SM22α在TGFβ诱导的血管平滑肌细胞表型重塑中的作用
发布时间：2018-12-10 15:37
【摘要】：目的:血管平滑肌细胞(vascular smooth muscle cells, VSMC)是非终末分化细胞,具有表型重塑的特性。SM22α(smooth muscle 22 alpha, SM22α)是VSMC分化早期的标志基因之一,其表达具有平滑肌组织特异性和细胞表型特异性。本研究利用小干扰RNA(small interfering RNA, siRNA)技术敲低内源性SM22α表达,观察对转化生长因子β(transforming growth factor, TGFβ)诱导的VSMC细胞骨架组构、氧化应激以及分化相关转录因子Krüppel样因子4(krüppel-like factor 4, KLF4)活化的影响。 方法:用贴块法分离、培养大鼠VSMC,取3～5代细胞进行实验;用免疫沉淀检测KLF4的磷酸化水平;Western blot分析检测TGFβ对SM22α和SMα-actin表达的影响;用免疫荧光染色显示细胞内SM22α表达和定位;用DHE染色确定SM22α在氧化应激中的作用。 结果: 1 TGFβ诱导血管平滑肌细胞表达SM22α用TGFβ(2 ng/ml)刺激VSMC不同时间,SM22α、SMα-actin的表达水平呈时间依赖性升高。表明TGFβ能诱导血管平滑肌细胞SM22α表达。 2 SM22α抑制TGFβ诱导的分化转录调节因子KLF4磷酸化Western blot结果显示,TGFβ处理的细胞,其KLF4的磷酸化水平明显升高。转染SM22α特异的siRNA敲低内源性SM22α表达后, TGFβ诱导的KLF4磷酸化较对照组更加明显。提示SM22α可抑制TGFβ诱导的KLF4磷酸化。 3 SM22α介导TGFβ诱导的actin应力纤维的形成 免疫荧光染色结果显示,TGFβ处理的VSMC应力纤维致密、成束,SM22α荧光强度明显增强,沿应力纤维分布;而敲低内源性SM22α,再给予TGFβ刺激,则细胞应力纤维稀疏,SM22α荧光强度减弱,弥散分布于细胞浆中。提示SM22α参与了TGFβ诱导的应力纤维形成。采用DHE荧光染色法观察VSMC O2?-的生成。结果显示,TGFβ刺激后,显示O2?-生成的红色荧光强度增加;敲低内源性SM22α,再给予TGFβ刺激,可见细胞内红色荧光强度更明显。说明TGFβ可诱导VSMC产生氧化应激,而SM22α对该过程可能具有抑制作用。结论: 1 TGFβ诱导血管平滑肌细胞表达SM22α; 2 SM22α抑制TGFβ诱导的分化转录调节因子KLF4磷酸化和氧化应激; 3 SM22α介导TGFβ诱导的actin应力纤维形成。 4 SM22α抑制TGFβ诱导的氧化应激 采用DHE荧光染色法观察VSMC O2?-的生成。结果显示,TGFβ刺激后,显示O2?-生成的红色荧光强度增加;敲低内源性SM22α,再给予TGFβ刺激,可见细胞内红色荧光强度更明显。说明TGFβ可诱导VSMC产生氧化应激,而SM22α对该过程可能具有抑制作用。 结论: 1 TGFβ诱导血管平滑肌细胞表达SM22α; 2 SM22α抑制TGFβ诱导的分化转录调节因子KLF4磷酸化和氧化应激; 3 SM22α介导TGFβ诱导的actin应力纤维形成。
[Abstract]:Objective: vascular smooth muscle cells (vascular smooth muscle cells, VSMC) are non-terminal differentiated cells and have phenotypic remodeling characteristics. SM22 伪 (smooth muscle 22 alpha, SM22 伪 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 伪 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 伪 and SM 伪-actin was detected by; Western blot analysis. The expression and localization of SM22 伪 in cells were demonstrated by immunofluorescence staining, and the role of SM22 伪 in oxidative stress was determined by DHE staining. Results: 1 TGF 尾 induced the expression of SM22 伪 in vascular smooth muscle cells stimulated by TGF 尾 (2 ng/ml) at different time points, and the expression of SM22 伪 and SM 伪-actin increased in a time dependent manner. The results showed that TGF 尾 could induce the expression of SM22 伪 in vascular smooth muscle cells. 2 SM22 伪 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 伪 -specific siRNA knockout, the phosphorylation of KLF4 induced by TGF 尾 was more obvious than that of control group. These results suggest that SM22 伪 can inhibit KLF4 phosphorylation induced by TGF 尾. 3The formation of actin stress fibers induced by TGF 尾 was mediated by SM22 伪. 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 伪 was obviously enhanced and distributed along the stress fibers. However, when the endogenous SM22 伪 was knocked down and then stimulated by TGF 尾, the stress fibers were sparse, the fluorescence intensity of SM22 伪 was weakened and distributed in the cytoplasm. It is suggested that SM22 伪 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 伪 was knocked down and then stimulated by TGF 尾. It was suggested that TGF 尾 could induce oxidative stress in VSMC, but SM22 伪 might inhibit the process. Conclusion: 1 TGF 尾 induces the expression of SM22 伪 in vascular