通过细胞质中的转导激活子Armadillo引起Wingless样的表现型。然而，如果Ago1是Wingless信号转导途径的保护成份，那么dAgo1的丢失也就不会产生所期望的表现型[20]。因此，Ago1可能在一并行途径起作用。而类似地，由于在卵巢Hedgehog过表达能弥补Piwi的缺乏，可见Piwi与Hedgehog相并行起作用。这些也就与Hedgehog是干细胞促进因子相一致[21、22]。当然，在人类Wingless信号转导途径与Agronaute家族间也存在联系。如人类Wilm肾肿瘤EIF2C1/hAgo1基因缺失，而这与激活β-磷酸苯丙胺的突变有关[23]。
综上所述：RNAi可能通过其两个必需基因—Dicer家族、Agronaute基因家族参与调控干细胞的增殖分化[2、3]。
三、RNAi技术在干细胞研究中的应用前景
最近通过比较研究胚胎干细胞、造血干细胞和神经干细胞等几类主要的干细胞，结果发现，这些干细胞都含有一组重要的基因，大约283个。同时还对老鼠和人类的造血干细胞进行了比较，也发现这两个不同的物种也都含有这283个基因。这揭示干细胞拥有自己的一套基因组。同期研究揭示至少216个基因是胚胎干细胞、神经干细胞和造血干细胞所独有的。这些基因可能是决定干细胞特性的最关键的实质因素．譬如：不同干细胞具有共同模式的基因（JAK/STAT和TGF-β途径的几个基因）参与干细胞的自我更新等基本功能，Cy28等特征的基因将有利于我们认识干细胞独立的起源位置和发育阶段；Foxd3、Oct4、Fgf4和Sox2基因是干细胞维持多能性所必需的基因.其它一部分分别参与转录调控、细胞周期调控、RNA加工、翻译、蛋白折叠、分子伴侣等过程。而此外大约100多个基因仍然未知。另有研究比较了干细胞周围的干细胞滋养细胞和非滋养细胞的基因活性，发现有4000多个基因在干细胞滋养细胞中表现活跃，而在非滋养性细胞中失去了活性。目前对这些周围细胞仍然缺乏充分认识 [24、25]。为此需要弄清上述每个基因的功能及其干细胞固有基因与周围滋养细胞基因之间的网络关系。由于RNAi干扰技术比传统方法（反义核苷酸技术、基因敲除等）更快更方便地使基因表达沉默或基因表达降低，从而为干细胞相关基因功能的研究提供了更有效地工具[1-3]。RNAi干扰技术不仅有利于干细胞基因调控机制的研究，而且有利于研究干细胞的分化及其细胞、基因治疗。例如：通过dsRNA使基因沉默研究干细胞的分化；利用RNAi技术对干细胞的基因做某些修饰如创建“万能供者细胞”，即破坏细胞中表达组织相容性复合物的基因，躲避受者免疫系统的监视，从而达到防止免疫排斥效应发生的目的 [2、26]。总之， RNAi技术为干细胞生物学研究提供一新的、实用的方法和手段。
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(12) Hutvagner G, McLachlan J, Pasquinelli AE, et al.A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science. 2001;293(5531):834-8.
(13) Ketting RF, Fischer SE, Bernstein E,et al.Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans.Genes Dev. 2001;15(20):2654-9.
(14) Provost P, Silverstein RA, Dishart D,et al.Dicer is required for chromosome segregation and gene silencing in fission yeast cells.Proc Natl Acad Sci U S A. 2002;99(26):16648-53.
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版主就是版主，水平不一般。2003年就注意到小分子RNA与干细胞的关系了。如果你2003年开始这个领域研究，4年后的今天也许nature、science、PNAS上会出现你的名字。不知道版主现在集中在哪个领域？目前来看，小分子RNA，尤其是miRNA在干细胞研究的研究条件已经基本成熟，相关背景也多起来了。技术门槛也不高。有兴趣的朋友们动起手来啊。将来回报一定丰厚。
楼上的兄弟,说来惭愧!做那玩意需要很多钱!当时毕业后忙于到处搞钱!也只搞到几万元!难呀!国家自然基金连续申请几年了!没有结果呀!哎!郁闷!
现在主要把硕士阶段的课题继续!也就是干细胞迁移归巢到损伤组织部位.已经获得很好的结果!准备投稿到stem cell还不知咋样!现在回过头想.当时还是应该坚持做.可惜呀!不过现在
干细胞迁移归巢到损伤组织部位也是很好的方向啊。我正在查这方面的资料。这与基因治疗关系密切啊。有空向你请教。另外，现在很多人认为stem cells杂志日渐衰微，不像刚开刊时水平了，好结果要发在好刊物上啊。下面是目前小RNA研究的重要策略：
1-抽RNA,合成probe做Northern
2-抽RNA,杂microarray
3-抽RNA,分离小片段,加linker, 做RT, 设计特异性primer-PCR
4-抽RNA,分离小片段,克隆, sequence(用于发现新miRNA)
5-信息学预测, 预测miRNA gene还是可以相信的.
功能学研究
1- 选定一个process, 如tumorgenesis, stem cell differentiation, stress condition, aging...联系上述detection手段, 检测目标miRNA expression pattern.
2-研究与目标miRNA结合的蛋白, 比如合成biotin-RNA probe做WCE pull-down. 最初的Ago-Dorsha就是这样发现的
3-overexpress/knock-down miRNA. 一是做knockout ES cell, 一是设计siRNA. 后者似乎曾经在哪里见过, 不知道普遍性如何.
4-in-situ, 通过检测spatial pattern给出一定的功能提示. 可以是animal level也可以是细胞水平.
5-RNA-IP 主要用于与研究目标蛋白是否结合RNA, 结合什么RNA的问题. 现在越来越得到重视, 因为该方法简单易行, 容易上手, 而且结果可信.
C) 寻找target mRNA
这部分是最头疼.
1)-overexpress/knockdown 目标miRNA, microarray分析mRNA的改变. 注:现在发现miRNA也可以通过siRNA的途径作用.
2) -信息学寻找3'-UTR的配对序列. 然后把这些3'-UTR接到reporter(GFP/Luciferase)ORF后面, 然后看reporter activity的改变. 这是比较通常鉴定target
mRNA的方法. 但是大家做过reporter assay的人都知道, 这是非常难以重复并且缺乏说服力的试验.
3)我提出一种假设, 是否可以结合-2中的biotin-miRNA和RNA-IP的方法鉴定目标miRNA与目标mRNA的结合, 如果结合是短暂的也可以先crosslink一
下, 然后再做IP. 我觉得这应该是比较可行而且是可信的方法.
原来《RNAi在干细胞研究中的应用及其进展》这篇文章就是版主的呀，曾经拜读过，很高深。小弟最近也在造干细胞迁移和归巢方面搞点小实验，可惜囊中羞涩啊。
见到许多国内外学者在讨论基因超表达。是否可以通过基因导入使干细胞抗原超表达促使其迁移呢？涉及干细胞迁移的表面抗原复杂，比如说CXC家族成员CXCR4，其趋化因子为SDF-1。假使能通过某种方法将CXCR4基因导入干细胞内，促使其表达，那势必会在趋化因子SDF-1的作用下促进干细胞迁移。
另外，在模拟内环境在体外进行细胞迁移的实验中多数学者采用Transwell装置，可惜偶做过几次最终还是以败失告终，查阅了很多资料也找不到一种好方法能模拟那环境来进行细胞迁移方面的实验。
我正在做小分子RNA在调亡过程中的作用及影响。结果不知道如何?哪位同道高手做过类似实验,可否赐教.谢谢.
