Klotho蛋白在子痫前期患者胎盘外泌体中表达及其对血管内皮细胞氧化应激的影响  

Expression of Klotho protein in placenta exosomes in patients with pre-eclampsia and its effect on oxidative stress in vascular endothelial cells

作　　者：薛筱蕾 胥保梅[1] XUE Xiaolei;XU Baomei(Department of Obstetrics,Fifth Affiliated Hospital,Xinjiang Medical University,Urumqi 830011,China)

机构地区：[1]新疆医科大学第五附属医院产科,新疆乌鲁木齐830011

出　　处：《吉林大学学报（医学版）》2023年第6期1528-1538,共11页Journal of Jilin University：Medicine Edition

基　　金：新疆维吾尔自治区科技厅自然科学基金项目(2020D01C224)。

摘　　要：目的:探讨Klotho在子痫前期(PE)患者来源的胎盘外泌体(Exo)中的表达,阐明其对血管内皮细胞氧化应激的影响。方法:收集40例孕产妇的临床资料,其中正常妊娠(NP)者20名,PE患者20例,设为NP组和PE组,分离2组研究对象外周血中胎盘Exo。将oe-Klotho和oe-NC质粒转染入人绒毛膜滋养层细胞HTR-8/SVneo中,作为oe-Klotho组和oe-NC组,收集HTR-8/SVneo细胞Exo。采用实时荧光定量PCR(RT-PCR)法和Western blotting法检测2组研究对象胎盘Exo和2组HTR-8/SVneo细胞Exo中Klotho mRNA及蛋白表达水平。取生长状态良好的人脐静脉内皮细胞(HUVECs),按Exo来源不同将HUVECs分为PE-Exo组(与PE患者胎盘Exo共培养)、NP-Exo组(与NP胎盘Exo共培养)、oe-Klotho-Exo组(与转染oe-Klotho的HTR-8/SVneo细胞Exo共培养)和oe-NC-Exo组(与转染oe-NC的HTR-8/SVneo细胞Exo共培养)。采用透射电子显微镜(TEM)观察Exo形态表现,Western blotting法检测Exo标记分子CD63、TSG101和胎盘Exo标记分子PLAP蛋白表达水平以鉴定Exo,荧光显微镜观察HUVECs对Exo的摄取情况。酶联免疫吸附试验(ELISA)法检测各组HUVECs中一氧化氮(NO)、活性氧(ROS)和丙二醛(MDA)水平及超氧化物歧化酶(SOD)活性,RT-qPCR法检测各组HUVECs中内皮型一氧化氮合酶(eNOS)mRNA表达水平,Western blotting法检测各组HUVECs中eNOS蛋白表达水平。结果:与NP组比较,PE组患者收缩压和舒张压升高(P<0.05),新生儿体质量和胎盘质量均明显降低(P<0.05或P<0.01)。TEM观察,来源于NP组和PE组研究对象的胎盘Exo均呈圆形和椭圆形的囊泡盘状结构,包膜完整,形状相似,直径为50~100 nm;2组研究对象胎盘Exo均高表达Exo标记蛋白CD63和TSG101及胎盘Exo特异性蛋白PLAP。纳米示踪分析(NTA)检测,胎盘Exo的粒径为50~200 nm,提示由外周血分离的囊泡均是胎盘来源Exo。TEM、Western blotting和NTA检测,来源于oe-NC组和oe-Klotho组滋养层细胞的囊泡具有Exo的典型结构、分子标志物和粒径大�Objective:To discuss the expression of Klotho in placenta exosomes(Exo)of the patients with pre-eclampsia(PE),and to clarify its effect on the oxidative stress in the vascular endothelial cells.Methods:The clinical data of 40 pregnant women including 20 with normal pregnancy(NP)women(NP group)and 20 PE patients(PE group)were collected.The placenta Exo in peripheral blood of the patients in two groups were isolated,and the oe-Klotho and oe-NC plasmids were transfected into the human chorionic trophoblast cells(HTR-8/SVneo),respectively,and were regarded as oe-Klotho group and oe-NC group.The expression levels of Klotho mRNA and protein in placenta Exo and the HTR8/SVneo cells in two groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)method and Western blotting method.The human umbilical vein endothelial cells(HUVECs)with good growth status were taken and divided into PE-Exo group(co-cultured with placenta Exo from the patients with PE),NP-Exo group(co-cultured with placenta Exo from the NP subjects),oe-Klotho-Exo group(co-cultured with Exo from the HTR-8/SVneo cells transfected with oe-Klotho),and oe-NC-Exo group(co-cultured with Exo from the HTR-8/SVneo cells transfected with oe-NC)according to the sources of Exo.The expression levels of exosomal marker proteins CD63,TSG101,and placenta Exo-specific marker PLAP protein were identified by transmission electron microscope(TEM)and Western blotting method;the levels of nitric oxide(NO),reactive oxygen species(ROS),and malondialdehyde(MDA)and the activities of superoxide dismutase(SOD)in the HUVECs in various groups were detected by enzymelinked immunosorbent assay(ELISA)method;the expression levels of endothelial nitric oxide synthase(eNOS)mRNA in the HUVECs in various groups was detected by RT-qPCR method;the expression levels of eNOS protein in the HUVECs in various groups were detected by Western blotting method.Results:Compared with NP group,the systolic and diastolic blood pressures of the patients in PE group were increased(P<0.05),the body

关 键 词：KLOTHO蛋白 子痫前期 外泌体 血管内皮细胞 氧化应激 

分 类 号：R394[医药卫生—医学遗传学]