smooth muscle cells, 2 SM22 伪 inhibits KLF4 phosphorylation and oxidative stress induced by TGF 尾, and 3 SM22 伪 mediates actin stress fiber formation induced by TGF 尾. 4 SM22 伪 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 伪 was knocked down and then stimulated by TGF 尾. It was suggested that TGF 尾 could induce oxidative stress in VSMC, but SM22 伪 might inhibit the process. Conclusion: 1 TGF 尾 induces the expression of SM22 伪 in vascular smooth muscle cells, 2 SM22 伪 inhibits KLF4 phosphorylation and oxidative stress induced by TGF 尾, and 3 SM22 伪 mediates actin stress fiber formation induced by TGF 尾.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
本文编号：2370821
[Abstract]:Objective: vascular smooth muscle cells (vascular smooth muscle cells, VSMC) are non-terminal differentiated cells and have phenotypic remodeling characteristics. SM22 伪 (smooth muscle 22 alpha, SM22 伪 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 伪 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 伪 and SM 伪-actin was detected by; Western blot analysis. The expression and localization of SM22 伪 in cells were demonstrated by immunofluorescence staining, and the role of SM22 伪 in oxidative stress was determined by DHE staining. Results: 1 TGF 尾 induced the expression of SM22 伪 in vascular smooth muscle cells stimulated by TGF 尾 (2 ng/ml) at different time points, and the expression of SM22 伪 and SM 伪-actin increased in a time dependent manner. The results showed that TGF 尾 could induce the expression of SM22 伪 in vascular smooth muscle cells. 2 SM22 伪 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 伪 -specific siRNA knockout, the phosphorylation of KLF4 induced by TGF 尾 was more obvious than that of control group. These results suggest that SM22 伪 can inhibit KLF4 phosphorylation induced by TGF 尾. 3The formation of actin stress fibers induced by TGF 尾 was mediated by SM22 伪. 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 伪 was obviously enhanced and distributed along the stress fibers. However, when the endogenous SM22 伪 was knocked down and then stimulated by TGF 尾, the stress fibers were sparse, the fluorescence intensity of SM22 伪 was weakened and distributed in the cytoplasm. It is suggested that SM22 伪 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 伪 was knocked down and then stimulated by TGF 尾. It was suggested that TGF 尾 could induce oxidative stress in VSMC, but SM22 伪 might inhibit the process. Conclusion: 1 TGF 尾 induces the expression of SM22 伪 in vascular smooth muscle cells, 2 SM22 伪 inhibits KLF4 phosphorylation and oxidative stress induced by TGF 尾, and 3 SM22 伪 mediates actin stress fiber formation induced by TGF 尾. 4 SM22 伪 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 伪 was knocked down and then stimulated by TGF 尾. It was suggested that TGF 尾 could induce oxidative stress in VSMC, but SM22 伪 might inhibit the process. Conclusion: 1 TGF 尾 induces the expression of SM22 伪 in vascular smooth muscle cells, 2 SM22 伪 inhibits KLF4 phosphorylation and oxidative stress induced by TGF 尾, and 3 SM22 伪 mediates actin stress fiber formation induced by TGF 尾.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
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