Ras超家族亚群A/Rho关联含卷曲螺旋结合蛋白激酶1在无机磷酸盐中诱导大鼠瓣膜间质细胞成骨分化的作用机制  

Mechanism of RhoA/Rock-1 in inorganic phosphate-induced osteogenic differentiation of rat valvular interstitial cells

作　　者：芦潮 余文朋 丁静丽[2] 林峰 朱国栋 胡发嘉 董啸[3] 周建良[3] Lu Chao;Yu Wenpeng;Ding Jingli;Lin Feng;Zhu Guodong;Hu Fajia;Dong Xiao;Zhou Jianliang(Graduate School of Nanchang University,Nanchang 330031,China;Department of Gastrology,the Second Affiliated Hospital of Nanchang University,Nanchang 330006,China;Department of Cardiovascular Surgery,the Second Affiliated Hospital of Nanchang University,Nanchang 330006,China)

机构地区：[1]南昌大学研究生院,330031 [2]南昌大学第二附属医院消化内科,330006 [3]南昌大学第二附属医院心脏大血管外科,330006

出　　处：《中华实验外科杂志》2020年第2期272-275,共4页Chinese Journal of Experimental Surgery

基　　金：江西省教育厅科学技术研究项目(150274);江西省自然基金项目(20171ACB21061、20192BAB205070);国家自然科学基金(81770388、81860079、81660070);国家“863”计划青年科学家专题项目(2014AA020539)。

摘　　要：目的探讨Ras超家族亚群A(RhoA)/Rho关联含卷曲螺旋结合蛋白激酶1(Rock-1)在无机磷酸盐中诱导大鼠瓣膜间质细胞(VICs)成骨分化的作用机制。方法取8只雄性大鼠(购自上海西普尔必凯动物实验有限公司)的主动脉瓣膜消化培养至第3代后免疫荧光鉴定。小干扰RNA(siRNA)-RhoA转染VICs后用无机磷酸盐成骨培养基(IP-OIM)分别诱导转染组(RNAi),阴性对照组(NC)及空白对照组(CON)的成骨分化,并通过蛋白质印迹法(Western blot)与聚合酶链反应(PCR)评估转染后RhoA、Rock-1、Runt相关转录因子2(Runx-2)及骨钙素(OST)的变化;7 d行碱性磷酸酶(ALP)活性相关检测,14 d行钙含量相关检测评估其早期与晚期成骨分化趋势。用IP-OIM刺激转染后的VICs 60 min,并通过Western blot检测SMAD1/5/9的磷酸化变化。采用单因素方差分析。结果鉴定后的VICs转染siRNA-RhoA并用IP-OIM培养,经过Western blot及PCR检测后可知RNAi的RhoA(Western blot:0.330±0.020;PCR:0.357±0.060)及Rock-1(Western blot:0.366±0.020;PCR:0.383±0.040)比CON均出现表达下调(RhoA Western blot:t=36.680,P<0.05 PCR:t=10.610,P<0.05;Rock-1 Western blot:t=31.240,P<0.05 PCR:t=13.210,P<0.05),差异有统计学意义,RNAi的OST(Western blot:0.520±0.030;PCR:0.510±0.070)和Runx-2(Western blot:0.573±0.020;PCR:0.57±0.060)比CON也出现了下降(OST Western blot:t=16.630,P<0.05;PCR:t=6.970,P<0.05;Runx-2 Western blot:t=21.040,P<0.05;PCR:t=6.710,P<0.05),差异有统计学意义。在7 d的ALP染色中RNAi的颜色最浅,ALP活性检测中RNAi[(1.005±0.101)U/mg]活性比较NC[(2.042±0.116)U/mg]和CON[(1.931±0.136)U/mg]明显下降(t=10.570,P<0.05;t=9.400,P<0.05),差异有统计学意义。在14 d的相对钙含量测定中RNAi[(22.290±1.981)ng/L]钙含量比较NC[(41.100±2.440)ng/L]和CON[(41.760±1.215)ng/L]明显下降(t=10.530,P<0.05;t=14.510,P<0.05),差异有统计学意义;同样茜素红钙沉积染色中,也是RNAi颜色最浅。经IP-OIM分别刺激60 min,RNAi的SMAD1/5/9的磷酸化水平(Objective To investigate the action mechanism of RhoA/Rock-1 in inorganic phosphate-induced osteogenic differentiation of rat valvular interstitial cells(VICs).Methods The rat aortic valve(n=8,male,Shanghai Sppir BK)was digested and cultured to the third generation,and immunofluorescence identification was performed.Then VICs were transfected with small interfering RNA(siRNA)-RhoA,and in the transfection group(RNAi),the negative control group(NC)and blank control group(CON)the osteogenic differentiation was induced by inorganic phosphate-osteogenic inducing medium(IP-OIM).Western blotting and polymerase chain reaction(PCR)were used to evaluate whether there were significant changes in RhoA,Rock-1,Runx-2 and Osteocalcin(OST).Changes in alkaline phosphatase(ALP)activity on the 7th day,and calcium assay on the 14th day were used to assess osteogenic differentiation trend.Transfected VICs were stimulated by IP-OIM for 60 min,and the SMAD1/5/9 level of phosphorylation was detected by Western blotting.Results Identified VICs were transfected with siRNA-RhoA and then cultured with IP-OIM.Western blotting and PCR showed that RhoA(Western blotting:0.330±0.020;PCR:0.357±0.060)and Rock-1(Western blotting:0.366±0.020;PCR:0.383±0.040)in the RNAi decreased significantly compared to CON(RhoA,for Western blotting:t=36.680,P<0.05;for PCR:t=10.610,P<0.05.Rock-1,for Western blotting:t=31.240,P<0.05;for PCR:t=13.210,P<0.05),while OST(Western blotting:0.520±0.030;PCR:0.510±0.070)and Runx-2(Western blotting:0.573±0.020;PCR:0.570±0.060)in the RNAi also decreased(OST,for Western blotting:t=16.630,P<0.05;for PCR:t=6.970,P<0.05.Runx-2,for Western blotting:t=21.040,P<0.05;for PCR:t=6.710,P<0.05).The RNAi was the lightest in ALP staining on day 7,while compared with NC[(2.042±0.116)U/mg]and CON[(1.931±0.136)U/mg],the activity of RNAi[(1.005±0.101)U/mg]in ALP activity test was significantly decreased(t=10.570,P<0.05;t=9.400,P<0.05).On the 14th day,the calcium content assay revealed that the RNAi[(22.290±1.981)ng/L]was significant

关 键 词：瓣膜间质细胞 钙化性心脏瓣膜病 无机磷酸盐 Ras超家族亚群A/Rho关联含卷曲螺旋结合蛋白激酶1 SMAD1/5/9 

分 类 号：R542[医药卫生—心血管疾病]