雷帕霉素通过促进细胞自噬抑制大鼠血管平滑肌细胞增殖活性效应  

Rapamycin inhibits proliferation of rat vascular smooth muscle cells by promoting autophagy

作　　者：邬文莉 卢志远 黎凡 王洪涛 郝建锁 高梓君 陈亦阳 Wu Wenli;Lu Zhiyuan;Li Fan;Wang Hongtao;Hao Jiansuo;Gao Zijun;Chen Yiyang(Department of Oral and Maxillofacial Surgery,Stomatology Medical Center,Guangzhou Women and Children′s Medical Center,Guangzhou 510623,China)

机构地区：[1]广州市妇女儿童医疗中心口腔颌面外科,广州510623

出　　处：《中华口腔医学研究杂志（电子版）》2022年第1期12-20,共9页Chinese Journal of Stomatological Research(Electronic Edition)

基　　金：广州市妇女儿童医疗中心科研基金(YIP⁃2019⁃038)。

摘　　要：目的:探讨雷帕霉素(RAP)抑制表现异常增殖活性的大鼠血管平滑肌细胞(VSMC)增殖活性的作用及其分子机制。方法:使用不同质量浓度的血小板衍生生长因子(PDGF)作为诱导剂处理大鼠主动脉VSMC,构建模拟脉管畸形的体外模型。实验分为对照组、PDGF组、PDGF+RAP组和PDGF+VEC(血管内皮细胞)组,观察对VSMC增殖活性的改变,检测不同处理方式对VSMC自噬水平的影响。两组间比较应用独立样本t检验,多组间比较应用重复测量方差分析,多组mRNA表达量、蛋白表达量比较应用单因素方差分析。结果:细胞增殖检测(CCK-8)和EdU实验结果显示:PDGF处理的VSMC活性呈时间依赖性上升。与PDGF组相比,在48和72 h,PDGF+RAP组细胞增殖活性分别降低了24.8%(0.129±0.010比0.172±0.012,t=4.787,P=0.009)和45.1%(0.170±0.012比0.292±0.046,t=4.431,P=0.011);PDGF+VEC组细胞增殖活性分别降低了10.0%(0.155±0.013比0.172±0.012,t=2.357,P=0.076)和8.9%(0.266±0.022比0.292±0.046,t=1.718,P=0.161)。实时荧光定量PCR和蛋白免疫印迹(Western blot)结果显示,与对照组比较,PDGF可抑制平滑肌蛋白抗体-B(Smoothelin-B)表达(0.486±0.057比1.005±0.067,t=10.192,P=0.001),促进细胞视黄醇结合蛋白-1(CRBP-1)表达(3.185±0.091比0.991±0.056,t=35.461,P<0.001);而在PDGF+RAP组和PDGF+VEC组,Smoothelin-B表达增加(0.857±0.091比0.486±0.057,t=5.943,P=0.004;0.563±0.067比0.486±0.057,t=1.501,P=0.208),CRBP-1表达减少(1.579±0.042比3.185±0.091,t=27.707,P<0.001;2.951±0.144比3.185±0.091,t=2.382,P=0.076)。与对照组比较,PDGF组LC3B(LC3-Ⅱ/Ⅰ比值)明显下降(0.175±0.003比1.020±0.020,t=72.263,P<0.001),p62表达显著上升(2.632±0.098比1.005±0.007,t=28.562,P=0.001)。与PDGF组比较,PDGF+RAP组的LC3B明显升高(0.316±0.037比0.175±0.003,t=6.529,P=0.022),p62蛋白显著降低(1.396±0.070比2.632±0.098,t=17.771,P<0.001);PDGF+VEC组LC3B稍有上升(0.206±0.014比0.175±0.003,t=3.687,P=0.021),而p62蛋白表达降低(2.400±0.076比Objective To investigate the effect and molecular mechanism of rapamycin(RAP)on inhibiting the proliferation of rat vascular smooth muscle cells(VSMCs)with abnormal proliferation activity.Methods Rat aortic VSMCs were treated with different concentrations of platelet⁃derived growth factor(PDGF)as an inducer to construct an in vitro model of vascular malformation.They were divided into Blank group,PDGF group,PDGF+RAP group and PDGF+VECs(vascular endothelial cells)group.The changes in the proliferation activity of VSMCs were observed and the effects of different treatments on the autophagy level of VSMCs were detected.The independent sample t test was used for comparison between two groups and the repeated measures analysis of variance was used for comparison among multiple groups.The mRNA and protein expression among multiple groups were compared via One⁃Way analysis of variance.Results The results of CCK⁃8 and EdU experiments showed that the activity of VSMCs treated with PDGF significantly increased in a time⁃dependent manner.Compared with the PDGF group,at 48 and 72 hours,the cell proliferation activity of the PDGF+RAP group was reduced by 24.8%(0.129±0.010 vs.0.172±0.012,t=4.787,P=0.009)and 45.1%(0.170±0.012 vs.0.292±0.046,t=4.431,P=0.011),respectively.The cell proliferation activity of PDGF+VECs group decreased by 10.0%(0.155±0.013 vs.0.172±0.012,t=2.357,P=0.076)and 8.9%(0.266±0.022 vs.0.292±0.046,t=1.718,P=0.161),respectively.Real⁃time quantitative PCR and Western blot results showed that,compared with the Blank group,PDGF could inhibit the expression of Smoothelin⁃B(0.486±0.057 vs.1.005±0.067,t=10.192,P=0.001)and promote the expression of cellular retinol binding protein⁃1(CRBP⁃1)(3.185±0.091 vs.0.991±0.056,t=35.461,P<0.001).In the PDGF+RAP group and PDGF+VECs group,the expression of Smoothelin⁃B increased(0.857±0.091 vs.0.486±0.057,t=5.943,P=0.004;0.563±0.067 vs.0.486±0.057,t=1.501,P=0.208)and the expression of CRBP⁃1 decreased(1.579±0.042 vs.3.185±0.091,t=27.707,P<0.0

关 键 词：西罗莫司(雷帕霉素) 脉管畸形 自噬 哺乳动物雷帕霉素靶蛋白 

分 类 号：R73[医药卫生—肿瘤]