SM22α在TGFβ誘導(dǎo)的血管平滑肌細(xì)胞表型重塑中的作用
發(fā)布時(shí)間：2018-12-10 15:37
【摘要】：目的:血管平滑肌細(xì)胞(vascular smooth muscle cells, VSMC)是非終末分化細(xì)胞,具有表型重塑的特性。SM22α(smooth muscle 22 alpha, SM22α)是VSMC分化早期的標(biāo)志基因之一,其表達(dá)具有平滑肌組織特異性和細(xì)胞表型特異性。本研究利用小干擾RNA(small interfering RNA, siRNA)技術(shù)敲低內(nèi)源性SM22α表達(dá),觀察對(duì)轉(zhuǎn)化生長(zhǎng)因子β(transforming growth factor, TGFβ)誘導(dǎo)的VSMC細(xì)胞骨架組構(gòu)、氧化應(yīng)激以及分化相關(guān)轉(zhuǎn)錄因子Krüppel樣因子4(krüppel-like factor 4, KLF4)活化的影響。 方法:用貼塊法分離、培養(yǎng)大鼠VSMC,取3～5代細(xì)胞進(jìn)行實(shí)驗(yàn);用免疫沉淀檢測(cè)KLF4的磷酸化水平;Western blot分析檢測(cè)TGFβ對(duì)SM22α和SMα-actin表達(dá)的影響;用免疫熒光染色顯示細(xì)胞內(nèi)SM22α表達(dá)和定位;用DHE染色確定SM22α在氧化應(yīng)激中的作用。 結(jié)果: 1 TGFβ誘導(dǎo)血管平滑肌細(xì)胞表達(dá)SM22α用TGFβ(2 ng/ml)刺激VSMC不同時(shí)間,SM22α、SMα-actin的表達(dá)水平呈時(shí)間依賴性升高。表明TGFβ能誘導(dǎo)血管平滑肌細(xì)胞SM22α表達(dá)。 2 SM22α抑制TGFβ誘導(dǎo)的分化轉(zhuǎn)錄調(diào)節(jié)因子KLF4磷酸化Western blot結(jié)果顯示,TGFβ處理的細(xì)胞,其KLF4的磷酸化水平明顯升高。轉(zhuǎn)染SM22α特異的siRNA敲低內(nèi)源性SM22α表達(dá)后, TGFβ誘導(dǎo)的KLF4磷酸化較對(duì)照組更加明顯。提示SM22α可抑制TGFβ誘導(dǎo)的KLF4磷酸化。 3 SM22α介導(dǎo)TGFβ誘導(dǎo)的actin應(yīng)力纖維的形成 免疫熒光染色結(jié)果顯示,TGFβ處理的VSMC應(yīng)力纖維致密、成束,SM22α熒光強(qiáng)度明顯增強(qiáng),沿應(yīng)力纖維分布;而敲低內(nèi)源性SM22α,再給予TGFβ刺激,則細(xì)胞應(yīng)力纖維稀疏,SM22α熒光強(qiáng)度減弱,彌散分布于細(xì)胞漿中。提示SM22α參與了TGFβ誘導(dǎo)的應(yīng)力纖維形成。采用DHE熒光染色法觀察VSMC O2?-的生成。結(jié)果顯示,TGFβ刺激后,顯示O2?-生成的紅色熒光強(qiáng)度增加;敲低內(nèi)源性SM22α,再給予TGFβ刺激,可見細(xì)胞內(nèi)紅色熒光強(qiáng)度更明顯。說明TGFβ可誘導(dǎo)VSMC產(chǎn)生氧化應(yīng)激,而SM22α對(duì)該過程可能具有抑制作用。結(jié)論: 1 TGFβ誘導(dǎo)血管平滑肌細(xì)胞表達(dá)SM22α; 2 SM22α抑制TGFβ誘導(dǎo)的分化轉(zhuǎn)錄調(diào)節(jié)因子KLF4磷酸化和氧化應(yīng)激; 3 SM22α介導(dǎo)TGFβ誘導(dǎo)的actin應(yīng)力纖維形成。 4 SM22α抑制TGFβ誘導(dǎo)的氧化應(yīng)激 采用DHE熒光染色法觀察VSMC O2?-的生成。結(jié)果顯示,TGFβ刺激后,顯示O2?-生成的紅色熒光強(qiáng)度增加;敲低內(nèi)源性SM22α,再給予TGFβ刺激,可見細(xì)胞內(nèi)紅色熒光強(qiáng)度更明顯。說明TGFβ可誘導(dǎo)VSMC產(chǎn)生氧化應(yīng)激,而SM22α對(duì)該過程可能具有抑制作用。 結(jié)論: 1 TGFβ誘導(dǎo)血管平滑肌細(xì)胞表達(dá)SM22α; 2 SM22α抑制TGFβ誘導(dǎo)的分化轉(zhuǎn)錄調(diào)節(jié)因子KLF4磷酸化和氧化應(yīng)激; 3 SM22α介導(dǎo)TGFβ誘導(dǎo)的actin應(yīng)力纖維形成。
[Abstract]:Objective: vascular smooth muscle cells (vascular smooth muscle cells, VSMC) are non-terminal differentiated cells and have phenotypic remodeling characteristics. SM22 偽 (smooth muscle 22 alpha, SM22 偽 is one of the marker genes in the early stage of VSMC differentiation. Its expression has smooth muscle tissue specificity and cell phenotypic specificity. In this study, we used small interfering RNA (small interfering RNA, siRNA) technique to knock down endogenous SM22 偽 expression, and observed the cytoskeletal fabric of VSMC cells induced by transforming growth factor 尾 (transforming growth factor, TGF 尾 (TGF- 尾). Oxidative stress and the effect of differentiation related transcription factor Kr 眉 ppel like factor 4 (KLF4) activation. Methods: the 3 ~ (th) ~ (th) passage of rat VSMC, was isolated by patch method, the phosphorylation level of KLF4 was detected by immunoprecipitation, and the effect of TGF 尾 on the expression of SM22 偽 and SM 偽-actin was detected by; Western blot analysis. The expression and localization of SM22 偽 in cells were demonstrated by immunofluorescence staining, and the role of SM22 偽 in oxidative stress was determined by DHE staining. Results: 1 TGF 尾 induced the expression of SM22 偽 in vascular smooth muscle cells stimulated by TGF 尾 (2 ng/ml) at different time points, and the expression of SM22 偽 and SM 偽-actin increased in a time dependent manner. The results showed that TGF 尾 could induce the expression of SM22 偽 in vascular smooth muscle cells. 