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

Effect and Mechanism of Rapamycin Inhibiting mTOR on Heart Valve Cell Calcification in Experimental Rats

目的:探讨雷帕霉素靶向抑制m TOR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:m TOR抑制剂雷帕霉素通过抑制m TOR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m TOR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 （roTOR） on heart valve cell calcification in experimental rats. Methods： The rat＇s valvular interstitial cells were isolated and the cells were cultured in 4 groups： （1） Normal control group,（2） Calcification group, （3） Rapamycin group and （4） Calcification ＋ 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 calcified nodule in Calcification group （0.471 ± 0.091） was more than Normal control group （0.104 ± 0.023）, while the nodule in Calcification ＋ rapamycin group （0.237 ± 0.039） was less than Calcification group, all P〈0.05. Compared with Normal control group, the protein expression levels of bmp-2, osteopontin and smad-I were obviously increased in Calcificationgroup, P〈0.05, and compared with Calcification group, the above indexes were obviously lower in Calcification ＋ 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 roTOR, therefore, reducing the valvular interstitial cell calcification which might be related to roTOR pathway suppression in experimental rats.