<jinsdjn
我正在做小分子RNA在调亡过程中的作用及影响。结果不知道如何?哪位同道高手做过类似实验,可否赐教.谢谢。
现在有没有眉目了？我正在做成骨细胞的调亡研究，如果可行的话，咱们可以好好讨论一下，qq380597813,msn mhqsir0827@hotmail.com
首先，和楼主讨论一下“小分子RNA”这个中文名称。
仔细看看了大家讨论的内容，不难看出大家的目光都集中与长度在19~22nt的RNA上，有microRNA，piRNA，siRNA等，这些RNA在英文中统称为"small RNA"；我想这个大家不会有什么异议，所以我认为对应的中文名称应该是“小RNA“而不是"小分子RNA“。这是从RNA的长度来以区别其他类型的RNA；
对于“小分子RNA”，这个概念应该是不存在的，起码在这里使用是有歧义的，因为我们通常说“生命大分子有三类：糖、蛋白质和核酸”；其中核酸包括核糖核酸(RNA)和脱氧核糖核酸(DNA)，也就是说RNA是属于一种生物大分子物质。
综上所述，我们应该使用small RNA--小RNA这个称谓。
欢迎探讨！
接着说说我知道的small RNA的种类：
small RNA:
目前大家比较熟悉的应该是microRNA，endogenous siRNA(内源性的siRNA)；
siRNA还有其他分类，如ta-siRNA,secondary siRNAs;
2006年Nature上报道的piRNA;
最新发现的应该是2007年发表在cell上的21-U RNA:
下边转贴一个报道
============================================
小RNA在动物和其它真核细胞中发挥重要的基因调节作用。最近，来自 Whitehead 生物医学研究所、霍华德休斯医学研究所、麻省和霍华德Broad研究所的研究人员，利用高通量测序技术彻底地对已知的秀丽隐杆线虫（Caenorhabditis elegans）约40万小RNA进行了测序。研究结果刊登于最新一期《CELL》封面。
除了出18个microRNA(miRNA)，使在线虫中检测到的miRNA数量上升到112个外，还发现了上千种内源性siRNA，这些siRNA由RNA指导的RNA聚合酶（RNA-directed RNA polymerases）作用于与精子发生和转位子有关的转录本（transcripts）产生。
第三大突破是发现了第三种线虫类（nematode）小RNA——21U-RNAs。21U-RNAs被精确限定于21个核苷长度，有共同的uridine 5′-monophosphate起始端，其它20个核苷酸不同，3′核糖被修饰。21U-RNAs起源于第IV染色体两个broad regions上的分布的5700多个基因座中——主要位于蛋白编码基因之间或者是内含子中。这些基因座有相同的上游基序（motif），有助于精确寻找21U-RNAs。这些基序在线虫类进化过程中比较保守，可能是因为它们在产生差异、自我表达的小RNA（diverse, autonomously expressed, small RNAs ，dasRNAs）过程中有重要作用。
麻省理工Phillip Sharp教授说这项研究的重大意义勿庸置疑，“过去的30年中我们没有对非编码RNA给予足够重视，这是原则性错误，RNA干扰和microRNAs告诉我们20个核苷左右的小RNA，包含了足够多的信息，有助于特异的基因调控。”
Bartel实验室前博士后Scott Baskerville说“这次发现进一步证实我们还没有完全弄清RNA的功能，利用相同的测略（深度测序）在其它物种中寻找新型小RNA将是一件很有意思的事情。”
多年来，Bartel小组致力于对线虫的RNA进行系统性普查。2001年，他们利用传统的Sanger测序法对330个小RNA进行测序，鉴别出55个microRNAs；2003年，利用深度测序法（deeper sequencing effort）进行后续实验，阅读4000小RNA，又获得了40个microRNAs；最近一次是采用454公司大规模焦磷酸测序系统（massively parallel pyrosequencing system）对400，000个小RNA（包括18个新的microRNAs）测序。
“我们知道一定存在未经测序的microRNAs，但不知道有多少，”Bartel说。他发现的一个被遗漏的microRNAs是lsy-6，只是被分离出来，但没经过测序。尽管lsy-6只是表达在线虫的少数几个细胞种，但表达量是那些在所有细胞都表达的microRNA的十万倍。
利用上游基序为指导，Bartel推测线虫基因组中大约有12，000个21U-RNA基因座。“如果把这些基因座作为基因，那么线虫中的基因数量会翻倍。”奇怪的是，尽管这些基因的基因座在C. elegans和C. briggsae是保守的，但是它们的转录体序列并不保守。“好像是进化过程加大了这些小RNA之间的差异，而不是加大了它们的保守性。”
一个公开的问题是21U-RNAs的功能，尽管它们在C. elegans 和C. briggsae之间序列并不保守，但是Bartel推测它们仍保留了通过影响组蛋白的分布，控制局部染色体结构和基因表达。其它问题还包括：用于21U-RNAs合成的聚合酶是哪种？合成的机制是怎样的？Bartel透露其目前正在和2006年诺贝尔奖获得者、马萨诸塞州立大学医学中心Craig Mello 合作，验证RNAi造成的突变线虫中， 21U-RNA产物是否有缺陷。
仔细看了biozy版主的帖子，发现称呼小分子RNA确实不合适，我又查了一些资料，发现还是称呼“小RNA”合适。当时没细究这个称呼，网站上铺天盖地都是用“小分子RNA”这个称呼，所以就顺手写成“小分子RNA”。感谢biozy战友对待科学的严禁态度。
呵呵 推荐两年多前 生物信息版的【专题讨论】
★ JUNK DNA && NON-CODING RNA [精华]
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关注中!
很希望我也能做出一些东西来,现在才明白什么是烧钱呀!