2 SM22 偽 inhibited the phosphorylation of KLF4, a transcription regulator induced by TGF 尾, and the results showed that the KLF4 phosphorylation level of the cells treated with TGF 尾 was significantly higher than that of the cells treated with TGF 尾. After transfection of SM22 偽 -specific siRNA knockout, the phosphorylation of KLF4 induced by TGF 尾 was more obvious than that of control group. These results suggest that SM22 偽 can inhibit KLF4 phosphorylation induced by TGF 尾. 3The formation of actin stress fibers induced by TGF 尾 was mediated by SM22 偽. The results of immunofluorescence staining showed that the VSMC stress fibers treated with TGF 尾 were compact and bunched, and the fluorescence intensity of SM22 偽 was obviously enhanced and distributed along the stress fibers. However, when the endogenous SM22 偽 was knocked down and then stimulated by TGF 尾, the stress fibers were sparse, the fluorescence intensity of SM22 偽 was weakened and distributed in the cytoplasm. It is suggested that SM22 偽 is involved in the formation of stress fibers induced by TGF 尾. The formation of VSMC O _ 2-was observed by DHE fluorescence staining. The results showed that after TGF 尾 stimulation, the red fluorescence intensity of O _ 2 + -producing was increased, and the red fluorescence intensity was more obvious when the endogenous SM22 偽 was knocked down and then stimulated by TGF 尾. It was suggested that TGF 尾 could induce oxidative stress in VSMC, but SM22 偽 might inhibit the process. Conclusion: 1 TGF 尾 induces the expression of SM22 偽 in vascular smooth muscle cells, 2 SM22 偽 inhibits KLF4 phosphorylation and oxidative stress induced by TGF 尾, and 3 SM22 偽 mediates actin stress fiber formation induced by TGF 尾. 4 SM22 偽 inhibited oxidative stress induced by TGF 尾. DHE fluorescence staining was used to observe the formation of VSMC O _ 2 -. The results showed that after TGF 尾 stimulation, the red fluorescence intensity of O _ 2 + -producing was increased, and the red fluorescence intensity was more obvious when the endogenous SM22 偽 was knocked down and then stimulated by TGF 尾. It was suggested that TGF 尾 could induce oxidative stress in VSMC, but SM22 偽 might inhibit the process. Conclusion: 1 TGF 尾 induces the expression of SM22 偽 in vascular smooth muscle cells, 2 SM22 偽 inhibits KLF4 phosphorylation and oxidative stress induced by TGF 尾, and 3 SM22 偽 mediates actin stress fiber formation induced by TGF 尾.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329
本文編號(hào)：2370821
[Abstract]:Objective: vascular smooth muscle cells (vascular smooth muscle cells, VSMC) are non-terminal differentiated cells and have phenotypic remodeling characteristics. SM22 偽 (smooth muscle 22 alpha, SM22 偽 is one of the marker genes in the early stage of VSMC differentiation. Its expression has smooth muscle tissue specificity and cell phenotypic specificity. In this study, we used small interfering RNA (small interfering RNA, siRNA) technique to knock down endogenous SM22 偽 expression, and observed the cytoskeletal fabric of VSMC cells induced by transforming growth factor 尾 (transforming growth factor, TGF 尾 (TGF- 尾). Oxidative stress and the effect of differentiation related transcription factor Kr 眉 ppel like factor 4 (KLF4) activation. Methods: the 3 ~ (th) ~ (th) passage of rat VSMC, was isolated by patch method, the phosphorylation level of KLF4 was detected by immunoprecipitation, and the effect of TGF 尾 on the expression of SM22 偽 and SM 偽-actin was detected by; Western blot analysis. The expression and localization of SM22 偽 in cells were demonstrated by immunofluorescence staining, and the role of SM22 偽 in oxidative stress was determined by DHE staining. Results: 1 TGF 尾 induced the expression of SM22 偽 in vascular smooth muscle cells stimulated by TGF 尾 (2 ng/ml) at different time points, and the expression of SM22 偽 and SM 偽-actin increased in a time dependent manner. The results showed that TGF 尾 could induce the expression of SM22 偽 in vascular smooth muscle cells. 