沉默Dnmt1基因表达诱导大白鼠骨髓间充质干细胞向心肌样细胞分化

骨髓间充质干细胞(MSCs)具有向心肌样细胞分化的潜能.本室前期研究发现,MSCs在体外经DNA甲基转移酶(Dnmt)抑制剂5-氮胞苷诱导可分化为心肌样细胞.本研究证明,沉默DNA甲基化转移酶1（Dnmt1）基因表达,可诱导大鼠MSCs向心肌样细胞分化.本文采用表达Dnmt1 siRNA 慢病毒感染MSCs,沉默Dnmt1表达.DNA甲基化分析显示,随着沉默Dnmt1时间延长（7-28 d）,Gata-4基因上游DNA调控序列的CpG甲基化水平明显降低,而Gata-4 mRNA的转录水平明显上调,说明敲减Dnmt1表达导致Gata-4基因激活.蛋白质印迹和/或免疫细胞化学揭示,与对照组比较,心肌相关基因MHC 和cTnT表达上调, 而骨髓干细胞标志物CD90和CD29随转染时间延长表达下调.同时,实时定量PCR显示,心肌早期发育调控基因Nkx2.5 mRNA水平与Gata-4 mRNA相同,随表达Dnmt1 siRNA的慢病毒感染而上调.上述结果提示,敲减Dnmt1可降低心肌发育调控基因Gata-4启动子CpG岛的甲基化水平,上调Gata-4基因的表达,诱导骨髓间充质干细胞向心肌样分化.     

hhlim促进DMSO诱导的P19细胞向心肌分化

为了确定hhlim是否参与胚胎期的心肌分化和发育过程,用可表达hhlim蛋白和hhlim反义RNA的真核表达质粒转染P19胚胎干细胞,经G418筛选得到稳定表达hhlim和hhlim反义RNA的P19细胞克隆后,观察hhlim对P19细胞向心肌分化和发育的影响.结果显示,Nkx2.5和GATA-4在未被外源性hhlim基因转染的P19细胞中不表达.DMSO刺激细胞2天后,GATA-4开始表达,3天后Nkx2.5的表达活性显著升高.hhlim的过表达不但有利于P19细胞的存活和生长,而且还可以使Nkx2.5和GATA-4的表达比对照细胞提前1天.反义hhlim细胞株被DMSO诱导5天后,细胞仍呈集落化生长.同时,Nkx2.5和GATA-4开始表达的时间明显延滞.结果表明,hhlim能促进P19细胞向心肌细胞分化,其作用是通过促进转录因子GATA-4和Nkx2.5的表达而实现的.     

沉默Dnmt1基因表达诱导大鼠骨髓间充质干细胞向心肌样细胞分化

骨髓间充质干细胞(MSCs)具有向心肌样细胞分化的潜能.本室前期研究发现,MSCs在体外经DNA甲基转移酶(Dnmt)抑制剂5-氮胞苷诱导可分化为心肌样细胞.本研究证明,沉默DNA甲基化转移酶1(Dnmt1)基因表达,可诱导大鼠MSCs向心肌样细胞分化.本文采用表达Dnmt1 siRNA慢病毒感染MSCs,沉默Dnmt1表达.DNA甲基化分析显示,随着沉默Dnmt1时间延长(7-28 d),Gata-4基因上游DNA调控序列的Cp G甲基化水平明显降低,而Gata-4 mRNA的转录水平明显上调,说明敲减Dnmt1表达导致Gata-4基因激活.蛋白质印迹和/或免疫细胞化学揭示,与对照组比较,心肌相关基因MHC和c Tn T表达上调,而骨髓干细胞标志物CD90和CD29随转染时间延长表达下调.同时,实时定量PCR显示,心肌早期发育调控基因Nkx2.5 mRNA水平与Gata-4 mRNA相同,随表达Dnmt1 siRNA的慢病毒感染而上调.上述结果提示,敲减Dnmt1可降低心肌发育调控基因Gata-4启动子Cp G岛的甲基化水平,上调Gata-4基因的表达,诱导骨髓间充质干细胞向心肌样分化.     

心肌分化过程中经典Wnt信号促进Isl1表达北大核心CSCD

ISL1是第二生心区的分子标志,在心血管发育中发挥重要作用.在心脏发育过程中,Isl1的表达具有鲜明的时空特异性.本研究利用P19CL6畸胎瘤干细胞作为心肌分化模型,探讨了Isl1在心肌诱导分化过程中的时间特异性表达及经典Wnt信号通路对其的调控.研究发现,Isl1在心肌分化早期高表达,于诱导第4 d到达高峰,随后快速下调.其表达趋势与经典Wnt通路的激活模式具有时间上的同步性.通过加入Wnt3a蛋白及Li Cl激活经典Wnt通路,能够促进Isl1基因的表达,而Wnt通路抑制分子Frizzled-4/Fc和DKK1能够下调Isl1表达.β-catenin过表达及RNAi实验也获得相似的结果.染色质免疫共沉淀实验证实,Wnt通路效应分子LEF1,在细胞分化第4 d与其在Isl1基因启动子上游-2 300 bp处的结合增强,因而促进了Isl1基因的表达.本研究表明,经典Wnt信号能够通过LEF1/β-catenin与Isl1启动子特异结合,调控Isl1基因在心肌早期分化阶段的表达.     

