平滑肌细胞特异性Smad4基因敲除小鼠的建立
发布时间：2018-10-31 08:40
【摘要】： 转化生长因子-β(transforming growth factor-β,TGF-β)是TGF-β超家族的成员,包括TGF-β1.TGF-β2和TGF-β3.TGF-ps通过细胞膜上的Ⅰ型和Ⅱ型受体活化细胞内信号转导分子Smads而实现其广泛的功能,Smad4是TGF-β信号转导中的核心枢纽分子,它与受体依赖的Smads结合调节下游靶基因的表达。大量的研究显示它在心血管疾病中发挥着重要的作用。本研究的目的是利用Cre/LoxP系统建立平滑肌细胞特异性Smad4基因敲除小鼠并进行初步表型分析,期望获得一种可用于研究TGF-β在心血管疾病中的作用机制的动物模型以及建立一种分离原代平滑肌细胞的方法,为从基因敲除小鼠体内获得原代平滑肌细胞并在体外研究TGF-β对其功能影响提供技术手段。 本研究选择了平滑肌细胞特异性表达Cre重组酶的转基因小鼠(SMA-Cre)作为介导敲除的工具鼠,将其与ROSA26报告基因小鼠交配获得了SMA-Cre;ROSA26双转基因小鼠,通过LacZ染色显示在其主动脉壁的平滑肌细胞有特异性蓝染,说明SMA-Cre能在血管平滑肌细胞中特异的表达。接着用SMA-Cre转基因小鼠与Smad4条件基因打靶小鼠交配获得了在平滑肌细胞特异性Smad4基因敲除小鼠,通过扩增Smad4剔除条带和原位免疫荧光显示敲除小鼠中Smad4发生了敲除。Smad4基因敲除小鼠无胚胎致死,能够顺利出生,大约70%的基因敲除小鼠在6-8周龄死亡。Smad4基因敲除小鼠在6周的时候出现腹主动脉瘤。本研究用胶原酶消化法分离小鼠原代平滑肌细胞,免疫荧光鉴定此方法获得的平滑肌细胞。用这种方法分离平滑肌细胞特异性Smad4基因敲除小鼠的主动脉的原代平滑肌细胞,用免疫荧光,Western blot及Real-time PCR检测了敲除小鼠来源的原代平滑肌细胞中Smad4的表达,发现分离出来的原代平滑肌细胞获得了稳定遗传的Smad4敲除。 实验结果显示本研究成功建立了平滑肌细胞特异性Smad4基因敲除小鼠并能自发形成腹主动脉瘤。建立的改良原代平滑肌细胞分离方法可用于从平滑肌细胞特异性Smad4基因敲除小鼠中分离原代平滑肌细胞,为在体外环境下进一步研究TGF-p信号通路对平滑肌细胞功能影响打下基础。
[Abstract]:Transforming growth factor- 尾 (TGF- 尾) is a member of TGF- 尾 superfamily. It includes TGF- 尾 1. TGF- 尾 2 and TGF- 尾 3.TGF-ps, which activate the intracellular signal transduction molecule Smads by type I and type II receptors on the cell membrane. Smad4 is the core pivotal molecule in TGF- 尾 signal transduction. It binds to receptor-dependent Smads to regulate the expression of downstream target genes. Numerous studies have shown that it plays an important role in cardiovascular disease. The aim of this study was to establish smooth muscle cell-specific Smad4 knockout mice using Cre/LoxP system and to analyze the phenotypes. Looking forward to an animal model for studying the role of TGF- 尾 in cardiovascular disease and a method for isolating primary smooth muscle cells, In order to obtain primary smooth muscle cells from knockout mice in vivo and study the effect of TGF- 尾 on their function in vitro. In this study, transgenic mice (SMA-Cre), which expressed Cre recombinant enzymes specifically in smooth muscle cells, were selected as tools for mediated knockout, and the mice were mated with ROSA26 reporter gene mice to obtain SMA-Cre;. The expression of SMA-Cre in vascular smooth muscle cells (VSMCs) of ROSA26 double transgenic mice was demonstrated by LacZ staining, which indicated that SMA-Cre could be expressed in vascular smooth muscle cells (VSMC). The specific Smad4 gene knockout mice in smooth muscle cells were obtained by mating SMA-Cre transgenic mice with Smad4 conditioned gene targeting mice. Amplification of Smad4 knockout bands and in situ immunofluorescence showed that Smad4 knockout occurred in knockout mice. Smad4 gene knockout mice died without embryo and could be born smoothly. About 70% of the knockout mice died at the age of 6-8 weeks. Smad4 knockout mice developed abdominal aortic aneurysms at 6 weeks. The primary smooth muscle cells of mice were isolated by collagenase digestion and identified by immunofluorescence. The primary smooth muscle cells of smooth muscle cell specific Smad4 gene knockout mice were isolated by this method. Immunofluorescence, Western blot and Real-time PCR were used to detect the expression of Smad4 in the primary smooth muscle cells derived from knockout mice. A stable inherited Smad4 knockout was found in primary smooth muscle cells. The results showed that the smooth muscle cell specific Smad4 gene knockout mice were successfully established and the abdominal aortic aneurysms were formed spontaneously. The improved primary smooth muscle cell isolation method can be used to isolate primary smooth muscle cells from smooth muscle cell-specific Smad4 gene knockout mice. The results provide a basis for further study on the effects of TGF-p signaling pathway on smooth muscle cell function in vitro.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R-332