最近也准备做siRNA，请问高手，这个做下来大概需要多长时间呀，最难做的是哪一步压
“沉默”基因帮助实验鼠修复受损心脏
美国哥伦比亚大学医学中心研究人员研究发现，实验鼠体内一个原本“沉默”的基因得到表达后，实验鼠就能自我修复受损的心脏。这一发现有助于寻找心脏病的新疗法。
传统的心脏病疗法包括进行“搭桥”手术绕过受损心脏的坏死细胞进行治疗以及进行心脏移植手术等，而哥伦比亚大学研究人员在实验中采用的方法却迥然不同。
研究人员发现，实验鼠体内有一种名为cyclinA2的基因，它只在实验鼠处于胎盘的时期表现活跃，而在成年实验鼠体内保持“沉默”。cyclinA2与细胞再生有关。
在实验中，研究人员通过基因技术使一部分实验鼠体内原本“沉默”的cyclinA2基因得到表达，对另一部分实验鼠则不进行任何操作，然后使这些实验鼠都患上心脏病。结果发现，cyclinA2基因得到表达的实验鼠得以幸存，而对照组实验鼠则因心脏衰竭而奄奄一息。
这一成果刊登在最新一期美国《循环研究》杂志上，研究人员下一步打算在人身上进行此类研究。
cyclinA2启动心脏修复系统
心脏病一直是威胁人类的主要疾病之一宾夕法尼亚医学院研究人员正在研究一种能“开启”受损心脏组织修复过程的蛋白。研究人员可以通过引发细胞周期信号控制动物模型中的细胞去再生受损心脏组织。如果这项研究在人体试验成功，就可以改善治疗心脏病的途径。研究结果发表于美国心脏协会杂志《Circulation》网络版。
这是不同于传统解决心脏病问题的另一新概念。传统的思维方法是替换或者是旁路移植手术，比如通过外科手术“搭桥”绕过受损心脏的坏死细胞、进行心脏移植手术等，Joseph Woo博士解释说：“什么将是最好的解决方案？最好的办法应该是简单的使受损组织再生。举个例子，切除部分肝脏，机体会自动修复和重新生长，使之恢复原样。”
然而，要想使受损心脏得到恢复，除非机体收到特殊的“信号”。受损心脏得不到修复，其后果是严重的。假如心脏中某部位坏死，
比如心脏病发作，就不能发挥向全身输送动脉血的有效功能，最终有可能导致心力衰竭和死亡。
为了寻找使心脏恢复的方法，Woo最先研究了大鼠心脏再生信号——最近有研究证实大鼠心脏有能力修复受损的心脏组织。
研究人员操控这些信号使得心脏得以再生。
Woo小组通过启动心脏细胞分化和复制，研究心肌细胞再生能力。实验中他们表达了一种叫做cyclinA2的细胞周期调控蛋白，这种蛋白的特殊性在于它控制细胞周期中的静止期和分裂期的转换；对于小鼠、大鼠和人类等来说，它是在出生后中唯一被完全沉默的细胞周期蛋白。
本项研究通过基因转移表达cyclinA2蛋白，使心肌细胞的功能得到很大改善。
“这是第一个激活肌体先天修复系统来治疗受损心脏的实验。”Woo说：”我们正在研究这种再生策略用于开发治疗坏死心脏的新方法的潜力，将来也许可以减少外科手术甚至药物治疗。研究成果还没有用于人类试验，这期间还需要数年的时间。”
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© 2006 American Heart Association, Inc. Volume 114(1) suppl I, 4 July 2006, pp I-206-I-213
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Outline
Abstract
Methods
Animal Care and Biosafety
Induction of Heart Failure
Adenoviral Vector Delivery
Confirmation of Protein Expression
Tissue Section Preparation
Immunohistochemical Assessment of Cardiomyocyte DNA Synthesis and Proliferation
Immunohistochemical Assessment of Mitosis
Measurement of Ventricular Geometry
Assessment of Myofilament Density
Assessment of Hemodynamic Function
Statistical Analysis
Statement of Responsibility
Results
Adenoviral Expression
Cardiomyocyte Proliferation
Myofilament Density
Ventricular Geometry
Cardiac Function
Discussion
Sources of Funding
Disclosures
References
Graphics
Figure 1
Table 1
Figure 2
Figure 3
Figure 4
Table 2
Figure 5
Recent History
Therapeutic Delivery of C...Therapeutic Delivery of C...
Therapeutic Delivery of Cyclin A2 Induces Myocardial Regeneration and Enhances Cardiac Function in Ischemic Heart Failure
[Myocardial Protection and Vascular Biology]
Woo, Y Joseph MD; Panlilio, Corinna M. BA; Cheng, Richard K. MD; Liao, George P. MB; Atluri, Pavan MD; Hsu, Vivian M. BA; Cohen, Jeffrey E. BA; Chaudhry, Hina W. MD
From Division of Cardiothoracic Surgery (Y.J.W., C.M.P., G.P.L., P.A., V.M.H., J.E.C.), Department of Surgery, University of Pennsylvania School of Medicine, Philadelphia, Pa; Division of Cardiology (R.K.C., H.W.C.), Department of Medicine, Columbia University School of Medicine, New York, NY.
Presented at the American Heart Association Scientific Sessions, Dallas, Tex, November 13–16, 2005.
Correspondence to Y. Joseph Woo, Division of Cardiothoracic Surgery, University of Pennsylvania, 3400 Spruce St, Philadelphia PA 19104.E-mail: wooy@uphs.upenn.edu
Abstract
Background—: Heart failure is a global health concern. As a novel therapeutic strategy, the induction of endogenous myocardial regeneration was investigated by initiating cardiomyocyte mitosis by expressing the cell cycle regulator cyclin A2.
Methods and Results—: Lewis rats underwent left anterior descending coronary artery ligation followed by peri-infarct intramyocardial delivery of adenoviral vector expressing cyclin A2 (n =32) or empty adeno-null (n =32). Cyclin A2 expression was characterized by Western Blot and immunohistochemistry. Six weeks after surgery, in vivo myocardial function was analyzed using an ascending aortic flow probe and pressure-volume catheter. DNA synthesis was analyzed by proliferating cell nuclear antigen (PCNA), Ki-67, and BrdU. Mitosis was analyzed by phosphohistone-H3 expression. Myofilament density and ventricular geometry were assessed. Cyclin A2 levels peaked at 2 weeks and tapered off by 4 weeks. Borderzone cardiomyocyte cell cycle activation was demonstrated by increased PCNA (40.1±2.6 versus 9.3±1.1; P<0.0001), Ki-67 (46.3±7.2 versus 20.4±6.0; P<0.0001), BrdU (44.2±13.7 versus 5.2±5.2; P<0.05), and phosphohistone-H3 (12.7±1.4 versus 0±0; P<0.0001) positive cells/hpf. Cyclin A2 hearts demonstrated increased borderzone myofilament density (39.8±1.1 versus 31.8±1.0 cells/hpf; P=0.0011). Borderzone wall thickness was greater in cyclin A2 hearts (1.7±0.4 versus 1.4±0.04 mm; P<0.0001). Cyclin A2 animals manifested improved hemodynamics: Pmax (70.6±8.9 versus 60.4±11.8 mm Hg; P=0.017), max dP/dt (3000±588 versus 2500±643 mm Hg/sec; P<0.05), preload adjusted maximal power (5.75±4.40 versus 2.75±0.98 mWatts/µL2; P<0.05), and cardiac output (26.8±3.7 versus 22.7±2.6 mL/min; P=0.004).
Conclusions—: A therapeutic strategy of cyclin A2 expression via gene transfer induced cardiomyocyte cell cycle activation yielded increased borderzone myofilament density and improved myocardial function. This approach of inducing endogenous myocardial regeneration provides proof-of-concept evidence that cyclin A2 may ultimately serve as an efficient, alternative therapy for heart failure.
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Heart failure has become a major global health concern. Current therapies ranging from revascularization to remodeling to replacement are limited and variably effective. Cellular cardiomyoplasty, with its myriad forms of cell types and delivery routes, has shown great experimental promise and some benefit in early clinical application.
An effective endogenous repair strategy would be a theoretically ideal therapy. However, because of the post-mitotic state of adult cardiomyocytes, the predominant myocardial response to injury is cardiomyocyte hypertrophy. Although recent research has identified a putative population of resident cardiac progenitor stem cells, the native role of these cells during injury is clearly clinically insufficient for maintaining cardiac function and the potential role of the manipulation of these cells is still highly uncertain.1–5 A mechanistically more attractive approach is to attempt to induce native cardiomyocytes to reenter the cell cycle and replicate.6–9 Multiple potential cell cycle regulators such as cyclins and cyclin-dependent kinases (cdk) serve as potential therapeutic targets.10 Manipulation of the restriction point control cyclin D has shown initial promise.11
Cyclin A2 possesses a unique role in its 2-point control of the cell cycle, first by interacting with cdk2 in controlling the G1/S transition into DNA synthesis and then by interacting with cdks 1 and 2 to control the G2/M entry into mitosis.12 We have previously demonstrated that constitutive expression of the cell cycle regulator cyclin A2 in a transgenic mouse yields robust postnatal cardiomyocyte mitosis and hyperplasia.13 To examine the potential role of this regenerative strategy as a therapy for heart failure, a cyclin A2-expressing adenoviral vector was constructed and delivered to rat hearts after left anterior descending coronary artery infarction and subsequent evidence of cardiomyocyte proliferation, peri-infarct geometric enhancement, and cardiac functional improvement were observed.