心肌分化过程中经典Wnt信号促进Isl1表达

ISL1是第二生心区的分子标志,在心血管发育中发挥重要作用.在心脏发育过程中,Isl1的表达具有鲜明的时空特异性.本研究利用P19CL6畸胎瘤干细胞作为心肌分化模型,探讨了Isl1在心肌诱导分化过程中的时间特异性表达及经典Wnt信号通路对其的调控.研究发现,Isl1在心肌分化早期高表达,于诱导第4 d到达高峰,随后快速下调.其表达趋势与经典Wnt通路的激活模式具有时间上的同步性.通过加入Wnt3a蛋白及Li Cl激活经典Wnt通路,能够促进Isl1基因的表达,而Wnt通路抑制分子Frizzled-4/Fc和DKK1能够下调Isl1表达.β-catenin过表达及RNAi实验也获得相似的结果.染色质免疫共沉淀实验证实,Wnt通路效应分子LEF1,在细胞分化第4 d与其在Isl1基因启动子上游-2 300 bp处的结合增强,因而促进了Isl1基因的表达.本研究表明,经典Wnt信号能够通过LEF1/β-catenin与Isl1启动子特异结合,调控Isl1基因在心肌早期分化阶段的表达.     

文昌鱼一个GATA基因在胚胎发育中的表达图式(英文)

GATA基因在脊椎动物和非脊椎动物的发育中行使重要的功能,该家族的成员在进化上也足非常保守的.脊椎动物的GATA基因分为两个亚群:GATA1/2/3和GATA4/5/6.通过生物信息分析,在文吕鱼的基因缓中找到了3个GATA基因:一个GATA1/2/3业家族基因,两个GATA4/5/6亚家族基因:还找到一个类GATA基因.还克隆了白氏文昌鱼(Branchiostoma belcheri)GATA123的一段序列,并研究了它在早期胚胎发育中的表达图式.结果表明GATA123在原肠胚的中内胚层表达,而在神经胚晚期和幼体早期,GATA123在脑泡和消化道中部区域表达.这种表达模式与头部发育的重要基因Otx相类似.结果提示在文吕鱼脑泡的发育过程中GATA123和Otx很可能共同发挥着重要的作用.     

锌指转录因子GATA4的功能研究进展

先天性心脏病(congenital heart disease,CHD)是常见的先天性缺陷之一,严重危害儿童和成人的健康。锌指转录因子GATA4是心脏形态正常发育的调节因子,贯穿胚胎发育和心脏发育阶段多个表达过程。GATA4的突变会导致多种先天性心脏病的发生,新型GATA4突变(c.A899C,p.K300T)揭示了与房间隔缺陷(ASD)相关的GATA4甲基化位点的突变,拓宽了GATA4基因的突变图谱。GATA4作为心肌细胞生长和心肌发育中的关键参与者,可以调节心肌基因的表达,以应对肥厚性刺激。GATA4通过TRAF3IP2和IL1A激活NF-κB通路,将自噬和DDR联系到衰老和炎症反应。本综述围绕GATA4的结构与功能进行综述,为先天性心脏病的治疗、抗衰老提供一些病理基础或理论参考。     

microRNA499 慢病毒载体转染诱导大鼠骨髓间充质干细胞 向心肌样细胞分化*

目的:探讨microRNA 499(miR-499)慢病毒转染对诱导大鼠骨髓来源间充质干细胞(BM-MSCs)向心肌样细胞分化的作用。方法:取第四代Wistar大鼠骨髓来源间充质干细胞进行流式细胞检测,鉴定干细胞表面特异标记物。使用符合干细胞鉴定标准的细胞批次用于后续实验。实验设置miR499慢病毒转染、慢病毒空白转染2个处理组,分别于处理后即日、1d,3d,5d,7d收集细胞进行下列实验:实时荧光定量PCR检测心肌重要转录因子GATA4、NKx2.5和MEF2C的mRNA表达,western-blot检测心肌特异蛋白I(cTnI)的表达。结果:培养第四代Wistar大鼠骨髓来源间充质干细胞表达干细胞表面特异标记物,可用于实验。大鼠骨髓来源间充质干细胞microRNA 499慢病毒载体转染后microRNA 499表达明显升高,且转染后1d,3d,5d,7d,GATA4、NKx2.5和MEF2C的mRNA表达逐渐增强。慢病毒空白转染组未见明显变化。western-blot检测自第3天开始可见cTnI阳性表达条带,慢病毒空白转染组未检测到明显阳性表达条带。结论:microRNA 499可诱导大鼠骨髓来源间充质干细胞向心肌样细胞分化。     

