液泡膜蛋白1通过影响活性氧介导心脏成纤维细胞激活与增殖  

Vacuole membrane protein 1 mediates cardiac fibroblast activation and proliferation by influencing reactive oxygen species

作　　者：李雅 冯翠娜[2] 祖玉刚[2] 汪煜 江永正 LI Ya;FENG Cuina;ZU Yugang;WANG Yu;JIANG Yongzheng(Department of Cardiology,Sijing Hospital of Songjiang District,Shanghai 201601,China;Department of Cardiology,Affiliated Hospital of Hebei University,Hebei Province,Baoding 071000,China)

机构地区：[1]上海市松江区泗泾医院心内科,上海201601 [2]河北大学附属医院心内科,河北保定071000

出　　处：《中国医药导报》2024年第36期13-17,31,共6页China Medical Herald

基　　金：河北省自然科学基金资助项目(H2021201046)。

摘　　要：目的探讨液泡膜蛋白1(VMP1)对心脏成纤维细胞激活、增殖的影响。方法分离小鼠乳鼠心脏成纤维细胞,将细胞分为对照组、转化生长因子β(TGF-β)诱导组、VMP1敲减组、VMP1敲减+TGF-β诱导组。采用Western blot法明确TGF-β激活的成纤维细胞VMP1的变化情况。采用免疫荧光比较各组细胞α-SMA的表达情况及细胞形态;培养24、48、72 h后采用CCK-8法检测各组细胞增殖情况,采用碘化丙啶(PI)染色及流式细胞术检测细胞周期情况;采用活性氧探针及流式细胞术检测各组活性氧(ROS)的含量。结果与对照组比较,TGF-β诱导组α-平滑肌肌动蛋白(α-SMA)表达水平升高、VMP1表达水平降低(P<0.05)。与对照组比较,TGF-β诱导组心脏成纤维细胞α-SMA的免疫荧光强度增加(P<0.01),与TGF-β诱导组比较,VMP1敲减+TGF-β诱导组α-SMA免疫荧光强度增加(P<0.01)。CCK-8实验发现,TGF-β诱导组各时间点光密度(OD)值高于对照组(P<0.05)。与TGF-β诱导组比较,VMP1敲减+TGF-β诱导组各时间点OD值低于TGF-β诱导组。流式细胞术检测发现,与对照组比较,TGF-β诱导组细胞G2/M期的细胞比例增加(P<0.05)。与TGF-β诱导组比较,VMP1敲减+TGF-β诱导组的细胞G2/M期的细胞比例增加(P<0.01)。ROS探针显示,TGF-β诱导组ROS荧光阳性细胞数与对照组比较,差异无统计学意义(P>0.05)。与TGF-β诱导组比较,VMP1敲减+TGF-β诱导组ROS荧光阳性细胞数增加(P<0.05)。结论在TGF-β诱导的心脏成纤维细胞转化中,VMP1蛋白表达降低,通过增加成纤维细胞ROS累积,促进细胞增殖,并参与成纤维细胞活化为肌成纤维细胞,最终促进心脏纤维化。Objective To explore the effects of vacuole membrane protein 1(VMP1)on the activation and proliferation of cardiac-fibroblasts.Methods The cardiac fibroblasts from neonatal rat were isolated and classified into four groups:control group,transforming growth factor-β(TGF-β)induced group,VMP1 knockdown group and VMP1 knockdown+TGF-βinduced group.Western blot method was used to determine the changes of TGF-βinduced cardiac fibroblasts VMP1.Immunofluorescence was adopted to analyze the express ofα-SMA and cell morphology among different groups.After 24,48,and 72 h of cultivation,CCK-8 was applied to detect the cell proliferation and propylene iodide(PI)staining and flow cytometry were employed to detect cell cycle.The reactive oxygen species(ROS)probe and flow cytometry were used to investigate the accumulation of ROS in each groups.Results Compared with the control group,the expression level ofα-smooth muscle actin(α-SMA)was increased and the expression level of VMP1 was decreased in TGF-βinduced group(P<0.05).Compared with the control group,the immunofluorescence intensity ofα-SMA in cardiac fibroblasts of TGF-βinduced group was increased(P<0.01),and compared with TGF-βinduced group,the immunofluorescence intensity ofα-SMA in VMP1 knockdown+TGF-βinduction group was increased(P<0.01).CCK-8 test showed that TGF-βinduced group opticla density(OD)value was higher than control group at each time point(P<0.05).Compared with TGF-βinduction group,OD value of VMP1 knockdown+TGF-βinduced group was lower than that of TGF-βinduced group at all time points.Flow cytometry showed that compared with the control group,the proportion of G2/M phase cells in TGF-β-induced group was increased(P<0.05).Compared with TGF-βinduction group,the proportion of G2/M phase cells in VMP1 knockdown+TGF-βinduced group was increased(P<0.01).ROS probe showed that there was no significant difference in the number of ROS fluorescence positive cells between TGF-β-induced group and control group(P>0.05).Compared with TGF-βinduced gro

关 键 词：液泡膜蛋白1 心脏成纤维细胞 心脏纤维化 心脏重构 

分 类 号：R542.23[医药卫生—心血管疾病]