摘要
目的:通过下调和上调c-Jun在人血管平滑肌细胞(hVSMCs)中的表达,研究c-Jun对hVSMCs增殖的调控作用及其机制。方法:①l-Jun基因低表达实验:体外培养hVSMCs,将细胞分为空白对照组、阴性对照组(转染阴性对照siRNA)和siRNA干扰组(转染靶向c-Jun的siRNA)三组,用构建好的siRNA转染各组细胞并培养24 h。②c-Jun基因过表达实验:体外培养hVSMCs,将细胞分为空白对照组、阴性对照组(转染阴性对照病毒载体)和慢病毒组(转染过表达c-Jun的慢病毒载体)三组,用构建好的慢病毒转染各组细胞并培养48 h。完成上述的细胞转染后,光镜观察各组细胞的生长情况,加入CCK-8试剂检测各组细胞的0D值。Western Blot检测c-Jun基因、线粒体融合基因2(Mfn2)的表达以及RAS-RAF-MEK-ERK1/2信号通路蛋白的表达。结果:siRNA干扰c-Jun表达后,与阴性对照组(1.301±0.027)或空白对照组(1.223±0.016)相比,siRNA干扰组(1.759±0,023)的0D值显著增加(P<0.05),Mfn2的表达下调,RAS、RAF、MEK以及ERK1/2蛋白的表达显著增加(P<0.05)。相反,慢病毒过表达c-Jun后,与阴性对照组(1.660±0,009)或空白对照组(1.862±0,123)相比,慢病毒组(1.101±0,101)的0D值显著减少(P<0.05),Mfn2的表达上调,RAS、RAF、MEK以及ERK1/2蛋白的表达显著减少(P<0.05)。结论:c-Jun可能通过调控Mfn2-RAS-RAF-MEK-ERK1/2信号通路调节hVSMCs的增殖。
Objective:To explore the regulation effect and mechanism of c-Jun on human vascular smooth muscle cells(hVSMCs)proliferation by down-regulating and up-regulating the expression of c-Jun in hVSMCs.Methods:①c-Jun gene low expression experiment:hVSMCs were cultured in vitro,and the cells were divided into three groups:blank control group,negative control group(transfected negative control siRNA)and siRNA interference group(transfected c-Jun targeting siRNA).The cells of each group were transfected with constructed siRNA and cultured for 24 hours.②c-Jun gene overexpression experiment:hVSMCs were cultured in vitro,and the cells were divided into three groups:blank control group,negative control group(transfected with negative control virus vector)and lentivirus group(transfected with lentivirus vector overexpressing c-Jun).The cells of each group were transfected with constructed lentivirus and cultured for 48 hours.After the above cell transfection,the growth of cells in each group was observed under light microscope,and the OD value of cells was detected by CCK-8 assay.The expression of c-Jun,Mitofusin 2(Mfn2)and RAS-RAFMEK-ERK1/2 signaling pathway proteins were detected by Western Blot.Results:In the siRNA interference experiment,compared with negative control group(1.301±0.027)or blank control group(1.223±0.016),the OD of hVSMCs in siRNA interference group(1.759±0.023)was significantly increased(P<0.05),indicating c-Jun silence promoted hVSMCs proliferation,while the expression of Mfn2 was down-regulated,and the expression levels of RAS,RAF,MEK and ERK1/2 protein were significantly increased(P<0.05).On the contrary,in lentivirus overexpression experiment,compared with negative control group(1.660±0.009)or blank control group(1.862±0.123),the OD of hVSMCs in the lentivirus group(1.101±0.101)was significantly decreased(P<0.05),indicating that the overexpression of c-Jun suppressed hVSMCs proliferation,while the expression of Mfn2 was up-regulated,and the expression levels of RAS,RAF,MEK,and ERK1/2 protein
作者
吴琼
刘洁琳
刘雅
辛毅
曾荣
程文立
王佐广
WU Qiong;LIU Jielin;LIU Ya;XIN Yi;ZENG Rong;CHENG Wenli;WANG Zuoguang(Department of Hypertension,Beijing Anzhen Hospital,Capital Medical University,Beijing Institute of Heart,Lung and Blood Vessel Diseases,Beijing 100029,China)
出处
《心肺血管病杂志》
CAS
2021年第4期376-380,共5页
Journal of Cardiovascular and Pulmonary Diseases
基金
国家自然科学基金(81370229,81270216)
北京市自然科学基金(7102045)。
