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目的以RNA干扰抑制血管平滑肌细胞(vascular smooth muscle cells,VSMCs)Geminin基因的表达观测VSMCs增殖情况及表型标志物。方法分别利用RT-PCR,Western blot检测VSMCs中Geminin基因、蛋白表达的变化,筛选出干扰效果最为显著的序列用于构建慢病毒包裹的稳定干扰模型。应用[3H]-TdR、EdU掺入试验检测VSMCs增殖的情况;同时使用Western blot分别检测干扰前后相关标志性基因的表达。结果①siRNA转染后,以第1亚组干扰效果最为显著,与另外2个阳性亚组及对照组比较差异具有统计学意义(P<0.05)。②[3H]-TdR、EdU掺入实验显示:阳性干扰组的掺入量较两对照组明显升高(P<0.05)。③siRNA转染使Geminin基因表达减弱后,阳性组VSMCs合成型标志物骨桥蛋白(osteopontin,OPN)表达水平显著升高,收缩型标志物平滑肌肌动蛋白(smooth muscle actin,α-Actin)表达水平明显降低,与对照组间比较,差异均有统计学意义(P<0.05)。结论 RNA干扰介导的Geminin基因沉寂可显著增强VSMCs的增殖能力,并有助于VSMCs的表型由收缩型向合成型转化。
OBJECTIVE: To observe the proliferation of VSMCs and the phenotypic markers by using RNA interference to suppress the expression of Geminin gene in vascular smooth muscle cells (VSMCs). Methods The changes of Geminin gene and protein expression in VSMCs were detected by RT-PCR and Western blot, respectively. The sequences with the most significant interference effect were screened out to construct the stable interference model of lentivirus parvovirus. The [3H] -TdR and EdU incorporation assays were used to detect the proliferation of VSMCs. Western blot was used to detect the expression of related landmark genes before and after interference. Results (1) After siRNA transfection, the interference effect of the first subgroup was the most significant, and the difference was statistically significant compared with the other two positive subgroups and the control group (P <0.05). ② [3H] -TdR, EdU incorporation experiments showed that the amount of positive interference group was significantly higher than the two control groups (P <0.05). ③ The expression of osteopontin (OPN), a synthetic marker of VSMCs in positive group, was significantly increased after the expression of Geminin gene was reduced by siRNA transfection. The expression level of contractile markers smooth muscle actin (α-Actin) Significantly lower than the control group, the difference was statistically significant (P <0.05). Conclusion RNA interference mediated silencing of Geminin gene can significantly enhance the proliferation of VSMCs and help VSMCs transform from contractile to synthetic.