血管紧张素转换酶2基因转染抑制大鼠平滑肌细胞血管紧张素Ⅱ1型受体表达及下游转录激活子3信号通路

摘要

Objective To explore the effects of recombinated lentiviral angiotensin-converting enzyme 2 ( ACE2 ) vector transfer on the expression of angiotnsin Ⅱ type 1 ( AT1 ) receptor in cultured vascular smooth muscle cells (VSMCs).Methods VSMCs were divided into 7 groups:( 1 ) Control:serumfree culture medium; (2) Lentiviral-GFP vector group:Lentiviral-GFP vector ( MOI =10) ; ( 3 ) Ang Ⅱ group ( 10-7 mol/L) ;(4)Ang Ⅱ ( 10-7 mol/L) + Lentiviral-ACE2 ( MOI =10) group; (5)Ang Ⅱ ( 10-7 mol/L) +Irbesartan (10-7 mol/L) group ; (6) Ang Ⅱ ( 10-7 mol/L) + irbesartan (10-7 mol/L) + Lentiviral-ACE2 ( MOI =10) group ; ( 7 ) Lentiviral-ACE2 ( MOI =10 ) group.Ninety-six hours later,the proliferation of VSMCs was determined with CCK-8 Kit.AT1 receptor mRNA and protein expressions were detected with quantitative real-time PCR and Western blot,the signaling pathway of signal transducer and activator of transcription 3 (STAT3) was also detected.Results ACE2 gene transfer significantly inhibited the VSMCs proliferation in the absence or presence of Ang Ⅱ.AT1 receptor mRNA and protein expressions were also significantly downregulated in the absence or presence of Ang Ⅱ.Similar to AT1 receptor mRNA and protein expression changes, STAT3 phosphorylation was also significantly inhibited by ACE2 overexpression.Conclusion Our results suggest that overexpression of ACE2 gene could inhibit the VSMCs proliferation by downregulating AT1 receptor expression and STAT3 phosphorylation. ACE2 could also directly inhibit AT1 receptor in cultured VSMCs.%目的 利用构建的血管紧张素转化酶(ACE)2慢病毒表达载体转染平滑肌细胞,探讨ACE2对血管紧张素Ⅱ1型受体(AT1R)表达的影响.方法 培养平滑肌细胞,并进行分组及干预:(1)空白对照组:仅加入无血清培养基.(2)慢病毒重组绿色荧光蛋白表达空载体( Lentiviral-GFP,GFP)组:加入感染复数(MOI)为10的Lentiviral-GFP空载体.(3)血管紧张素(Ang)Ⅱ组:加入终浓度为10-7 mol/L的AngⅡ.(4) AngⅡ+慢病毒重组ACE2表达载体(Lentiviral-ACE2,ACE2)组:同时加入浓度为10-7 mol/L的AngⅡ和MOI为10的Lentiviral-ACE2.(5) AngⅡ+厄贝沙坦组:同时加入终浓度为10-7 mol/L的AngⅡ和厄贝沙坦.(6) AngⅡ+ACE2+厄贝沙坦组:同时加入终浓度为10-7 mol/L的AngⅡ和厄贝沙坦以及MOI为10的Lentiviral-ACE2.(7)ACE2组:仅加入MOI为10的Lentiviral-ACE2.将上述干预细胞提取RNA后运用实时PCR检测细胞ATIR mRNA的表达,Western blot技术分别检测细胞ATIR蛋白表达,以及下游信号通路信号转导子和转录激活子3(STAT3)蛋白磷酸化水平.运用CCK-8试剂盒检测上述干预组的细胞增殖水平.结果 (1)AngⅡ+ACE2组和AngⅡ+ACE2+厄贝沙坦组的ATIR mRNA表达均低于AngⅡ组(分别为0.39±0.02和0.24±0.01比1.80±0.08,P分别＜0.05和0.01).(2) AngⅡ+ACE2组和AngⅡ+ACE2+厄贝沙坦组的ATIR蛋白表达均低于AngⅡ组(分别为0.83±0.07和0.52±0.07比1.10±0.13,P分别＜0.05和0.01).(3)AngⅡ+ACE2组和AngⅡ+ACE2+厄贝沙坦组的STAT3蛋白磷酸化水平均低于AngⅡ组(分别为0.09±0.01和0.02 ±0.01比0.50±0.10,P分别＜0.05和0.01).(4) AngⅡ+ACE2组和AngⅡ+ ACE2+厄贝沙坦组的细胞增殖水平均低于AngⅡ组(分别为0.84±0.08和0.77±0.10比1.16±0.11,P分别＜0.05和0.01).结论 ACE2通过抑制AT1R和下游的STAT3信号通路抑制AngⅡ诱导的平滑肌细胞增殖,并提示ACE2对AT1R有直接的抑制作用.