Methods
Animal Care and Biosafety
Adult, male Lewis rats weighing 250 to 300 grams were obtained from Charles River Laboratories (Boston, Mass). Food and water were provided ad libitum. This study was performed in accordance with the standard humane care guidelines of the Guide for the Care and Use of Laboratory Animals and the Institutional Animal Care and Use Committee of the University of Pennsylvania, which conform to federal guidelines.
Induction of Heart Failure
Male Lewis rats (n =64) were anesthetized with intraperitoneal ketamine (75 mg/kg) and xylazine (7.5 mg/kg), endotracheally intubated with a 14-gauge angiocatheter, mechanically ventilated (Hallowell EMC, Pittsfield, Mass) and supplemented with 1.5% isoflurane as maintenance anesthesia. A left fourth intercostal space thoracotomy was performed and the pericardium was incised. The left anterior descending coronary artery (LAD) was identified and encircled with a 7-0 prolene suture at the level of the left atrial appendage. The suture was briefly snared to confirm adequate consistent ligation, as evidenced by blanching of the arterial region of distribution, and then permanently ligated. This highly reproducible method causes an infarction of 30% of the left ventricle. The animals were closed in 3 layers over a temporary thoracostomy tube and allowed to recover for six weeks. Over the course of 6 weeks, the animals predictably progressed into ischemic cardiomyopathy.14–16 A subset of animals received serial intraperitoneal injections of bromodeoxyuridine (BrdU; BD Pharmingen San Diego, Calif) at a concentration of 80 µg/kg at 4-day intervals to examine DNA synthesis.
Adenoviral Vector Delivery
Replication-deficient (E1, E3 deleted) adenoviral vectors containing murine cyclin A2 (Pubmed gene bank ID X75483) driven by the cytomegalovirus promoter were made by the University of Iowa Gene Transfer Vector Core (Iowa City, Iowa). Corresponding empty replication-deficient adenovirus with null content was used as a control. At the time of coronary ligation, animals were randomized to either therapy with adeno-cyclin A2 or adeno-null injection. Immediately after LAD ligation, a total of 3×109 plaque forming units were injected into 5 predetermined regions in the peri-infarct borderzone as previously described.17
Confirmation of Protein Expression
In vivo expression of the cyclin A2 protein was confirmed in a subset of normal noninfarct animals (n =8/group) by direct intramyocardial injections of adeno-cyclin A2 or adeno-null vector into the cardiac apex. Myocardial tissue was harvested at 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks and frozen immediately in liquid nitrogen. Myocardium was subsequently dounced in homogenization buffer consisting of 50 mmol/L tris/HCl (pH 7.5), 100 mmol/L NaCl, 5 mmol/L EDTA, 1% v/v Triton X-100, 1 mmol/L NaF, 1 mmol/L Na2VO4, 0.2 mmol/L phenymethylsulfonyl fluoride, 10 µg/mL leupeptin, and 10 µg/mL aprotinin. Lysates were cleared by centrifugation at 12 000 rpm for 10 minutes at 4°C and analyzed for protein content via the Bradford method (BioRad, Hercules, Calif). Then, 40 µg of each protein sample was then denatured at 70°C for 10 minutes and electrophoresed onto a 4% to 12% sodium dodecyl sulfate-polyacrylamide gel. Equal loading of protein was verified by Coomassie blue staining. Proteins were transferred to Immobilon-P PVDF membranes (Millipore, Bedford, Mass) and immunoblotting was performed using a mouse anti-cyclin A monoclonal antibody (Abcam, Cambridge, Mass). This antibody has been tested in mice and has demonstrated cross-reactivity with murine cyclin A. Proteins were visualized via horseradish peroxidase conjugated anti-mouse antibody (Amersham Biosciences, Piscataway, NJ) and chemiluminescence detection (Amersham Biosciences).
Immunohistochemisty was used to confirm protein expression within the harvested hearts; 10-µm thin myocardial sections were fixed in 4% paraformaldehyde, permeabilized with Triton X-100, and blocked with 5% normal goat serum for 2 hours at room temperature, followed by incubation with rabbit anti-human cyclin A (1:600; Abcam, Cambridge, Mass) overnight at 4°C. Antibody cross-reactivity with mouse and rat cyclin A has been demonstrated by the manufacturer. The slides were washed 3 times in phosphate-buffered saline and incubated with FITC-conjugated goat anti-rabbit secondary antibody (1:1000; Abcam) for 45 minutes at room temperature. Cyclin A2 expression was quantitated in 4 fields/specimen of the region of interest of both cyclin A2 and null hearts by fluorescence microscopy (40× air magnification; Leica Microsystems, Wetzlar, Germany).
Tissue Section Preparation
Six weeks after infarction and before explantation, the right atrium was incised and the hearts were perfused and rinsed with 10 mL phosphate-buffered saline via the aortic root with resultant blanching of the myocardium. The hearts were arrested in diastole, distended with Optimum Cutting Temperature embedding compound at a fixed distending pressure, and snap-frozen in liquid nitrogen. Explanted hearts were analyzed in a blinded fashion. Sequential transverse 10-µm tissue sections were made at the level of the papillary muscles. Sections were immediately fixed in 4% paraformaldehyde, washed, and blocked in 5% normal goat serum/phosphate-buffered saline.
Immunohistochemical Assessment of Cardiomyocyte DNA Synthesis and Proliferation
Myocardial thin sections were fixed, permeabilized, and blocked as described. Immunohistochemical analysis of cardiomyocyte DNA synthesis was performed by staining for PCNA (1:600 rabbit anti-human PCNA; Abcam) and Ki-67 (1:500 goat anti-mouse Ki-67; Santa Cruz Biotechnology, Santa Cruz, Calif) as indicators of DNA synthesis and cellular proliferation. Positive nuclei were co-localized to [alpha]-sarcomeric actin (1:700 mouse anti-rabbit [alpha]-sarcomeric actin; Sigma Aldrich, St. Louis, Mo) to confine PCNA and Ki-67–positive staining specifically to cardiomyocytes. The [alpha]-isoform of sarcomeric actin will label only [alpha]-cardiac muscle actins and [alpha]-skeletal muscle actins, and will not stain smooth muscle actin. Dual-positive cells in 4 high-power peri-infarct fields were then quantitated in a blinded fashion for each specimen and averaged (40× air magnification; Leica Microsystems, Wetzlar, Germany).
From the subset of animals that had received serial injections of BrdU, tissue sections were processed as detailed and BrdU staining was analyzed. After fixation and antigen retrieval, tissue sections were incubated with a biotinylated mouse anti-BrdU antibody (1:600; BD Pharmingen, San Diego, Calif). Streptavidin-horseradish peroxidase was used to detect the presence of antibody via the Diaminobenzidine (DA substrate system. (BrdU detection assay; BD Pharmingen).
Immunohistochemical Assessment of Mitosis
Sections were analyzed for mitosis using the nuclear mitosis marker, phosphohistone-H3 (1:600 mouse anti-human phosphohistone-H3; Upstate Biotechnology, Lake Placid, NY). Dual-positive staining for both phosphohistone-H3 and cardiomyocytes localized to DAP-stained nuclei was performed as described. The number of dual-positive stained cells was counted in a blinded fashion in 4 high-power fields per specimen.
