基质交感分子1对大鼠内皮祖细胞细胞周期的影响  

Impact of stromal interaction molecule 1 silencing on cell cycle of endothelial progenitor cells

作　　者：况春燕[1] 黄岚[1] 喻杨[1] 邓梦杨[1] 王逵[1] 钱德慧[1] 

机构地区：[1]第三军医大学附属新桥医院全军心血管疾病研究所,重庆400037

出　　处：《中华心血管病杂志》2011年第7期649-653,共5页Chinese Journal of Cardiology

基　　金：国家自然科学基金（30770852）

摘　　要：目的 探讨RNA干扰技术沉默基质交感分子1（STIM1）基因后对内皮祖细胞（EPC）细胞周期的影响.方法 实验分为腺病毒阴性对照组（NSC组）、大鼠STIM1干扰腺病毒载体转染组（si/rSTIM1组）及大鼠STIM1干扰腺病毒载体与人重组STIM1腺病毒载体共转染组（si/rSTIM1＋hSTIM1组）.分离培养大鼠骨髓源性EPC,用构建好的STIM1干扰腺病毒载体和人重组STIM1腺病毒载体进行转染后,采用逆转录PCR检测细胞基质交感分子1表达,流式细胞技术检测细胞周期,激光共聚焦测量钙离子浓度,免疫共沉淀检测STIM1与瞬时受体电位通道1（TRPC1）之间的相互作用.酶联免疫吸附测定法检测大鼠1,4,5-三磷酸肌醇（IP3）含量.结果 细胞转染后48 h,si/rSTIM1组细胞内STIM1 mRNA表达明显低于NSC组 （0.37±0.02比1.00±0.02,P〈0.05）,钙离子浓度低于NSC组（34.07±4.10 比86.51±14.12,P〈0.05）,EPC的细胞周期停止在G1期[si/rSTIM1组：（90.91±1.10）%,NSC组：（77.10±0.56）%,P〈0.05],而si/rSTIM1＋hSTIM1组细胞STIM1 mRNA表达、钙离子浓度及分布在G1期的细胞数均恢复到NSC组水平.免疫共沉淀实验显示STIM1与TRPC1蛋白分子在EPC上相互作用,IP3浓度在三组之间比较差异无统计学意义（P〉0.05）.结论 STIM1基因沉默通过降低EPC的钙离子浓度,导致细胞周期停滞在G1期,调控EPC细胞增殖。Objective To investigate the effect of stromal interaction molecule 1 （STIM1） silencing on EPCs cell cycle. Methods Rat bone marrow derived endothelial progenitor cells （EPCs） were isolated and cultured in L-DMEM with 20% FBS. Ad-si/rSTIM1 and Ad-hSTIM1 were then transfected into EPCs and the expression of STIM1 mRNA was detected by RT-PCR. The cell cycle was determined using flow cytometry analysis and intracellular free Ca2＋ was measured using LSCM. Co-immunoprecipitation was performed to examine the interaction between STIM1 and TRPC1. Protein levels of inositol 1, 4, 5-trisphosphate were analyzed with ELISA assay. Results Forty-eight hours after transfection, the expression of STIM1 mRNA was significantly downregulated （0.37±0.02 vs.1.00±0.02, P〈0.05） and intracellular free Ca2＋ level was significantly reduced （34.07±4.10 vs. 86.51±14.12,P〈0.05） in Ad-si/rSTIM1 group compared with control group. The cell cycle was arrested at G1 phase[（90.91±1.10）% vs. （77.10±0.56）%, P〈0.05]and the store-operated channel entry was strikingly inhibited in EPCs after treatment with Ad-si/rSTIM1. However, cotransfection of Ad-hSTIM1 with Ad-si/rSTIM1 significantly reversed these responses. Interestingly, co-immunoprecipitation study showed that STIM1 co-precipitated with TRPC1, and IP3 levels measured by ELISA were similar among three groups （P〉0.05）. Conclusion siRNA-mediated knockdown of STIM1 inhibited EPCs proliferation by reducing intracellular free Ca2＋ through TRPC1-SOC signaling pathway

关 键 词：干细胞 细胞周期 RNA干扰 瞬时受体电位通道 基质交感分子1 

分 类 号：R363[医药卫生—病理学]