本文编号：2301545
[Abstract]:Transforming growth factor- 尾 (TGF- 尾) is a member of TGF- 尾 superfamily. It includes TGF- 尾 1. TGF- 尾 2 and TGF- 尾 3.TGF-ps, which activate the intracellular signal transduction molecule Smads by type I and type II receptors on the cell membrane. Smad4 is the core pivotal molecule in TGF- 尾 signal transduction. It binds to receptor-dependent Smads to regulate the expression of downstream target genes. Numerous studies have shown that it plays an important role in cardiovascular disease. The aim of this study was to establish smooth muscle cell-specific Smad4 knockout mice using Cre/LoxP system and to analyze the phenotypes. Looking forward to an animal model for studying the role of TGF- 尾 in cardiovascular disease and a method for isolating primary smooth muscle cells, In order to obtain primary smooth muscle cells from knockout mice in vivo and study the effect of TGF- 尾 on their function in vitro. In this study, transgenic mice (SMA-Cre), which expressed Cre recombinant enzymes specifically in smooth muscle cells, were selected as tools for mediated knockout, and the mice were mated with ROSA26 reporter gene mice to obtain SMA-Cre;. The expression of SMA-Cre in vascular smooth muscle cells (VSMCs) of ROSA26 double transgenic mice was demonstrated by LacZ staining, which indicated that SMA-Cre could be expressed in vascular smooth muscle cells (VSMC). The specific Smad4 gene knockout mice in smooth muscle cells were obtained by mating SMA-Cre transgenic mice with Smad4 conditioned gene targeting mice. Amplification of Smad4 knockout bands and in situ immunofluorescence showed that Smad4 knockout occurred in knockout mice. Smad4 gene knockout mice died without embryo and could be born smoothly. About 70% of the knockout mice died at the age of 6-8 weeks. Smad4 knockout mice developed abdominal aortic aneurysms at 6 weeks. The primary smooth muscle cells of mice were isolated by collagenase digestion and identified by immunofluorescence. The primary smooth muscle cells of smooth muscle cell specific Smad4 gene knockout mice were isolated by this method. Immunofluorescence, Western blot and Real-time PCR were used to detect the expression of Smad4 in the primary smooth muscle cells derived from knockout mice. A stable inherited Smad4 knockout was found in primary smooth muscle cells. The results showed that the smooth muscle cell specific Smad4 gene knockout mice were successfully established and the abdominal aortic aneurysms were formed spontaneously. The improved primary smooth muscle cell isolation method can be used to isolate primary smooth muscle cells from smooth muscle cell-specific Smad4 gene knockout mice. The results provide a basis for further study on the effects of TGF-p signaling pathway on smooth muscle cell function in vitro.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R-332
【参考文献】
相关期刊论文 前2条
1 吴建芳;;小鼠血管平滑肌细胞原代培养方法的改良[J];青海医学院学报;2008年01期
2 杨蕾蕾,吴壮,程萱,徐军,杨晓;平滑肌细胞特异表达Cre重组酶转基因小鼠Cre重组酶的表达分布[J];生物技术通讯;2005年04期
，本文编号：2301545
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2301545.html
最近更新
教材专著
