低强度脉冲聚焦超声上调Vimentin蛋白表达抑制软骨细胞凋亡  

Focused low-intensity pulsed ultrasound upregulates Vimentin protein expression to inhibit chondrocyte apoptosis

作　　者：李东倩 叶海霞 虞乐华[1] 贾朗[1] LI Dongqian;YE Haixia;YU Lehua;JIA Lang(Department of Rehabilitation Medicine,the Second Affiliated Hospital of Chongqing Medical University,Chongqing,400010;Department of Rehabilitation,Chongqing City Management College,Chongqing,401331,China)

机构地区：[1]重庆医科大学附属第二医院康复医学科,重庆400010 [2]重庆城市管理职业学院康复教研室,重庆401331

出　　处：《陆军军医大学学报》2023年第12期1292-1300,共9页Journal of Army Medical University

基　　金：国家自然科学基金青年科学基金(81802234);重庆市自然科学基金面上项目(cstc2020jcyj-msxmX0134);重庆医科大学未来医学青年创新团队发展支持计划项目(W0196);重庆医科大学附属第二医院“宽仁英才”项目(kryc-gg-2116)。

摘　　要：目的探讨低强度脉冲聚焦超声(focused low-intensity pulsed ultrasound,FLIPUS)通过机械效应抑制软骨细胞凋亡的分子机制。方法用胰蛋白酶联合Ⅱ型胶原酶提取C57BL/6J乳鼠膝关节原代软骨细胞进行体外实验,IL-1β处理细胞模拟骨关节炎样软骨细胞损伤并进行验证,同时检测不同处理时间下相关蛋白表达变化。细胞分为Control组、IL-1β组和IL-1β+FLIPUS组,流式细胞术检测各组软骨细胞凋亡率,细胞免疫荧光检测各组细胞中Vimentin的结构分布。Western blot检测各组Col2α1、MMP13、Bcl-2、Vimentin及p-Tyr397表达。结果提取软骨细胞并模拟了骨关节炎样软骨细胞损伤模型。与Control组相比,随着IL-1β处理时间的延长,Col2α1、Vimentin、p-Tyr397、Bcl-2和p-AKT蛋白表达降低(P<0.05),MMP13、Bax和cleaved-Caspase3蛋白表达升高(P<0.05)。与IL-1β组相比,IL-1β+FLIPUS组软骨细胞凋亡水平降低(P<0.001),MMP13表达下降(P<0.05);Col2α1(P<0.01)、Bcl-2、Vimentin及p-Tyr397表达上升(P<0.05);Vimentin排列分布发生重塑,荧光强度增加。结论FLIPUS上调软骨细胞Vimentin表达,促进FAK的Tyr397位点磷酸化,抑制软骨细胞凋亡。Objective To investigate the molecular mechanism of focused low-intensity pulsed ultrasound(FLIPUS)to inhibit chondrocyte apoptosis through mechanical effects.MethodsPrimary chondrocytes were isolated from the knee of suckling C57BL/6J mice with trypsin combined with type II collagenase and then cultured with IL-1βstimulation to mimic a cellular model of osteoarthritis-like chondrocyte injury.The model was validated and related protein expression changes were detected after different treatment times.The cells were divided into Control group,IL-1βgroup and IL-1β+FLIPUS group.The apoptotic rate of chondrocytes was detected by flow cytometry,structural distribution of Vimentin was observed with immunofluorescence assay,and expression of Col2α1,MMP13,Bcl-2,Vimentin and p-Tyr397 was measured with Western blotting.ResultsPrimary mouse chondrocytes were isolated and cultured,and simulated to be a cellular model of osteoarthritis-like chondrocyte injury.With elapse of IL-1βtreatment time,the protein levels of Col2α1,Vimentin,p-Tyr397,Bcl-2 and p-AKT were decreased(P<0.05),and those of MMP13,Bax and cleaved-Caspase3 were increased(P<0.05)compared to the levels in the Control group.FLIPUS led to reduced apoptosis(P<0.001),decreased MMP13 expression(P<0.05)and enhanced levels of Col2α1(P<0.01),and Bcl-2 and Vimentin and p-Tyr397(P<0.05)when compared with the IL-1βgroup.Vimentin distribution was remodeled,with increased fluorescence intensity.ConclusionFLIPUS up-regulates Vimentin expression,promotes phosphorylation at the Tyr397 locus of FAK,and inhibits apoptosis in chondrocyte.

关 键 词：低强度脉冲聚焦超声 膝骨关节炎 软骨细胞凋亡 机械转导 VIMENTIN FAK 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学] R454.3[医药卫生—基础医学] R684.3