Transforming growth factor- (cid:1) 1 signaling contributes to development of smooth muscle cells from embryonic stem cells
Transforming growth factor- (cid:1) 1 signaling contributes to development of smooth muscle cells from embryonic stem cells
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转化生长因子-1信号通路有助于胚胎干细胞向平滑肌细胞的发育。美国C1560-C1568，2004年。敲除转化生长因子（TGF）-（cid：1）1或其信号通路的组分可导致小鼠胚胎死亡，原因是在显著的平滑肌细胞（SMC）成熟之前卵黄囊血管发育受损。因此，TGF-（cid：1）1在SMC发育中的作用仍不清楚。胚胎干细胞（ESC）衍生的胚状体（EB）重演了许多早期胚胎发育的事件，并代表了一个更生理的背景下，研究SMC的发展比大多数其他体外系统。目前的研究表明，在小鼠ESC-EB模型系统中诱导SMC选择性基因平滑肌（cid：2）-肌动蛋白（SM（cid：2）A）、SM 22（cid：2）、心肌蛋白、平滑肌肽-B和平滑肌肌球蛋白重链（SMMHC）。显著地，SM 2，与完全分化的SMC相关的SMMHC同种型，被表达。重要的是，结果表明，SMMHC表达细胞的聚集体表现出可见的收缩活性，表明所有对收缩性SMC发育至关重要的调节途径在该体外模型系统中是功能性的。用表达可溶性截短型TGF-（cid：1）II型受体的腺病毒抑制内源性TGF-（cid：1），减弱了ESC-EB中SMC选择性基因表达的增加，TGF-（cid：1）1特异性抗体也是如此。感兴趣的是，小干扰（si）RNA实验的结果提供了早期与晚期SMC标记基因的差异TGF-（cid：1）-Smad信号传导的证据，因为SM（cid：2）A启动子活性依赖于Smad 2和Smad 3两者，而SMMHC活性依赖于Smad 2。这些结果首次提供了直接证据，证明TGF-（cid：1）1通过Smad 2和Smad 3信号传导在从全能ESC向SMC发育中起重要作用。（Upstate，Waltham，MA）以1：1，000稀释度施用，并且以1：3，000使用抗兔辣根过氧化物酶二级。根据制造商的指导，用增强的化学发光试剂盒（Amer-sham Biosciences，皮斯卡特维，NJ）使印迹可视化。统计分析mRNA表达水平的定量数据与非配对t检验进行比较。用方差分析比较多组中的荧光素酶活性。选择P（cid：9）0.05的概率值作为所有检验的显著性。
Transforming growth factor- 1 signaling contributes to development of smooth muscle cells from embryonic stem cells. Am C1560–C1568, 2004.—Knockout of transforming growth factor (TGF)- (cid:1) 1 or components of its signaling pathway leads to embryonic death in mice due to impaired yolk sac vascular development before significant smooth muscle cell (SMC) maturation occurs. Thus the role of TGF- (cid:1) 1 in SMC development remains unclear. Embryonic stem cell (ESC)-derived embryoid bodies (EBs) recapitulate many of the events of early embryonic development and represent a more physiological context in which to study SMC development than most other in vitro systems. The present studies showed induction of the SMC-selective genes smooth muscle (cid:2) -actin (SM (cid:2) A), SM22 (cid:2) , myocardin, smooth-elin-B, and smooth muscle myosin heavy chain (SMMHC) within a mouse ESC-EB model system. Significantly, SM2, the SMMHC isoform associated with fully differentiated SMCs, was expressed. Importantly, the results showed that aggregates of SMMHC-express-ing cells exhibited visible contractile activity, suggesting that all regulatory pathways essential for development of contractile SMCs were functional in this in vitro model system. Inhibition of endogenous TGF- (cid:1) with an adenovirus expressing a soluble truncated TGF- (cid:1) type II receptor attenuated the increase in SMC-selective gene expression in the ESC-EBs, as did an antibody specific for TGF- (cid:1) 1. Of interest, the results of small interfering (si)RNA experiments provided evidence for differential TGF- (cid:1) -Smad signaling for an early vs. late SMC marker gene in that SM (cid:2) A promoter activity was dependent on both Smad2 and Smad3 whereas SMMHC activity was Smad2 dependent. These results are the first to provide direct evidence that TGF- (cid:1) 1 signaling through Smad2 and Smad3 plays an important role in the development of SMCs from totipotential ESCs. (Upstate, Waltham, MA) were applied at 1:1,000 dilution, and an anti-rabbit-horseradish peroxidase secondary was used at 1:3,000. The blot was visualized with an enhanced chemiluminescence kit (Amer-sham Biosciences, Piscataway, NJ) according to the manufacturer’s guidelines. Statistical analysis. Quantitative data on mRNA expression levels were compared with unpaired t -tests. Luciferase activity in multiple groups was compared with ANOVA. A probability value of P (cid:9) 0.05 was selected as significant for all tests.