肌苷通过抑制线粒体通透性转换孔开放缓解缺氧/复氧诱导的人绒毛膜滋养层细胞凋亡  

Inosine inhibits opening of mitochondrial permeability transition pore to alleviate hypoxic/reoxygenationinduced apoptosis of human chorionic trophoblast cells

作　　者：钟雅雯 王煜 王海臻 黄莉萍[1] Yawen Zhong;Yu Wang;Haizhen Wang;Liping Huang(Department of Obstetrics and Gynecology,Nanfang Hospital,Southern Medical University,Guangzhou 510000,Guangdong Province,China;Department of Obstetrics and Gynecology,The Fifth Affiliated Hospital,Southern Medical University,Guangzhou 510000,Guangdong Province,China)

机构地区：[1]南方医科大学南方医院妇产科,广州510000 [2]南方医科大学第五附属医院妇产科,广州510000

出　　处：《中华妇幼临床医学杂志（电子版）》2024年第5期525-533,共9页Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition)

基　　金：国家自然科学基金青年基金项目(81703078);广东省自然科学基金面上项目(2022A1515011730、2023A1515011074)。

摘　　要：目的探讨肌苷抑制线粒体通透性转换孔(mPTP)开放,对缺氧/复氧(H/R)诱导的人绒毛膜滋养层细胞(HTR-8/SVneo细胞株)凋亡的影响及机制。方法采用人绒毛膜滋养层细胞HTR-8/Svneo细胞株为材料,建立H/R诱导的HTR-8/Svneo体外细胞模型。按照细胞培养方式,将HTR-8/SVneo细胞分为4组:H/R组(n=3,仅予H/R诱导),H/R+肌苷组(n=3,采用H/R诱导+肌苷共培养处理24 h),H/R+环孢素A(Cs A)组(n=3,采用H/R诱导+Cs A共培养处理24 h),以及阴性对照(NC)组(n=3,细胞常规培养)。分别采用calcein/PI染色检测HTR-8/SVneo细胞活性(PI染色相对阳性率);DHE探针检测细胞活性氧含量(活性氧相对荧光强度);JC-1探针检测细胞线粒体膜电位(绿色与红色荧光强度比值);Fluo-4AM探针检测细胞中Ca^(2+)含量(Fluo-4AM相对荧光强度);mPTP检测试剂盒检测mPTP开放程度(mPTP相对荧光强度);流式细胞术检测细胞凋亡率;实时荧光定量酶联免疫吸附法(RT-qPCR)检测BAX、BCL2的mRNA表达水平;Western blotting检测cleaved-Caspase3蛋白相对表达水平及BAX与BCL2比值(BAX/BCL2)。采用成组t检验,对H/R组分别与其余3组的上述指标进行统计学比较。结果H/R组细胞PI染色相对阳性率,活性氧相对荧光强度,JC-1探针检测的绿色与红色荧光强度比值,Fluo-4AM相对荧光强度,细胞凋亡率,BAX m RNA水平,cleaved-Caspase3相对表达量及BAX/BCL2,均分别高于NC组、H/R+肌苷组、H/R+Cs A组,而mPTP相对荧光强度及BCL2 m RNA水平,则均分别低于这3组,并且差异均有统计学意义(均为P<0.05)。结论肌苷可以通过抑制HTR-8/SVneo细胞mPTP开放,缓解线粒体功能障碍,进而抑制H/R诱导的HTR-8/SVneo细胞凋亡。Objective To investigate the effect and mechanism of inosine inhibiting the opening of mitochondrial permeability transition pore(mPTP)on hypoxic/reoxygenation(H/R)-induced apoptosis of human chorionic trophoblast cells(HTR-8/SVneo cell line).Methods Human chorionic trophoblast cells(HTR-8/Svneo cell line)was used as research material,and H/R-induced HTR-8/SVneo cell model was established invitro.According to the cell culture mode,HTR-8/SVneo cells were divided into 4 groups:H/R group(n=3,only H/R-induced),H/R+inosine group(n=3,H/R+inosine co-coculture treatment for 24 h),H/R+cyclosporine A(Cs A)group(n=3,H/R+Cs A co-culture treatment for 24 h),negative control(NC)group(n=3,conventional cell culture).Calcein/PI staining was used to detect the activity of HTR-8/SVneo cells(relative positive rate of PI staining).DHE probe was used to detect the content of active oxygen species(relative fluorescence intensity of active oxygen species).JC-1 probe was used to detect mitochondrial membrane potential(ratio of green to red fluorescence intensity).Fluo-4AM probe was used to detect Ca^(2+)content in cells(Fluo-4AM relative fluorescence intensity).mPTP assay kit was used to detect mPTP openness(mPTP relative fluorescence intensity).The apoptosis rate was detected by flow cytometry.Real-time fluorescence quantitative enzyme-linked immunosorbent assay(RT-qPCR)was used to detect the m RNA expression levels of BAX and BCL2.Western blotting detected cleaved Caspase3 protein relative expression level and the ratio of BAX to BCL2(BAX/BCL2).The above indexes were statistically compared between H/R group and the other three groups,respectively by independent-samples t test.Results PI staining relative positive rate,reactive oxygen species relative fluorescence intensity,ratio of green to red fluorescence intensity detected by JC-1 probe,Fluo-4AM relative fluorescence intensity,apoptosis rate,BAX mRNA level,cleaved Caspase3 relative expression and BAX/BCL2 in H/R group was respectively higher than those in NC group,H/R+inosine group

关 键 词：肌苷 缺氧/复氧 绒毛外滋养层细胞 线粒体通透性转换孔 细胞凋亡 膜电位 线粒体 体外研究 细胞培养技术 活性氧 

分 类 号：R714[医药卫生—妇产科学] R329.2[医药卫生—临床医学]