CAJ | 학술논문

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.
목적 이용구건적혈관긴장소전화매(ACE)2만병독표체재체전염평활기세포,탐토ACE2대혈관긴장소Ⅱ1형수체(AT1R)표체적영향.방법 배양평활기세포,병진행분조급간예:(1)공백대조조:부가입무혈청배양기.(2)만병독중조록색형광단백표체공재체( Lentiviral-GFP,GFP)조:가입감염복수(MOI)위10적Lentiviral-GFP공재체.(3)혈관긴장소(Ang)Ⅱ조:가입종농도위10-7 mol/L적AngⅡ.(4) AngⅡ+만병독중조ACE2표체재체(Lentiviral-ACE2,ACE2)조:동시가입농도위10-7 mol/L적AngⅡ화MOI위10적Lentiviral-ACE2.(5) AngⅡ+액패사탄조:동시가입종농도위10-7 mol/L적AngⅡ화액패사탄.(6) AngⅡ+ACE2+액패사탄조:동시가입종농도위10-7 mol/L적AngⅡ화액패사탄이급MOI위10적Lentiviral-ACE2.(7)ACE2조:부가입MOI위10적Lentiviral-ACE2.장상술간예세포제취RNA후운용실시PCR검측세포ATIR mRNA적표체,Western blot기술분별검측세포ATIR단백표체,이급하유신호통로신호전도자화전록격활자3(STAT3)단백린산화수평.운용CCK-8시제합검측상술간예조적세포증식수평.결과 (1)AngⅡ+ACE2조화AngⅡ+ACE2+액패사탄조적ATIR mRNA표체균저우AngⅡ조(분별위0.39±0.02화0.24±0.01비1.80±0.08,P분별＜0.05화0.01).(2) AngⅡ+ACE2조화AngⅡ+ACE2+액패사탄조적ATIR단백표체균저우AngⅡ조(분별위0.83±0.07화0.52±0.07비1.10±0.13,P분별＜0.05화0.01).(3)AngⅡ+ACE2조화AngⅡ+ACE2+액패사탄조적STAT3단백린산화수평균저우AngⅡ조(분별위0.09±0.01화0.02 ±0.01비0.50±0.10,P분별＜0.05화0.01).(4) AngⅡ+ACE2조화AngⅡ+ ACE2+액패사탄조적세포증식수평균저우AngⅡ조(분별위0.84±0.08화0.77±0.10비1.16±0.11,P분별＜0.05화0.01).결론 ACE2통과억제AT1R화하유적STAT3신호통로억제AngⅡ유도적평활기세포증식,병제시ACE2대AT1R유직접적억제작용.
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.