靶向沉默Toll样受体4基因对血管平滑肌细胞增殖的影响  

Effect of silencing of toll like receptor-4 gene by RNA interference on vascular smooth muscle cells proliferation

作　　者：崔丽 郭兆增 富华颖[2] 王兴华[2] 李广平[2] 曹月娟[3] Cui Li;Guo Zhaozeng;Fu Huaying;Wang Xinghua;Li Guangping;Cao Yuejuan(Department of Cardiology,the Second Hospital of Tianjin Medical University,Tianjin 300211,China;Tianjin Institute of Cardiology/Department of Cardiology,the Second Hospital of Tianjin Medical University,Tianjin 300211,China;Department of Vascular Surgery,Tianjin Union Medical Center,Tianjin 300121,China)

机构地区：[1]天津市人民医院心内科,300121 [2]天津心脏病学研究所天津医科大学第二医院心内科,300211 [3]天津市人民医院血管诊疗中心,300121

出　　处：《中华实验外科杂志》2019年第6期1016-1019,共4页Chinese Journal of Experimental Surgery

基　　金：天津市人民医院科研基金项目(2018YJZD003).

摘　　要：目的应用小干扰RNA(siRNA)靶向沉默血管平滑肌细胞(VSMC)的Toll样受体4(TLR4)基因,观察抑制TLR4基因对VSMC生长增殖的影响并探讨其机制。方法以TLR4基因靶向的小发夹RNA表达质粒(pSilence2.1-siTLR4)及阴性对照质粒(pSilence2.1-NC)转染VSMC,并筛选稳定表达的细胞,反转录-聚合酶链反应(RT-PCR)及蛋白质印迹法(Western blot)检测TLR4mRNA和蛋白相对表达,应用脂多糖(LPS)刺激,检测TLR4及核因子-κB(NF-κB)的mRNA和蛋白表达变化;噻唑蓝(MTT)法绘制细胞生长曲线,流式细胞学检测细胞周期变化。组间比较采用单因素方差分析。结果siTLR4组较正常VSMC细胞TLR4mRNA及蛋白表达明显降低(FmRNA=111.300,F蛋白=274.500,P<0.01),差异有统计学意义。LPS刺激24h时各组细胞的TLR4及NF-κB mRNA及蛋白表达明显增加,siTLR4组表达明显低于正常组及NC组(mRNA:FTLR4=79.900,FNF-κB=211.900,蛋白:FTLR4=33.350,FNF-κB=136.900,P<0.05),差异有统计学意义。LPS刺激后,siTLR4组VSMC细胞生长增殖能力明显低于正常组及NC组,在72h和120h差异有统计学意义(F72h=5.920,F120h=9.747,P<0.05);流式细胞学结果提示siTLR4组G0/G1期明显高于正常组及NC组,差异有统计学意义(F=14.770,P<0.05)。结论siTLR4转染靶向沉默TLR4基因可使LPS诱导TLR4/NF-κB表达上调的作用明显减弱,通过抑制NF-κB的表达而使细胞更多的停滞于G0/G1期,从而抑制VSMC细胞增殖生长。Objective To explore the effect and possible mechanisms on vascular smooth muscle cells (VSMC) proliferation of silencing of toll like receptor-4 (TLR4) gene using small interfering RNA (siRNA). Methods The recombinants of pSilence2.1-siTLR4 and the negative control plasmid pSilence2.1-NC were transfected to VSMC. VSMC expressing plasmid stably was collected and TLR4 mRNA and protein expression were detected. The VSMC was treated with lipopolysaccharide (LPS) as specific ligand of TLR4, the mRNA and protein expression lever of TLR4, Nuclear factor κB (NF-κB) was examined. Cell proliferation and cell cycle distribution were evaluated by methyl thiazol tetrazolium (MTT) colorimetry and flow cytometry.The statistical significance of differences between groups was calculated using one-way analysis of variance (ANOVA). Results The TLR4 mRNA and protein expression of siTLR4 group was significantly down-regulated to compare with the control and negative control plasmid (NC) group (FmRNA=111.300, FPro=274.500, P<0.01). VSMC growth in siTLR4 group was significantly slower compared with the control and the NC group when incubated with LPS, there were statistically significant in 72 h and 120 h (F72 h=5.920, F120 h=9.747, P<0.05). The proportion increased in G0/G1 phase in siTLR4 group compared with the control and NC group stimulating by LPS. The mRNA and protein expression of TLR4 and NF-κB in all groups were increased when incubated with LPS, which of the siTLR4 group was still significantly lower than the normal group and NC group (mRNA FTLR4=79.900, FNF-κB=211.900, Protein FTLR4=33.350, F NF-κB=136.900, P<0.05). Conclusion SiRNA-TLR4 reduced TLR4 mRNA and protein expression in VSMC and down-regulated LPS-induced NF-κB activation. siRNA-TLR4 can inhibit proliferation and growth of VSMC by arresting cell cycle in G0/G1 phase.

关 键 词：TOLL样受体4 RNA干扰 血管平滑肌细胞 增殖 细胞周期 

分 类 号：R54[医药卫生—心血管疾病]