Measurement of Ventricular Geometry
Tissue sections were obtained at the level of the papillary muscle. The sections were stained with hematoxylin and eosin to delineate morphology. Digitized photomicrographs were taken with a Nikon Coolpix 4300 camera using standardized imaging distances. Geometric measurements were then computed in a blinded fashion using Scion Image Beta Release 4 (Scion Corporation, Frederick, Mass) from 2 representative tissue sections for each animal. The left ventricular diameter was measured in two perpendicular axes and averaged for each animal. The wall thickness of the left ventricular borderzone was measured and analyzed from 2 separate areas of each tissue section and averaged for each animal. The borderzone was defined as 1 field lateral to myocardial scar.18,19 The wall thickness index was calculated for each specimen and was defined as the ratio of borderzone wall thickness to remote normal myocardial wall thickness ×100.
Assessment of Myofilament Density
Myocardial borderzone hematoxylin and eosin-stained tissue sections were imaged and myofilament density was analyzed per high-power field. Myofilament density was defined as the total myocytes per high-power field. Four high-power fields were quantitated and averaged per tissue section in both borderzone and remote noninfarcted areas. Data analysis was performed in a blinded fashion, recorded, and analyzed for statistical significance.
Assessment of Hemodynamic Function
Six weeks after initial LAD ligation, a median sternotomy was performed and hemodynamic measurements were obtained using an ascending aortic Doppler flow probe and an intraventricular pressure-volume catheter. A 2.5-mm peri-aortic flow probe (Model 2.5PSL492; Transonic Systems Ithaca, NY) was placed around the ascending aorta to measure the cardiac output. A 2-French pressure-volume conductance microcatheter (model SPR838; Millar Instruments Houston, Tex) was volume and pressure calibrated 20 and placed via the left ventricular apex into the left ventricular cavity. Hemodynamic measurements were analyzed in a blinded fashion utilizing Chart v4.1.2 software (AD Instruments, Colorado Springs, Colo) and ARIA1 Pressure Volume Analysis software (Millar Instruments). In addition to steady-state hemodynamic measurements, contractility was measured from pressure-volume relationships obtained via preload reduction after occlusion of the inferior vena cava. Volume measurements were calibrated by 2-point linear interpolation with fixed-volume cuvettes of heparinized rat blood, and parallel conductance was excluded with the hypertonic saline injection technique.
Statistical Analysis
Statistical analysis was performed using JMP IN 5.1 software using 2-way ANOVA to test for differences among means. All results were expressed as means±SEM. Statistical significance was determined as a P<0.05.
Statement of Responsibility
The authors had full access to the data and take full responsibility for their integrity. All authors have read and agree to the manuscript as written.
Results
Adenoviral Expression
Western blot analysis of cyclin A2 protein levels demonstrated statistically significant cyclin A2 levels in the adeno-cyclin A2 samples as compared with adeno-null controls. As expected, adeno-null control samples did not contain measurable amounts of cyclin A2 protein. In the experimental animals, cyclin A2 protein reached peak levels at 14 days and tapered off by 28 days (Figure 1). Fluorescent immunocytochemical labeling confirmed cyclin A2 protein levels as early as 3 days after adenoviral injection. Immunocytochemical labeling for cyclin A2 was absent in the adeno-null animals at all time points, and absent in the adeno-cyclin A2 animals at 28 days.
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[Email Jumpstart To Image] Figure 1. Adenoviral expression. A, Western blot showing adenoviral mediated expression of cyclin A2 up to 21 days compared with null adenovirus. 3×109 plaque forming units of adenovirus were injected into 5 distinct areas around area of infarct created by LAD ligation during initial surgery. B, Animals were recovered and euthanized at each time point to generate a corresponding adenoviral expression curve. C, Immunohistochemical analysis yielded statistically significant values of cyclin molecule in only the cyclin A2 adenovirally treated tissue sections.
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Cardiomyocyte Proliferation
PCNA expression colocalized to [alpha]-sarcomeric actin stained cells was significantly upregulated in cyclin A2 animals compared with null controls, suggesting borderzone cardiomyocyte cell cycle activation. Ki-67 expression colocalized to [alpha]-sarcomeric actin-stained cells was likewise significantly upregulated in adeno-cyclin A2 animals compared with null controls, also suggesting borderzone cardiomyocyte cell cycle activation. Furthermore, BrdU uptake studies demonstrated a significant increase in the proportion of BrdU-positive cells per 1000 cardiomyocytes with adeno-cyclin A2 therapy, thereby confirming active DNA synthesis (Table 1). Colocalization of phosphohistone-H3 expression to [alpha] -sarcomeric actin stained cells in cyclin A2 animals compared with null controls was significantly elevated, confirming cardiomyocyte mitosis (Figure 2). These cellular proliferation studies provided multiple independent measures of cell cycle activation and cardiomyocyte mitosis with adeno-cyclin A2 therapy. These data demonstrate convincing evidence for cardiomyocyte proliferation with cyclin A2 activation.
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[Email Jumpstart To Image] TABLE 1. Immunohistochemical Analysis of Proliferation in Cardiac Tissue Samples
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[Email Jumpstart To Image] Figure 2. Immunohistochemical analysis of mitosis in tissue samples by phosphohistone-H3 staining. Phosphohistone-H3 was stained for as an indicator of active mitosis. A, Corresponding null-treated tissue sections did not stain positive for phosphohistone-H3. B, Representative cyclin-treated tissue section showing active intranuclear mitosis. Phosphohistone-H3 appears green, alpha-sarcomeric actin appears red, and DAPI-stained nuclei appear blue. C, Graphical representation of phosphohistone-H3 ratios in cardiomyocyte nuclei. Cyclin tissue sections (n =13) had significantly greater phosphohistone-H3 positive cells localized to cardiomyocyte nuclei than null controls (n =12) in the borderzone (12.7±1.4 vs 0±0; P<0.0001)
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Myofilament Density
Myofilament density analysis demonstrated a statistically significant increase in peri-infarct borderzone myofilaments in the adeno-cyclin A2 treatment animals as compared with null control animals. Quantitation of myofilament density in the remote, normal myocardium demonstrated no change in myofilament density or structure between the treatment and control groups. The similarity in remote myofilament composition and structure validated the assay and denoted similar baseline myocardial structure (Figure 3).
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[Email Jumpstart To Image] Figure 3. Myofilament density. Myofilament density was analyzed from respective tissue sections. A, Cyclin-treated tissue sections showed significantly greater myofilament density in the borderzone as compared with null-treated sections (n =12/subset). Cyclin and null-treated sections did not differ in myofilament density in the remote right ventricular areas that were not treated with adenovirus. B, Representative photomicrograph of remote myofilament density in null tissue sections. C. Representative photomicrograph of remote myofilament density in cyclin-treated tissue sections. D, Representative photomicrograph borderzone myofilament density in null tissue sections. E, Representative photomicrographs borderzone myofilament density in cyclin-treated tissue sections.
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Ventricular Geometry
Computerized planimetric analysis of ventricular geometry demonstrated a statistically significant increase in borderzone wall thickness in the adeno-cyclin A2 treated animals as compared with the adeno-null treated animals (1.8±0.04 versus 1.4±0.04 mm; P<0.001). The calculated wall thickness index (defined as the percent ratio of borderzone wall thickness to remote normal myocardial wall) was significantly increased in cyclin A2 experimental tissue sections as compared with controls (73.9±6.7 versus 62.4±5.4; P<0.001). Enhanced preservation of left ventricular geometry and diameter (8.8±0.2 versus 9.9±0.1 mm; P<0.001) was noted in the adeno-cyclin A2 group compared with control animals (Figure 4).