2 SM22 偽 inhibited the phosphorylation of KLF4, a transcription regulator induced by TGF 尾, and the results showed that the KLF4 phosphorylation level of the cells treated with TGF 尾 was significantly higher than that of the cells treated with TGF 尾. After transfection of SM22 偽 -specific siRNA knockout, the phosphorylation of KLF4 induced by TGF 尾 was more obvious than that of control group. These results suggest that SM22 偽 can inhibit KLF4 phosphorylation induced by TGF 尾. 3The formation of actin stress fibers induced by TGF 尾 was mediated by SM22 偽. The results of immunofluorescence staining showed that the VSMC stress fibers treated with TGF 尾 were compact and bunched, and the fluorescence intensity of SM22 偽 was obviously enhanced and distributed along the stress fibers. However, when the endogenous SM22 偽 was knocked down and then stimulated by TGF 尾, the stress fibers were sparse, the fluorescence intensity of SM22 偽 was weakened and distributed in the cytoplasm. It is suggested that SM22 偽 is involved in the formation of stress fibers induced by TGF 尾. The formation of VSMC O _ 2-was observed by DHE fluorescence staining. The results showed that after TGF 尾 stimulation, the red fluorescence intensity of O _ 2 + -producing was increased, and the red fluorescence intensity was more obvious when the endogenous SM22 偽 was knocked down and then stimulated by TGF 尾. It was suggested that TGF 尾 could induce oxidative stress in VSMC, but SM22 偽 might inhibit the process. Conclusion: 1 TGF 尾 induces the expression of SM22 偽 in vascular smooth muscle cells, 2 SM22 偽 inhibits KLF4 phosphorylation and oxidative stress induced by TGF 尾, and 3 SM22 偽 mediates actin stress fiber formation induced by TGF 尾. 4 SM22 偽 inhibited oxidative stress induced by TGF 尾. DHE fluorescence staining was used to observe the formation of VSMC O _ 2 -. The results showed that after TGF 尾 stimulation, the red fluorescence intensity of O _ 2 + -producing was increased, and the red fluorescence intensity was more obvious when the endogenous SM22 偽 was knocked down and then stimulated by TGF 尾. It was suggested that TGF 尾 could induce oxidative stress in VSMC, but SM22 偽 might inhibit the process. Conclusion: 1 TGF 尾 induces the expression of SM22 偽 in vascular smooth muscle cells, 2 SM22 偽 inhibits KLF4 phosphorylation and oxidative stress induced by TGF 尾, and 3 SM22 偽 mediates actin stress fiber formation induced by TGF 尾.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329
【參考文獻(xiàn)】
相關(guān)期刊論文 前4條
1 史建紅;溫進(jìn)坤;韓梅;;SM22α在細(xì)胞骨架組構(gòu)及血管重塑中的作用[J];生理科學(xué)進(jìn)展;2006年03期
2 程云會(huì),韓梅,溫進(jìn)坤;SM22α對(duì)血管平滑肌細(xì)胞骨架及收縮功能的影響[J];細(xì)胞生物學(xué)雜志;2003年06期
3 彭炎強(qiáng),紀(jì)玉蓮,姜宗培,余學(xué)清,杜勇;抑制NAD(P)H氧化酶表達(dá)和活性對(duì)大鼠糖尿病腎病的影響[J];中山大學(xué)學(xué)報(bào)(醫(yī)學(xué)科學(xué)版);2004年03期
4 張海燕;姜宗培;常潔;李曉艷;朱恒梅;余學(xué)清;;NADPH氧化酶在TGF-β_1誘導(dǎo)大鼠小管上皮細(xì)胞表達(dá)炎癥因子中的作用[J];中山大學(xué)學(xué)報(bào)(醫(yī)學(xué)科學(xué)版);2008年03期
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