Kindlin-2对血管平滑肌细胞迁移和黏附的影响及其机制的实验研究

Kindlin-2 regulates migration and adhesion of vascular smooth muscle cells via β1-integrin

目的 探讨Kindlin-2 RNA干扰对血管平滑肌细胞（VSMC）迁移、黏附以及β1整合素的影响,并进一步探讨Kindlin-2与β1整合素之间的关系.方法 构建Kindlin-2小干扰RNA（siRNA）慢病毒载体感染原代培养的VSMC.实验分为空白对照组、阴性对照组和Kindlin-2 siRNA组.通过Transwell和细胞划痕实验检测VSMC的迁移能力.细胞基质黏附实验检测VSMC黏附细胞外基质的能力.实时定量PCR和Western blot分别检测Kindlin-2与β1整合素的mRNA和蛋白表达水平.流式细胞技术检测VSMC表面总β1整合素和激活β1整合素的表达.结果 Kindlin-2siRNA慢病毒感染VSMC的效率达90％以上.Kindlin-2 siRNA组VSMC迁移数量低于空白对照组和阴性对照组（P均＜0.05）,且VSMC迁移的距离较空白对照组和阴性对照组短（P均＜0.05）.Kindlin-2 siRNA组VSMC黏附胶原Ⅰ的数量较空白对照组和阴性对照组少（P均＜0.05）,另外A590nm也较空白对照组和阴性对照组低（P均＜0.05）.Kindlin-2 siRNA组Kindlin-2 mRNA表达水平较空白对照组低了47％ （P＜0.05）,但β1整合素mRNA表达水平与空白对照组比较差异无统计学意义.Kindlin-2 siRNA组Kindlin-2蛋白表达水平较空白对照组和阴性对照组低（P均＜0.05）,3组间β1整合素蛋白表达水平差异无统计学意义.Kindlin-2 siRNA组VSMC表面激活β1整合素表达水平低于空白对照组和阴性对照组（P均＜0.05）,而细胞表面总β1整合素表达水平与空白对照组和阴性对照组比较差异均无统计学意义.结论 Kindlin-2可以调节VSMC的迁移和黏附,激活VSMC表面β1整合素.

Objective To explore the effects of Kindlin-2 RNA interference on vascular smooth muscle cells （VSMCs） migration,adhesion and β1-integrin as well as the relationship between Kindlin-2 and β1-integrin.Methods Primary VSMCs were cultured,infected with Kindlin-2 siRNA lentiviral vectors.VSMCs were divided into three groups：the blank control group,the negative control group and the Kindlin-2 siRNA group.The ability of VSMCs migration was measured by Transwell experiment and wound healing assay.The ability of VSMCs adhesion to extracelluar matrix was determined by cell-extracelluar matrix adhesion assay.The Kindlin-2 and β1-integrin mRNA and protein expression levels were detected by real-time quantitative PCR and Western blot.Total β1-integrin and active β1-integrin expression on the surface of VSMCs was evaluated by flow cytometry.Results The efficiency of Kindlin-2 siRNA lentivirus infected VSMCs was more than 90％.The number of VSMCs migration in the Kindlin-2 siRNA group was significantly lower than that of the blank control group and the negative group （all P ＜ 0.05）.Moreover,the distance of VSMCs migration was shorter than that of the blank control group and the negative group （all P ＜0.05）.The number of VSMCs adhesion to collagen Ⅰ was less than that of the blank control group and the negative group （all P ＜ 0.05）.A590nm of the Kindlin-2 siRNA group was also lower than that of the blank control group and the negative group （all P ＜ 0.05）.Compared with the blank control group,the expression level of Kindlin-2 mRNA in the Kindlin-2 siRNA group decreased 47％ （P ＜ 0.05）,but the expression level of β1-integrin mRNA remained unchanged.The Kindlin-2 protein level in the Kindlin-2 siRNA group was lower than that of the blank control group and the negative group （all P ＜ 0.05）.β1-integrin protein level was similar among the three groups.Activated β1-integrin on the surface of VSMCs in the Kindlin-2siRNA group was lower than that of the blank control group and the negative group （all P ＜ 0.05）.However,the expression level of total β1-integrin on the VSMCs surface was similar among the three groups.Conclusion Kindin-2 can regulate VSMCs migration and adhesion and activate β1-integrin on the surface of VSMCs.