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[Email Jumpstart To Image] Figure 4. Ventricular geometry. Ventricular geometry was measured from hematoxylin and eosin-stained sections of cyclin-treated and control tissue samples and displayed graphically. A, Quantitative analysis of ventricular wall thinning in tissue sections revealed that cyclin-treated tissue sections had greater left ventricular wall thickness. B, Quantitative analysis of intracavitary dimension in tissue sections revealed that cyclin-treated tissue sections had diminished left ventricular dilatation.
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This combination of enhanced borderzone wall thickness and preservation of ventricular geometry increases the potential for enhanced myocardial function.
Cardiac Function
Myocardial functional analysis 6 weeks after LAD ligation indicated a statistically significant increase in cardiac output as measured by Doppler analysis of ascending aortic blood flow in the adeno-cyclin A2 animals as compared with adeno-null animals. Dynamic pressure-volume analysis showed a statistically significant improvement in maximum generated ventricular pressure (Pmax), maximum dP/dT, elastance, and preload-adjusted maximal power with adeno-cyclin A2 therapy. Also, the ventricular volumes appear to be smaller in the cyclin hearts. There was no difference in the baseline heart rate between the treatment and control groups. Therefore, it appears that the adeno-cyclin A2-treated animals had statistically significant preservation of cardiac function compared with control animals (Table 2). Cyclin A2-treated animals also had significantly improved cardiac contractility, as indicated by an increased slope of the pressure-volume relationship during caval occlusion as compared with controls (0.75±0.19 versus 0.57±0.18; P=0.048). Representative pressure-volume loops from each group are shown in Figure 5.
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[Email Jumpstart To Image] TABLE 2. In Vivo Hemodynamic Measurements
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[Email Jumpstart To Image] Figure 5. Pressure-volume loops. Improved contractility was measured in cyclin A2-treated animals as compared with control animals (P-V slope 0.75±0.19 mm Hg/µL vs 0.57±0.18 mm Hg/µL). Representative pressure-volume loops are shown.
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Discussion
In infarcted hearts, targeted expression of the cell cycle regulator cyclin A2 induced cardiomyocyte mitotic activity, increased borderzone myofilament density, and improved ventricular function.
Controlling myocardial cell cycle regulation is a complex interaction between cyclins, cyclin-dependent kinases, cdk inhibitors such as p57, p21, p27, and other regulators including retinoblastoma protein, PCNA, and E2F.21–23 The cyclins, which play a central role in cell cycle control, form an appealing therapeutic target for inducing cardiomyocyte replication. Subsequent endogenous myocardial regeneration would seem an intuitively well-matched therapy to address necrotic and apoptotic cardiomyocyte loss in heart failure.
Cyclin A2 is unique in its control of both major transitions of the cell cycle at G1/S and G2/M. Cyclin A2 is the only cyclin that is completely silenced after birth in mice,13 rats,24 and humans.24 This disappearance of cyclin A2 occurs at a rate consistent with the rate of withdrawal of cardiomyocytes from the cell cycle.24 A targeted deletion of cyclin A2 in the mouse exhibited embryonic lethality at embryonic day 5.5.25 A recent study has demonstrated that the development of the majority of mouse tissues can occur independently of all 3 D-type cyclins.26 It has also been shown with a double knockout that cdk4 and cdk6 are not essential for cellular proliferation.27 It has also been reported that E-type cyclins are largely dispensable for normal development in mice.28 Both E1 and E2 knockout mice (-/-) developed normally. The D-type cyclins control restriction point movement into DNA synthesis and have been studied as a potential therapeutic target; however, the potential for cell cycle arrest before mitosis may be a limitation.29 Thus, cyclin A2 expression offers enhanced potential for inducing cell cycle re-entry given its control of the G2/M transition in addition to G1/S and makes cyclin A2 a particularly attractive therapeutic target.
We have previously demonstrated biologic marker evidence of cardiomyocyte mitosis and histomorphologic evidence of cardiomyocyte hyperplasia in a transgenic mouse expressing cyclin A2.13 Up to 70% increase in the calculated number of cardiomyocytes was noted, as well as a corresponding increase in heart weight-to-body weight ratio. These encouraging findings have prompted us to develop a therapeutic strategy based on inducing cardiomyocyte cyclin A2 expression.
In our model, cyclin A2 adenoviral expression peaked at 2 weeks and tapered off by 4 weeks. This limited duration of adenoviral expression may be of utility; providing cyclin A2 expression during an active period of adverse post-infarction ventricular remodeling when the presence of additional functional cardiomyocytes may be of particular benefit. Potential detrimental hypertrophy from excessive hyperplasia or perhaps neoplasia from prolonged cyclin A2 expression consequently may be avoided, because we did not observe any nests of aberrant appearing cells in any of the specimens. Furthermore, our examinations of transgenic animals constitutively expressing cyclin A2 for as long as 1.5 years did not reveal any neoplastic transformation.13
We examined multiple biologic markers of cellular proliferation in this study. While PCNA and Ki-67 are well-accepted indicators of cellular proliferation, phosphohistone-H3 expression is a very precise assay of mitosis and thus serves as the ultimate marker of the desired product of cell cycle re-entry.30 The complete absence of phosphohistone-H3 in borderzone and remote myocardium in our control animals confirmed the post-mitotic state of the adult rat heart. The expression levels in the remote uninjured myocardium were equivalent between null and cyclin A2 groups, thereby excluding any systemic factors to which differences could potentially be attributed. Additionally, each heart was matched to an internal control to confirm that the therapy was localizable and contained within the region of administration. BrdU labeling further verified that DNA synthesis actively occurred within cardiomyocytes.
This rationale was extended to the comparison of myofilament density. The remote myocardium revealed equivalent myofilament density among groups. Ischemic injury resulted in a notable decrement in myofilament density which was partially rescued with cyclin A2 therapy, yielding a 25% increase in density. Interestingly, the observed increase in borderzone wall thickness in cyclin A2 hearts equaled a 23% improvement. Although the absolute values may appear less than significant, on a relative scale, a 25% increase in myocytes is substantive. Additionally, in a heart which at baseline is only millimeters thick, any increase may be sufficient to yield considerable improvement in functional capacity. These histologic improvements were mirrored in multiple functional parameters.
Two theoretical limitations of this study warrant discussion. In using an adenoviral expression system to effect a nuclear process, a question arises as to whether an adenoviral gene product will undergo appropriate processing and targeting to attain appropriate intranuclear localization. One study reported that the fusion of a nuclear localization signal to an adenoviral expressed cyclin D1 was necessary to target the cyclin D1 to the nucleus and prevent cytoplasmic accumulation, which precluded cell cycle activation.31 A more recent study 11 questioned the necessity of such a nuclear localization signal and postulated that the observation of cytoplasmic accumulation had more to do with the adenoviral delivery protocol and an overwhelming multiplicity of infection with the in vitro cardiomyocyte model used in the Tamamori-Adachi study.31 Furthermore, 3 other studies of adenoviral-mediated expression of indirect cell-cycle regulators E2F,21 E1A,32 and FGF-5 33 have all demonstrated cell cycle re-entry to some degree. These studies imply that adenovirally transcribed and translated proteins can be appropriately processed by intrinsic post-translational modification and subcellular localizing machinery and thus can target and impact nuclear events.
The second limitation of this study pertains to the proposed existence of a putative resident cardiac progenitor stem cell population. Although the data in this study are consistent with the induction of native cardiomyocyte cell cycle activity and replication, there exists the theoretical possibility that it is in fact this stem cell population that may have been activated, thus providing a second potential mechanism to explain the degree of cardiac repair noted as a result of cyclin A2 administration. This putative mechanism is being investigated further. If such a population of stem cells is in fact being stimulated with this cyclin expression strategy, this may provide an unexpected but added benefit.
