siRNA沉默Rac1表达促进增生期血管瘤内皮细胞内质网应激和抑制细胞增殖的作用与机制研究

目的:研究慢病毒介导Rac1沉默对增生期血管瘤内皮细胞内质网应激及Caspase-12活化水平的影响。方法:用Rac1 siRNA慢病毒载体和阴性对照慢病毒载体感染人增生期血管瘤内皮细胞，用Realtime PCR和Western blot检测细胞中Rac1表达变化。MTT法测定细胞增殖能力，PI单染法检测细胞周期变化，Annexin V-FITC/PI双染法检测细胞凋亡变化，Western blot检测细胞中周期蛋白p21、细胞周期蛋白D1（Cyclin D1）和凋亡蛋白活化的Caspase-3（C-Caspase-3）表达水平，同时检测细胞中内质网应激相关蛋白CCAAT/增强子结合蛋白同源蛋白(CHOP)、活化的Caspase-12（C-Caspase-12）表达水平。结果:Rac1 siRNA慢病毒载体感染后的血管瘤内皮细胞中Rac1表达水平明显降低，阴性对照慢病毒对细胞中Rac1表达水平没有影响。沉默Rac1后的增生期血管瘤内皮细胞增殖能力下降，细胞G0/G1比例升高，p21蛋白水平升高，Cyclin D1蛋白水平下降，凋亡率升高，C-Caspase-3蛋白水平升高，CHOP、C-Caspase-12蛋白水平也升高，与没有感染慢病毒的细胞比较，差异有统计学意义（P＜0.05）。阴性对照慢病毒感染对血管瘤内皮细胞增殖、凋亡、细胞周期及细胞中p21、Cyclin D1、C-Caspase-3、CHOP、C-Caspase-12蛋白水平没有影响。结论:慢病毒介导Rac1沉默诱导增生期血管瘤内皮细胞凋亡，阻滞细胞周期，诱导内质网应激发生，诱导Caspase-12活化。

siRNA silencing Rac1 expression promotes endothelial cell endoplasmic reticulum stress and inhibits cell proliferation in proliferative hemangioma

Objective:To investigate the effect of lentivirus mediated Rac1 silencing on endoplasmic reticulum stress and Caspase-12 activation in proliferative hemangioma endothelial cells.Methods:Human proliferative hemangioma endothelial cells were infected by Rac1 siRNA lentiviral vector and negative control lentivirus vector.The expression of Rac1 in cells was detected by Realtime PCR and Western blot.MTT assay was used to determine cell proliferation,and PI single staining was used to detect cell cycle changes.Annexin V-FITC/PI double staining was used to detect apoptosis,and the expression levels of cyclin p21,Cyclin D1 and apoptotic protein C-Caspase-3 were detected by Western blot.Meanwhile,the expression levels of endoplasmic reticulum stress related protein CHOP and C-Caspase-12 were detected.Results:The expression level of Rac1 in the endothelial cells of hemangioma after Rac1 siRNA lentivirus carrier infection was significantly decreased,and the negative control lentivirus had no effect on the expression of Rac1 in the cells.The proliferative ability of proliferative hemangioma after Rac1 silence decreased,the proportion of G0/G1 in cells increased,the level of p21 protein elevated,the level of Cyclin D1 protein decreased,the rate of apoptosis increased,the level of C-Caspase-3 protein elevated,the levels of CHOP and C-Caspase-12 protein also increased.Compared with the cells that have not infected the lentivirus,the difference was statistically significant (P＜0.05).Negative control lentivirus infection has no effect on the proliferation,apoptosis,cell cycle and the level of p21,Cyclin D1,C-Caspase-3,CHOP and C-Caspase-12 in the endothelial cells of hemangioma.Conclusion:Lentivirus mediated Rac1 silencing induces apoptosis in proliferative hemangioma endothelial cells,blocks cell cycle,induces endoplasmic reticulum stress,and the activation of Caspase-12 is induced.