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目的以RNA干扰抑制血管平滑肌细胞(vascular smooth muscle cells,VSMCs)ORC1基因,探讨ORC1基因表达抑制后对VSMCs表型的影响。方法实验设置正常对照组、阴性siRNA组及阳性siRNA组。应用Western blot检测ORC1基因表达的变化;免疫荧光染色后激光共聚焦显微镜观察细胞形态结构;应用流式细胞仪检测细胞表型标志蛋白表达。结果①siRNA转染后,阳性siRNA组ORC1基因表达水显著降低,而正常对照组及阴性siRNA组间ORC1基因表达水平无显著差异。②siRNA转染后,阳性转染组细胞呈现收缩型形态特征,空白对照组及阴性对照组细胞呈现合成型形态特征。③siRNA转染使ORC1表达减弱后,VSMCs收缩型标志物α-平滑肌肌动蛋白(α-SM actin)和平滑肌肌动蛋白重链2(SM-2)表达水平明显升高,合成型标志物骨桥蛋白(osteopontin)表达水平明显降低,空白对照组及阴性对照组间3种标志物表达水平无显著差异。结论RNA干扰介导的ORC1基因沉寂可促使VSMCs由合成型向收缩型转化。
OBJECTIVE: To investigate the effect of RNA interference on the expression of ORC1 in vascular smooth muscle cells (VSMCs) and to explore the effect of ORC1 gene knockdown on VSMCs phenotype. Methods The experiment set up normal control group, negative siRNA group and positive siRNA group. The changes of ORC1 gene expression were detected by Western blot. The morphology of cells was observed by confocal laser scanning microscopy after immunofluorescence staining. The expression of phenotypic marker protein was detected by flow cytometry. Results ① After siRNA transfection, the expression of ORC1 mRNA in the positive siRNA group was significantly decreased, while the expression level of ORC1 gene in the normal control group and the negative siRNA group was not significantly different. ② After siRNA transfection, the positive transfected cells showed contractile morphological characteristics, and the blank control group and negative control group showed synthetic morphological features. ③ The expression of VSMCs contractile markers α-SM actin and SM-2 in VSMCs was significantly increased after siRNA transfection reduced ORC1 expression. The expression of synthetic marker bone The expression of osteopontin was significantly decreased, while there was no significant difference in the expression of three markers between blank control group and negative control group. Conclusions RNA interference-mediated silencing of ORC1 can induce VSMCs to transform from systolic to contractile.