In this report, the therapeutic strategy of myocardial gene transfection to express cyclin A2 induced cardiomyocyte cell cycle activation. This activation yielded increased borderzone myofilament density and improved myocardial function. This approach details proof-of-concept evidence that can be further used to design a novel therapeutic strategy to administer or activate cyclin A2 and, hence, endogenous myocardial regeneration may be an attainable goal in the near future to limit the morbidity and mortality of human heart failure.
Sources of Funding
This work was supported in part by grants from the NIH National Heart Lung and Blood Institute/Thoracic Surgery Foundation for Research and Education HL072812 (Y.J.W.), NIH National Heart Lung and Blood Institute HL067048-03 (H.W.C.), National Heart Lung and Blood Institute, Ruth L. Kirschstein National Research Service Award, Individual Fellowship 1 F32 HL79769-01 (P.A.) and AHA Heritage Affiliate Medical Student Research Fellowship (R.K.C. while in medical school at Columbia University).
Disclosures
None.
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Key Words: cyclin; heart failure; myocardial regeneration
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Accession Number: 00003017-200607041-00034
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Acknowledgment of Reviewers
Circulation.2007; 115: e20-e30
1: J Thorac Cardiovasc Surg. 2007 Apr;133(4):927-33. Epub 2007 Feb 22.
Myocardial regeneration therapy for ischemic cardiomyopathy with cyclin A2.
Woo YJ, Panlilio CM, Cheng RK, Liao GP, Suarez EE, Atluri P, Chaudhry HW.
Division of Cardiothoracic Surgery, Division of Cardiology, Department of
Medicine, Columbia University School of Medicine, New York, New York, USA.
wooy@uphs.upenn.edu
OBJECTIVE: Heart failure therapies ranging from revascularization to remodeling
to replacement are variably effective. Theoretically, endogenous repair via
myocardial regeneration would be an ideal therapy. This study examined the
ability to initiate regeneration by adenoviral-mediated expression of the cell
cycle regulator cyclin A2. Our prior studies have demonstrated robust cyclin A2
transgene expression and marked antiphosphorylated histone H3 activity with this
strategy, indicating the induction of cardiomyocyte mitosis. METHODS: Adult
male, Lewis rats underwent left anterior descending coronary artery ligation
followed by intramyocardial delivery of either cyclin A2 adenoviral vector (n =
8) or empty adeno-null vector as a control (n = 8) into the peri-infarct border
zone. In vivo myocardial function was analyzed by echocardiography and invasive
left ventricular pressure catheter at 6 weeks, when the animals are
traditionally in heart failure. Hearts were explanted for immunoblotting and
left ventricular geometric analysis. Cellular proliferation was assessed by
proliferating cellular nuclear antigen expression. RESULTS: Cyclin A2 hearts
exhibited improved left ventricular function as compared with controls including
enhanced cardiac output (32 +/- 3.3 vs 26 +/- 5.0 mL/min, P < .05), stroke
volume (0.16 +/- 0.04 vs 0.11 +/- 0.04 mL, P < .05), ejection fraction (72% +/-
7.4% vs 46.% +/- 8.5%, P < .05), fractional shortening (35% +/- 5.4% vs 19% +/-
4.3%, P < .002), maximum pressure (72 +/- 9.3 vs 61 +/- 2.9 mm Hg, P < .05), and
end-systolic pressure (67 +/- 7.0 vs 55 +/- 7.0 mm Hg, P < .05). Enhanced
myocardial preservation was demonstrated by enhanced left ventricular border
zone wall thickness. Increased myocardial proliferation was evidenced by
increased expression of proliferating cell nuclear antigen expression in cyclin
A2-treated hearts. CONCLUSIONS: In failing hearts, targeted delivery of cyclin
A2 improves hemodynamic function, as measured by echocardiography and pressure
catheter analysis, preserves ventricular wall thickness, and may serve as an
ideal myocardial regenerative therapy.
PMID: 17382628 [PubMed - indexed for MEDLINE]
Related Links
Therapeutic delivery of cyclin A2 induces myocardial regeneration and
enhances cardiac function in ischemic heart failure. [Circulation. 2006]
PMID:16820573
Stromal cell-derived factor and granulocyte-monocyte colony-stimulating
factor form a combined neovasculogenic therapy for ischemic cardiomyopathy. [J
Thorac Cardiovasc Surg. 2005] PMID:16077394
Targeted overexpression of growth hormone by adenoviral gene transfer
preserves myocardial function and ventricular geometry in ischemic
cardiomyopathy. [J Mol Cell Cardiol. 2004] PMID:15081312
Local myocardial overexpression of growth hormone attenuates postinfarction
remodeling and preserves cardiac function. [Ann Thorac Surg. 2004] PMID:15172279
Targeted overexpression of leukemia inhibitory factor to preserve myocardium
in a rat model of postinfarction heart failure. [J Thorac Cardiovasc Surg. 2004]
PMID:15573071
1: Circ Res. 2007 May 10; [Epub ahead of print]
Cyclin A2 Induces Cardiac Regeneration After Myocardial Infarction and Prevents
Heart Failure.
Cheng RK, Asai T, Tang H, Dashoush NH, Kara R, Costa KD, Naka Y, Wu EX,
Wolgemuth DJ, Chaudhry HW.
Departments of Medicine, Surgery, Radiology, Biomedical Engineering, and
Genetics and Development, Columbia University, New York; and the Department of
Electrical and Electronics Engineering, University of Hong Kong, China.
Mammalian myocardial infarction is typically followed by scar formation with
eventual ventricular dilation and heart failure. Here we present a novel model
system in which mice constitutively expressing cyclin A2 in the myocardium
elicit a regenerative response after infarction and exhibit significantly
limited ventricular dilation with sustained and remarkably enhanced cardiac
function. New cardiomyocyte formation was noted in the infarcted zones as well
as cell cycle reentry of periinfarct myocardium with an increase in DNA
synthesis and mitotic indices. The enhanced cardiac function was serially
assessed over time by MRI. Furthermore, the constitutive expression of cyclin A2
appears to augment endogenous regenerative mechanisms via induction of side
population cells with enhanced proliferative capacity. The ability of cultured
transgenic cardiomyocytes to undergo cytokinesis provides mechanistic support
for the regenerative capacity of cyclin A2.
PMID: 17495221 [PubMed - as supplied by publisher]
2: J Thorac Cardiovasc Surg. 2007 Apr;133(4):927-33. Epub 2007 Feb 22.
Myocardial regeneration therapy for ischemic cardiomyopathy with cyclin A2.
Woo YJ, Panlilio CM, Cheng RK, Liao GP, Suarez EE, Atluri P, Chaudhry HW.
