CAJ | 학술논문

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,TRPC1的钙库操纵性钙通道参与STIM1对EPC钙离子浓度的调节.
목적 탐토RNA간우기술침묵기질교감분자1(STIM1)기인후대내피조세포(EPC)세포주기적영향.방법 실험분위선병독음성대조조(NSC조)、대서STIM1간우선병독재체전염조(si/rSTIM1조)급대서STIM1간우선병독재체여인중조STIM1선병독재체공전염조(si/rSTIM1+hSTIM1조).분리배양대서골수원성EPC,용구건호적STIM1간우선병독재체화인중조STIM1선병독재체진행전염후,채용역전록PCR검측세포기질교감분자1표체,류식세포기술검측세포주기,격광공취초측량개리자농도,면역공침정검측STIM1여순시수체전위통도1(TRPC1)지간적상호작용.매련면역흡부측정법검측대서1,4,5-삼린산기순(IP3)함량.결과 세포전염후48 h,si/rSTIM1조세포내STIM1 mRNA표체명현저우NSC조 (0.37±0.02비1.00±0.02,P<0.05),개리자농도저우NSC조(34.07±4.10 비86.51±14.12,P<0.05),EPC적세포주기정지재G1기[si/rSTIM1조:(90.91±1.10)%,NSC조:(77.10±0.56)%,P<0.05],이si/rSTIM1+hSTIM1조세포STIM1 mRNA표체、개리자농도급분포재G1기적세포수균회복도NSC조수평.면역공침정실험현시STIM1여TRPC1단백분자재EPC상상호작용,IP3농도재삼조지간비교차이무통계학의의(P>0.05).결론 STIM1기인침묵통과강저EPC적개리자농도,도치세포주기정체재G1기,조공EPC세포증식,TRPC1적개고조종성개통도삼여STIM1대EPC개리자농도적조절.
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.