| 平滑肌细胞特异性smad4基因敲除小鼠的建立 |
| Alternative Title | Establishing a mouse strain of specific deletion of smad4 in smooth muscle cells
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| 张鹏 |
| Subtype | 硕士
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| Thesis Advisor | 张迎梅
; 杨晓
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| 2010-05-19
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| Degree Grantor | 兰州大学
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| Place of Conferral | 兰州
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| Degree Name | 硕士
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| Keyword | TGF-β
smad4
平滑肌细胞
腹主动脉瘤
基因敲除
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| Abstract | 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-βs通过细胞膜上的І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-β信号通路对平滑肌细胞功能影响打下基础。 |
| Other Abstract | The TGF-β family belongs to a superfamily known as the transforming growth factor-β superfamily which has diverse functions in most cells.It comprises of TGF-β1，TGF-β2 and TGF-β3.TGF-β signals through receptor serine/threonine kinases and intracellular Smads proteins. Smad4 is an important factor of TGF-β signaling which can form complexes with the phosphorylated R-Smads and enter the nucleus, where they bind to DNA and interact with transcription factors to regulate gene expression.Many recent studies have demonstrated that the TGF-β signal pathway has an important role in Cadiovascular diseases. The technology of gene knock out is a new method to delete targeted gene in vivo. The aim of this study was to establish a smooth muscle cell specific Smad4 knockout mouse and to analysis its primary phenotype, intending to obtain a model of Cadiovascular disease to further study the effect of TGF-β in the development of the disease.Also the present study aims to establish a primary culture method of mouse vascular smooth muscle cell to obtain primary vascular smooth muscle cell from Smad4 gene knockout mice to further study the functions of TGF-β in vascular smooth muscle cell in vitro.
A mouse specifically expresses Cre reconbinase in smooth muscle cells was selected to mate with ROSA26 reporter strain to get a SMA-Cre;ROSA26 double transgenic mice.LacZ staining in the SMA-Cre;ROSA26 double transgenic mice demonstrated that the Cre reconbinase specifically expressed in the vascular smooth muscle cell within the vessel wall. Then the SMA-Cre mice was mated with a mouse strain that carries Smad4 conditional alleles (Smad4co/co) to obtain the smooth muscle cell specific Smad4 knockout mice (SMA-Cre; Smad4 flox/flox). The results manifested that Smad4 was indeed deleted in the vessel wall of the SMA-Cre; Smad4 flox/flox mice. The deletion of Smad4 in smooth muscle cell did not result in embryonic lethality, approximately 70% of the SMA-Cre; Smad4 flox/flox mice dead between 6 to 8 weeks of age. The mutant mice exhibited abdominal aortic aneurysm in 6 weeks of age. Reformed enzymatic technique was established in the present study to obtain primary smooth muscle cell culture which was characterized by immunofluorescenc. Aftardward, the primary smooth muscle cells was isolated from the SMA-Cre; Smad4 flox/flox mice,Western blot and Real-time PCR were used to detect the expression of Smad4. It demonstrated that the isolated cells were stable heredity w... |
| URL | 查看原文
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| Language | 中文
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| Document Type | 学位论文
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| Identifier | https://ir.lzu.edu.cn/handle/262010/220548
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| Collection | 生命科学学院
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Recommended Citation
GB/T 7714 |
张鹏. 平滑肌细胞特异性smad4基因敲除小鼠的建立[D]. 兰州. 兰州大学,2010.
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