Division of Cardiothoracic Surgery, Division of Cardiology, Department of
Medicine, Columbia University School of Medicine, New York, New York, USA.
wooy@uphs.upenn.edu
OBJECTIVE: Heart failure therapies ranging from revascularization to remodeling
to replacement are variably effective. Theoretically, endogenous repair via
myocardial regeneration would be an ideal therapy. This study examined the
ability to initiate regeneration by adenoviral-mediated expression of the cell
cycle regulator cyclin A2. Our prior studies have demonstrated robust cyclin A2
transgene expression and marked antiphosphorylated histone H3 activity with this
strategy, indicating the induction of cardiomyocyte mitosis. METHODS: Adult
male, Lewis rats underwent left anterior descending coronary artery ligation
followed by intramyocardial delivery of either cyclin A2 adenoviral vector (n =
8) or empty adeno-null vector as a control (n = 8) into the peri-infarct border
zone. In vivo myocardial function was analyzed by echocardiography and invasive
left ventricular pressure catheter at 6 weeks, when the animals are
traditionally in heart failure. Hearts were explanted for immunoblotting and
left ventricular geometric analysis. Cellular proliferation was assessed by
proliferating cellular nuclear antigen expression. RESULTS: Cyclin A2 hearts
exhibited improved left ventricular function as compared with controls including
enhanced cardiac output (32 +/- 3.3 vs 26 +/- 5.0 mL/min, P < .05), stroke
volume (0.16 +/- 0.04 vs 0.11 +/- 0.04 mL, P < .05), ejection fraction (72% +/-
7.4% vs 46.% +/- 8.5%, P < .05), fractional shortening (35% +/- 5.4% vs 19% +/-
4.3%, P < .002), maximum pressure (72 +/- 9.3 vs 61 +/- 2.9 mm Hg, P < .05), and
end-systolic pressure (67 +/- 7.0 vs 55 +/- 7.0 mm Hg, P < .05). Enhanced
myocardial preservation was demonstrated by enhanced left ventricular border
zone wall thickness. Increased myocardial proliferation was evidenced by
increased expression of proliferating cell nuclear antigen expression in cyclin
A2-treated hearts. CONCLUSIONS: In failing hearts, targeted delivery of cyclin
A2 improves hemodynamic function, as measured by echocardiography and pressure
catheter analysis, preserves ventricular wall thickness, and may serve as an
ideal myocardial regenerative therapy.
Publication Types:
Evaluation Studies
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
PMID: 17382628 [PubMed - indexed for MEDLINE]
3: Circulation. 2006 Jul 4;114(1 Suppl)206-13.
Therapeutic delivery of cyclin A2 induces myocardial regeneration and enhances
cardiac function in ischemic heart failure.
Woo YJ, Panlilio CM, Cheng RK, Liao GP, Atluri P, Hsu VM, Cohen JE, Chaudhry HW.
Division of Cardiothoracic Surgery, Department of Surgery, University of
Pennsylvania School of Medicine, 3400 Spruce St, Philadelphia PA 19104, USA.
wooy@uphs.upenn.edu
BACKGROUND: Heart failure is a global health concern. As a novel therapeutic
strategy, the induction of endogenous myocardial regeneration was investigated
by initiating cardiomyocyte mitosis by expressing the cell cycle regulator
cyclin A2. METHODS AND RESULTS: Lewis rats underwent left anterior descending
coronary artery ligation followed by peri-infarct intramyocardial delivery of
adenoviral vector expressing cyclin A2 (n =32) or empty adeno-null (n =32).
Cyclin A2 expression was characterized by Western Blot and immunohistochemistry.
Six weeks after surgery, in vivo myocardial function was analyzed using an
ascending aortic flow probe and pressure-volume catheter. DNA synthesis was
analyzed by proliferating cell nuclear antigen (PCNA), Ki-67, and BrdU. Mitosis
was analyzed by phosphohistone-H3 expression. Myofilament density and
ventricular geometry were assessed. Cyclin A2 levels peaked at 2 weeks and
tapered off by 4 weeks. Borderzone cardiomyocyte cell cycle activation was
demonstrated by increased PCNA (40.1+/-2.6 versus 9.3+/-1.1; P<0.0001), Ki-67
(46.3+/-7.2 versus 20.4+/-6.0; P<0.0001), BrdU (44.2+/-13.7 versus 5.2+/-5.2;
P<0.05), and phosphohistone-H3 (12.7+/-1.4 versus 0+/-0; P<0.0001) positive
cells/hpf. Cyclin A2 hearts demonstrated increased borderzone myofilament
density (39.8+/-1.1 versus 31.8+/-1.0 cells/hpf; P=0.0011). Borderzone wall
thickness was greater in cyclin A2 hearts (1.7+/-0.4 versus 1.4+/-0.04 mm;
P<0.0001). Cyclin A2 animals manifested improved hemodynamics: Pmax (70.6+/-8.9
versus 60.4+/-11.8 mm Hg; P=0.017), max dP/dt (3000+/-588 versus 2500+/-643 mm
Hg/sec; P<0.05), preload adjusted maximal power (5.75+/-4.40 versus 2.75+/-0.98
mWatts/microL2; P<0.05), and cardiac output (26.8+/-3.7 versus 22.7+/-2.6
mL/min; P=0.004). CONCLUSIONS: A therapeutic strategy of cyclin A2 expression
via gene transfer induced cardiomyocyte cell cycle activation yielded increased
borderzone myofilament density and improved myocardial function. This approach
of inducing endogenous myocardial regeneration provides proof-of-concept
evidence that cyclin A2 may ultimately serve as an efficient, alternative
therapy for heart failure.
Publication Types:
Evaluation Studies
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
PMID: 16820573 [PubMed - indexed for MEDLINE]
4: J Biol Chem. 2004 Aug 20;279(34):35858-66. Epub 2004 May 24.
Cyclin A2 mediates cardiomyocyte mitosis in the postmitotic myocardium.
Chaudhry HW, Dashoush NH, Tang H, Zhang L, Wang X, Wu EX, Wolgemuth DJ.
Department of Medicine, Columbia University College of Physicians and Surgeons,
New York, New York 10032, USA. hwc7@columbia.edu
Cell cycle withdrawal limits proliferation of adult mammalian cardiomyocytes.
Therefore, the concept of stimulating myocyte mitotic divisions has dramatic
implications for cardiomyocyte regeneration and hence, cardiovascular disease.
Previous reports describing manipulation of cell cycle proteins have not shown
induction of cardiomyocyte mitosis after birth. We now report that cyclin A2,
normally silenced in the postnatal heart, induces cardiac enlargement because of
cardiomyocyte hyperplasia when constitutively expressed from embryonic day 8
into adulthood. Cardiomyocyte hyperplasia during adulthood was coupled with an
increase in cardiomyoctye mitosis, noted in transgenic hearts at all time points
examined, particularly during postnatal development. Several stages of mitosis
were observed within cardiomyocytes and correlated with the nuclear localization
of cyclin A2. Magnetic resonance analysis confirmed cardiac enlargement. These
results reveal a previously unrecognized critical role for cyclin A2 in
mediating cardiomyocyte mitosis, a role that may significantly impact upon
clinical treatment of damaged myocardium.
PMID: 15159393 [PubMed - indexed for MEDLINE]
我个人感觉小RNA是很悬的东西，平均一个小RNA调控200个以上的基因，从这点上要搞清楚其作用以及判断其的实际应用价值（这点似乎更重要）似乎不是十分容易。
听在“Cell” “Nature”等上发相关文章的“牛人”的报告也感觉云山雾罩，许多关键问题也是回避再回避的。
想进入此领域的朋友要三思。
科学规律的发现就是在“云山雾罩”中逐渐被发现的。一个领域的发展是从无数的未知甚至是矛盾中中走出来的。小RNA的研究虽然还不很完善，但某些方面还是被确证了的。microRNA targets的研究是瓶颈，需要新的方法和技术的出现。目前通过computational approaches预测不尽人意；experimental methods又非常困难，只能就具体问题来研究。目前国际研究的主流是microRNA在某个生物学过程中的 regulatory minicircuitries，这方面研究往往发表在高分的杂志上，比如microRNA与蛋白信号、microRNA与转录因子等，也就是劈开microRNA targets不管，研究microRNA的功能。想进入microRNA的朋友可以从这方面入手。如果经费不足，建议不要碰用实验方法鉴定新microRNA的工作，可以考虑computational approaches结合experimental validation预测新microRNA genes。另外提醒朋友们，讨论的焦点要集中在干细胞中的小RNA。
