Smad4在维持胚胎心脏增殖中的作用研究

发布时间：2018-04-09 22:12

本文选题：Smad4　切入点：条件性基因敲除　出处：《南京大学》2010年博士论文

【摘要】：先天性心脏缺陷是胚胎致畸的主要原因之一,新生儿中约有1%有先天性心脏缺陷。影响胚胎心脏发育的因素很多,最近数年的研究也揭示了一些遗传方面的潜在原因,但其遗传机制仍并不十分明了。转化生长因子一β/骨形成蛋白(TGF-β/BMP)家族对胚胎心脏发育塑型及成体心脏病理改变均有重要作用,作为传递该信号通路核内信号唯一的Co-Smad,核内因子Smad4在此信号通路中具有中心作用。Smad4在成体心脏中敲除会导致心肌肥大及心衰的表型,但胚胎心脏发育中的作用尚无报道。本研究中我们发现Smad4在胚胎发育中期的心脏发育过程中起重要作用,能够调节BMP信号和一系列对心脏发育有调控作用的核心因子。因为Smad4全身敲除小鼠死于胚胎发育早期,我们利用Cre-Loxp系统,用胚胎心肌细胞特异表达的Cre转基因小鼠介导了Smad4基因在胚胎心肌细胞中的敲除。从胚胎期9.5天(E9.5)开始的Smad4基因敲除导致胚胎死于E12.5～15.5,突变胚胎表现出严重的心脏形态缺陷,包括显著变薄的心壁紧密层,结构紊乱的心小梁区和室间隔缺损(VSD)。极少量超过E15.5的幸存胚胎亦表现出VSD和流出道形态异常。条件性Smad4敲除导致极为显著的心肌细胞增殖下降,而细胞凋亡没有明显改变。指示心肌细胞分化的一系列肌动力蛋白在Smad4敲除心肌中的表达下调,包括肌球蛋白重链α和β,心室肌球蛋白轻链2,和心肌动蛋白α,提示心肌细胞分化能力的下降。此外,Smad4突变心脏中TGF-β/BMP通路也受到调控,BMP通路的配体中心脏特异性表达的BMP10的表达量和核因子Smad 1/5/8的活化程度均升高,提示Smad4和BMP10在心肌细胞发育中某种潜在的负反馈机制。一些心脏发育中关键的转录因子的表达有所降低,包括Nkx2.5, GATA4和MEF2c,这些因子在心脏发育中期对心肌细胞的分化有促进作用,从而其被Smad4引起的下调可能导致了心肌细胞分化的降低。以上数据从遗传学角度提供了直接证据,证明Smad4基因对调节胚胎期心肌细胞的增殖和分化均具有重要作用,同时也对先天性心脏缺陷可能的原因提供了新的见解。
[Abstract]:Congenital heart defect is one of the main reasons teratogeny, about 1% of newborns with congenital heart defects. Many factors affecting embryonic heart development in recent years, the research also reveals some genetic potential causes, but its genetic mechanism is still not very clear. A transforming growth factor beta / bone morphogenetic protein (TGF- /BMP) on the development of family shape and adult cardiac pathological changes play an important role in the embryonic heart, as the transmission of Co-Smad signal pathway in nuclear signal only, nuclear factor Smad4 in the signaling pathway has a central role of.Smad4 in a knockout phenotype will lead to cardiac hypertrophy and heart failure in the heart, but the role of embryo heart development has not been reported. In this study, we found that Smad4 plays an important role in the process of heart development in mid embryogenesis, can adjust the BMP signal and a series of heart development are The core factor of regulation.
Because the whole body Smad4 knockout mice died of early embryonic development, we use the Cre-Loxp system, Cre transgenic mice mediated expression of embryonic myocardial cells by specific Smad4 gene in embryonic cardiomyocytes knockout. From embryonic day 9.5 (E9.5) Smad4 gene knockout leads to embryonic deaths from E12.5 ~ 15.5, mutant embryos show a serious form of heart defects, including significant change in heart wall thin layer close, and trabecular area of heart septal defect structure disorder (VSD). A very small amount of more than E15.5 of the surviving embryos also showed abnormal morphology and VSD outflow tract.
Conditional knockout of Smad4 resulted in extremely significantly decreased myocardial cell proliferation, and cell apoptosis was not changed. A series of muscle indicative of myocardial cell differentiation in dynein Smad4 knockdown the expression of myocardial reduction, including alpha and beta myosin heavy chain, ventricular myosin light chain 2, and myocardial dynamic protein alpha, suggesting decreased differentiation myocardial cells. In addition, the Smad4 mutation in the heart of TGF- beta /BMP pathway is regulated by activation, expression and expression of nuclear factor Smad 1/5/8 BMP ligand pathway heart specific BMP10 were increased, suggesting that Smad4 and BMP10 in a potential negative feedback mechanism of myocardial cell development. Some transcription factors in heart development the key of the expression decreased, including Nkx2.5, GATA4 and MEF2c, these factors can promote the differentiation of heart development in mid myocardial cells, which is caused by the downregulation of Smad4 It may lead to a decrease in the differentiation of cardiac myocytes.
The above data provide direct evidence from genetic perspective, which proves that Smad4 gene plays an important role in regulating the proliferation and differentiation of embryonic cardiac myocytes, and also provides new insights into the possible causes of congenital heart defects.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R321

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