Slfn1对大鼠平滑肌细胞增殖的影响及机制  

Effect of Slfn1 on the proliferation of rat primary smooth muscle cells and its mechanism

作　　者：刘姿麟 肖森桐 况春燕[1] LIU Zilin;XIAO Sentong;KUANG Chunyan(Department of Cardiology,Guizhou Provincial People's Hospital,Guiyang 550003,Guizhou,China)

机构地区：[1]贵州省人民医院心内科,贵州贵阳550003

出　　处：《贵州医科大学学报》2024年第7期988-996,共9页Journal of Guizhou Medical University

基　　金：贵州省科技计划项目(黔科合基础〔2018〕1097)。

摘　　要：目的探讨睡眠因子1(Slfn1)对大鼠血管平滑肌细胞(VSMCs)增殖的影响及机制。方法麻醉处死56只雄性SD大鼠,剪取腹主动脉到主动脉弓的血管段、分离中膜组织块体外培养4~5 d,采用免疫荧光法检测α-平滑肌肌动蛋白(α-SMA)的表达鉴定平滑肌细胞;感染复数(MOI)为1×10^(2)、1×10^(3)、1×10^(4)浓度Slfn1的重组腺病毒干扰质粒转染VSMCs 48 h,通过荧光显微镜及流式细胞仪检测不同MOI组VSMCs中有绿色荧光蛋白(eGFP)的细胞/总细胞的百分比,确定最佳MOI用于后续实验,并分为Slfn1干扰组(用Slfn1的重组腺病毒干扰质粒转染)、Slfn1干扰对照组(用Slfn1的重组腺病毒干扰空载体质粒转染)及空白对照组(用正常血清培养),采用聚合酶链式反应(PCR)检测3组Slfn1信使RNA(mRNA)的表达,CCK-8检测VSMCs增殖;提取RNA进行高通量RNA测序、并通过R语言分析,寻找差异基因,对差异基因进行基因本体(GO)和京都基因和基因组数据库(KEGG)富集分析。结果原代培养VSMCs第10~15天时,细胞呈典型的“峰谷”样生长形态,细胞免疫荧光法显示VSMCs胞质内有α-SMA表达,鉴定为VSMCs;Slfn1的重组腺病毒干扰质粒转染VSMCs 48 h后,1×10^(3)MOI组VSMCs中质粒的转染效率高(90%);PCR结果示Slfn1干扰组VSMCs中Slfn1 mRNA表达减少(P<0.05),CCK8结果显示Slfn1干扰组VSMCs增殖增加(P<0.05);用Slfn1重组腺病毒干扰质粒转染VSMCs 48 h,提取RNA进行高通量RNA测序及R语言分析结果显示,差异基因中上调基因94个,下调基因181个;GO和KEGG富集分析显示,Slfn1基因可能通过蛋白质糖基化、mRNA监测通路、鞘脂类信号通路等调控VSMCs的增殖。结论基因干扰VSMCs的Slfn1可促进VSMCs的增殖,其机制可能与蛋白质糖基化、mRNA监测通路及鞘脂类信号通路等调控有关。Objective To investigate the effect of Slfn1 on the proliferation of vascular smooth muscle cells(VSMCs)and its mechanism.Methods A total of 56 male SD rats were sacrificed under anesthesia,and vascular segments were cut from the abdominal aorta to the aortic arch.Vascular medial membranes were isolated and cultured for 4-5 d.Immunofluorescent staining was used to detect the expression ofα-smooth muscle actin(α-SMA)to verify smooth muscle cells.VSMCs were infected with the recombinant adenoviruses for silencing Slfn1 with multiplicity(MOI)of 1×10^(2),1×10^(3),and 1×10^(4)for 48 hours,respectively.The percentages of VSMCs green fluorescent protein(GFP)in total VSMCs in different MOI groups was detected by fluorescence microscopy and flow cytometry to determine the optimal MOI for subsequent experiments.The cells were divided into Slfn1 interference group(infected with recombinant adenoviruses for silencing Slfn1),Slfn1 interference control group(infected with recombinant adenoviruses for silencing Slfn1 control)and blank control group(cultured with normal serum).Polymerase chain reaction(PCR)was used to detect Slfn1 messenger RNA expression(mRNA)in three groups.CCK-8 assay was used to detect VSMC proliferation.Total RNA was isolated for high-throughput RNA sequencing.R language analysis was performed on RNA sequencing data to identify differentially expressed genes.Gene Ontology(GO)and Kyoto Gene and Genome Database(KEGG)enrichment analyses were run on differentially expressed genes.Results On the 10^(th) to 15^(th) day of primary culture of VSMCs,the cells showed a typical"peak-valley"growth morphology.Immunofluorescent staining showedα-SMA expression were in VSMC cytoplasm,verifying that the cells were VSMCs.After 48 hours of infecting VSMCs with recombinant adenovirus for silencing Slfn1,the infection efficiency of VSMCs in the 1×10^(3)MOI group was high(90%).PCR results showed a decrease in Slfn1 mRNA expression in VSMCs in Slfn1 interference group(P<0.05),while CCK8 results showed an increase in VSMC p

关 键 词：大鼠 Sprgue-Dawley 细胞增殖 睡眠因子1 血管平滑肌细胞 蛋白质糖基化 

分 类 号：R341.6[医药卫生—基础医学]