Wnt通路中的部分基因在绒山羊胚胎皮肤毛囊中的表达分析

已知绒山羊毛囊的发育受Wnt等信号通路控制,但Wnt通路相关基因在绒山羊胚胎毛囊启动和生长发育过程中的表达及作用机制尚不清楚。本文采用RNA-Seq技术对45 d,55 d和65 d的绒山羊胚胎体侧皮肤进行了转录组测序,鉴定Wnt通路相关基因的表达。 RNA- Seq技术结合blast搜索,将转录组有效测序数据与云南黑山羊参考基因组序列(http://goat. kiz.ac. cn/GGD/download.htm)比对,获得了已知的Wnt通路（pathway hsa04310）中的123个相关基因（86.0%）。进而采用实时荧光定量PCR技术检测,验证了差异表达的Sfrp4、Wnt3、Wnt10a（上调）和Apc2（下调）基因在绒山羊胚胎不同时期皮肤中的表达量,初步探索了绒山羊毛囊在胚胎期启动、发育过程中,Wnt通路部分基因的表达模式,为进一步研究Wnt通路部分基因在绒山羊胚胎毛囊启动、发育过程中的作用机制提供了有意义的线索。     

BMP2 is required for early heart development during a distinct time period

BMP2, like its Drosophila homologue dpp, is an important signaling molecule for specification of cardiogenic mesoderm in vertebrates. Here, we analyzed the time-course of BMP2-requirement for early heart formation in whole chick embryos and in explants of antero-lateral plate mesoderm. Addition of Noggin to explants isolated at stage 4 and cultured for 24 h resulted in loss of NKX2.5, GATA4, eHAND, Mef2A and vMHC expression. At stages 5-8 the individual genes showed differential sensitivity to Noggin addition. While expression of eHAND, NKX2.5 and Mef2A was clearly reduced by Noggin vMHC was only marginally affected. In contrast, GATA4 expression was enhanced after Noggin treatment. The developmental period during which cardiac mesoderm required the presence of BMP signaling in vivo was assessed by implantation of Noggin expressing cells into stage 4-8 embryos which were then cultured until stage 10-11. Complete loss of NKX2.5 and eHAND expression was observed in embryos implanted at stages 4-6, and expression was still suppressed in stages 7 and 8 implanted embryos. GATA4 expression was also blocked by Noggin at stage 4, however increased at stages 5, 6 and 7. Explants of central mesendoderm, that normally do not form heart tissue were employed to study the time-course of BMP2-induced cardiac gene expression. The induction of cardiac lineage markers in central mesendoderm of stage 5 embryos was distinct for different genes. While GATA4, -5, -6 and MEF2A were induced to maximal levels within 6 h after BMP2 addition, eHAND and dHAND required 12 h to reach maximum levels of expression. NKX2.5 was induced by 6 h and accumulated over 48 h. vMHC and titin were induced at significant levels only after 48 h of BMP2 addition. These results indicate that cardiac marker genes display distinct expression kinetics after BMP2 addition and differential response to Noggin treatment suggesting complex regulation of myocardial gene expression in the early tubular heart.     

Cyclin‐dependent kinase 9 forms a complex with GATA4 and is involved in the differentiation of mouse ES cells into cardiomyocytes

The treatment of ES cells with trichostatin A (TSA), an HDAC inhibitor, induces the acetylation of GATA4 as well as histones, and facilitates their differentiation into cardiomyocytes. Recently, we demonstrated that cyclin‐dependent kinase 9 (Cdk9), a core component of positive elongation factor‐b, is a novel GATA4‐binding partner. The present study examined whether Cdk9 forms a complex with GATA4 in mouse ES cells and is involved in their differentiation into cardiomyocytes. Mouse ES cells and Nkx2.5/GFP ES cells, in which green fluorescent protein (GFP) is expressed under the control of the cardiac‐specific Nkx2.5 promoter, were induced to differentiate on feeder‐free gelatin‐coated plates. Immunoprecipitation/Western blotting in nuclear extracts from mouse ES cells demonstrated that Cdk9 as well as cyclin T1 interact with GATA4 during myocardial differentiation. TSA treatment increased Nkx2.5/GFP‐positive cells and endogenous mRNA levels of Nkx2.5 and atrial natriuretic factor. To determine the role of Cdk9 in myocardial cell differentiation, we examined the effects of a dominant‐negative form of Cdk9 (DN‐Cdk9), which loses its kinase activity, and a Cdk9 kinase inhibitor, 5,6‐dichloro‐1‐β‐ribofuranosyl‐benzimidazole (DRB) on TSA‐induced myocardial cell differentiation. The introduction of the DN‐Cdk9 inhibited TSA‐induced increase in GFP expression in Nkx2.5/GFP ES cells. The administration of DRB into ES cells significantly inhibited TSA‐induced increase of endogenous Nkx2.5 mRNA levels in ES cells as well as GFP expression in Nkx2.5/GFP ES cells. These findings demonstrate that Cdk9 is involved in the differentiation of mouse ES cells into cardiomyocytes by interacting with GATA4. J. Cell. Physiol. 226: 248–254, 2010. © 2010 Wiley‐Liss, Inc.