平滑肌細(xì)胞特異性Smad4基因敲除小鼠的建立
發(fā)布時(shí)間：2018-10-31 08:40
【摘要】： 轉(zhuǎn)化生長(zhǎng)因子-β(transforming growth factor-β,TGF-β)是TGF-β超家族的成員,包括TGF-β1.TGF-β2和TGF-β3.TGF-ps通過(guò)細(xì)胞膜上的Ⅰ型和Ⅱ型受體活化細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)分子Smads而實(shí)現(xiàn)其廣泛的功能,Smad4是TGF-β信號(hào)轉(zhuǎn)導(dǎo)中的核心樞紐分子,它與受體依賴的Smads結(jié)合調(diào)節(jié)下游靶基因的表達(dá)。大量的研究顯示它在心血管疾病中發(fā)揮著重要的作用。本研究的目的是利用Cre/LoxP系統(tǒng)建立平滑肌細(xì)胞特異性Smad4基因敲除小鼠并進(jìn)行初步表型分析,期望獲得一種可用于研究TGF-β在心血管疾病中的作用機(jī)制的動(dòng)物模型以及建立一種分離原代平滑肌細(xì)胞的方法,為從基因敲除小鼠體內(nèi)獲得原代平滑肌細(xì)胞并在體外研究TGF-β對(duì)其功能影響提供技術(shù)手段。 本研究選擇了平滑肌細(xì)胞特異性表達(dá)Cre重組酶的轉(zhuǎn)基因小鼠(SMA-Cre)作為介導(dǎo)敲除的工具鼠,將其與ROSA26報(bào)告基因小鼠交配獲得了SMA-Cre;ROSA26雙轉(zhuǎn)基因小鼠,通過(guò)LacZ染色顯示在其主動(dòng)脈壁的平滑肌細(xì)胞有特異性藍(lán)染,說(shuō)明SMA-Cre能在血管平滑肌細(xì)胞中特異的表達(dá)。接著用SMA-Cre轉(zhuǎn)基因小鼠與Smad4條件基因打靶小鼠交配獲得了在平滑肌細(xì)胞特異性Smad4基因敲除小鼠,通過(guò)擴(kuò)增Smad4剔除條帶和原位免疫熒光顯示敲除小鼠中Smad4發(fā)生了敲除。Smad4基因敲除小鼠無(wú)胚胎致死,能夠順利出生,大約70%的基因敲除小鼠在6-8周齡死亡。Smad4基因敲除小鼠在6周的時(shí)候出現(xiàn)腹主動(dòng)脈瘤。本研究用膠原酶消化法分離小鼠原代平滑肌細(xì)胞,免疫熒光鑒定此方法獲得的平滑肌細(xì)胞。用這種方法分離平滑肌細(xì)胞特異性Smad4基因敲除小鼠的主動(dòng)脈的原代平滑肌細(xì)胞,用免疫熒光,Western blot及Real-time PCR檢測(cè)了敲除小鼠來(lái)源的原代平滑肌細(xì)胞中Smad4的表達(dá),發(fā)現(xiàn)分離出來(lái)的原代平滑肌細(xì)胞獲得了穩(wěn)定遺傳的Smad4敲除。 實(shí)驗(yàn)結(jié)果顯示本研究成功建立了平滑肌細(xì)胞特異性Smad4基因敲除小鼠并能自發(fā)形成腹主動(dòng)脈瘤。建立的改良原代平滑肌細(xì)胞分離方法可用于從平滑肌細(xì)胞特異性Smad4基因敲除小鼠中分離原代平滑肌細(xì)胞,為在體外環(huán)境下進(jìn)一步研究TGF-p信號(hào)通路對(duì)平滑肌細(xì)胞功能影響打下基礎(chǔ)。
[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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R-332
本文編號(hào)：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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R-332
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 吳建芳;;小鼠血管平滑肌細(xì)胞原代培養(yǎng)方法的改良[J];青海醫(yī)學(xué)院學(xué)報(bào);2008年01期
2 楊蕾蕾,吳壯,程萱,徐軍,楊曉;平滑肌細(xì)胞特異表達(dá)Cre重組酶轉(zhuǎn)基因小鼠Cre重組酶的表達(dá)分布[J];生物技術(shù)通訊;2005年04期
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