探讨雷帕霉素靶向抑制mTOR对大鼠心瓣膜细胞钙化的影响及其作用机制

摘要

目的：探讨雷帕霉素靶向抑制mTOR对大鼠心瓣膜细胞钙化的影响及其作用机制。方法：体外分离大鼠心瓣膜间质细胞后培养并鉴定；细胞共分为4组：正常对照组、钙化诱导组、雷帕霉素组、钙化诱导+雷帕霉素组；采用流式细胞术检测各组细胞凋亡率；采用茜素红S染色并观察钙沉积的变化，对细胞进行钙化结节计数；蛋白免疫印迹法（Western blot）检测各组细胞中骨形成蛋白-2（bmp-2），骨钙素（osteocalcin），骨调素（osteopontin），smad同源物1（smad1）及半胱氨酸蛋白酶（caspase-3）的表达水平。结果：成功分离获得大鼠心瓣膜间质细胞；各组细胞凋亡率没有明显差异（P>0.05）；钙化诱导组细胞钙化结节数明显高于正常对照组（P<0.05）；钙化诱导组细胞钙化结节计数（0.471±0.091）较正常对照组（0.104±0.023）多，钙化诱导+雷帕霉素组细胞钙化结节计数（0.237±0.039）明显少于钙化诱导组，差异均有统计学意义（P<0.05）；钙化诱导组细胞的骨形成蛋白-2，骨钙素、smad同源物1和骨调素蛋白的表达水平明显高于正常对照组（P<0.05），钙化诱导+雷帕霉素组的骨钙素、smad同源物1和骨调素蛋白的表达水平明显低于钙化诱导组（P<0.05），但3个实验组间caspase-3蛋白表达水平差异均无统计学意义（P>0.05）。结论：mTOR抑制剂雷帕霉素通过抑制mTOR后下调其靶蛋白骨形成蛋白-2、骨钙素、smad同源物1和骨调素蛋白的表达水平，从而抑制大鼠心瓣膜细胞的钙化，即细胞钙化变缓很可能与mTOR通路被抑制有密切的关系。%Objective: To investigate the effect and mechanism of rapamycin inhibiting mammalian target of RAPA (mTOR) on heart valve cell calciifcation in experimental rats. Methods: The rat’s valvular interstitial cells were isolated and the cells were cultured in 4 groups:①Normal control group,②Calciifcation group,③Rapamycin group and ④Calciifcation + rapamycin group. The apoptosis rates of valvular interstitial cells were detected by flow cytometry, calcium deposition was observed by Alizarin S staining, the calcified nodules were counted and the protein expressions of bmp-2, osteocalcin, osteopontin, smad-1 and caspase-3 were examined by Western blot analysis. Results: The rat's valvular interstitial cells were suceessfully isolated; the cell apoptosis rates were similar among different groups,P>0.05. The calciifed nodule in Calciifcation group (0.471 ± 0.091) was more than Normal control group (0.104 ± 0.023), while the nodule in Calciifcation + rapamycin group (0.237 ± 0.039) was less than Calciifcation group, allP<0.05. Compared with Normal control group, the protein expression levels of bmp-2, osteopontin and smad-1 were obviously increased in Calciifcation group,P<0.05, and compared with Calciifcation group, the above indexes were obviously lower in Calciifcation + rapamycin group, P<0.05. The protein expression levels of caspase-3 were similar among 3 experimental groups,P>0.05. Conclusion: Rapamycin may down-regulate the targeting protein expressions of bmp-2, osteopontin and smad-1 via inhibiting mTOR, therefore, reducing the valvular interstitial cell calcification which might be related to mTOR pathway suppression in experimental rats.