摘要
目的 分析骨髓间充质干细胞来源的外泌体微小RNA-22-3p(miR-22-3p)对卵巢早衰的影响.方法 选取接受体外受精或者第二代试管婴儿治疗的卵巢早衰患者以及接受同样治疗的因男性因素不孕的正常志愿者提供卵泡液;通过差速离心法分离骨髓间充质干细胞来源外泌体;分离外泌体的形态学、粒子大小以及标志蛋白通过透射电子显微镜、NanoSight LM10分析仪以及蛋白免疫印迹方法进行分析.将颗粒细胞经环磷酰胺(CTX)、外泌体、miR-22-3p模拟物以及相应对照处理,分为CTX 组、Control 组、外泌体孵育组、PBS 处理组、CTX+miR-22-3p 组、CTX+miR-NC 组、CTX+Exosomes 组以及 CTX+PBS 组;采用蛋白免疫印迹法和定量实时聚合酶链反应(qRT-PCR)检测基因表达水平;利用二苯基四氮唑溴盐(MTT)试剂以及流式分析仪检测细胞活力和凋亡.结果 提取的外泌体具有典型的杯状囊泡,外泌体标志蛋白簇状分化抗原63(CD63)和簇状分化抗原9(CD9)表达在所提取的细胞上清外泌体中,外泌体粒子大小为80～150 nm;miR-22-3p在卵巢早衰患者和CTX诱导的卵巢颗粒细胞中显著低表达(P＜0.05),在骨髓间充质干细胞来源外泌体孵育卵巢颗粒细胞中显著高表达(P＜0.05);颗粒细胞活力在CTX组低于对照组,但在CTX+miR-22-3p组或CTX+Exosomes组高于对照组,差异有统计学意义(P＜0.05);颗粒细胞凋亡率在CTX组高于对照组,但在CTX+miR-22-3p组或CTX+Exosomes组低于对照组,差异有统计学意义(P＜0.05).结论 骨髓间充质干细胞来源的外泌体miR-22-3p抑制CTX诱导的卵巢颗粒细胞损伤.
Abstract
Objective To analyze the effect of microRNA-22-3p(miR-22-3p)of extracellular vesi-cles derived from bone marrow mesenchymal stem cells on premature ovarian failure.Methods Follicular fluids were provided by premature ovarian failure patients with in vitro fertilization or the second-gen-eration in vitro fertilization treatment and normal volunteers with the same treatment for infertility due to male factors;the exosomes derived from bone marrow mesenchymal stem cells were isolated by dif-ferential centrifugation;the morphology,particle size and marker proteins of isolated exosomes were analyzed by transmission electron microscopy,NanoSight LM10 analyzer and Western blotting.Granulosa cells were treated with cyclophosphamide(CTX),exosomes,miR-22-3 mimics and corresponding controls,and were divided into CTX group,control group,exosome incubation group,PBS treat-ment group,CTX+miR-22-3p group,CTX+miR-NC group,CTX+Exosomes group,and CTX+PBS group;gene expression was detected by Western blotting and quantitative real-time polymerase chain reaction(qRT-PCR).The 3-(4,5-Dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)reagent and flow cytometry were used to detect cell viability and apoptosis.Results The extracted exosomes had typical goblet vesicles,the exosome marker proteins C cluster of differentia-tion 63(CD63)and C cluster of differentiation 9(CD9)were expressed in the extracted exosomes,and exosome particle size was 80 to 150 nm;miR-22-3p was significantly lowly expressed in patients with premature ovarian failure and ovarian granulosa cells induced by CTX(P＜0.05),but was significantly highly expressed in ovarian granulosa cells incubated with exosomes derived from bone marrow mesenchymal stem cells(P＜0.05);the activity of granulosa cells in the CTX group was significantly lower than that in the control group,but was significantly higher in the CTX+miR-22-3p group or CTX+Exosomes group than that in the control group(P＜0.05);the apoptosis rate of granulosa cells in the CTX group was significantly higher than that in the control group,but was sig-nificantly lower in the CTX+miR-22-3p or CTX+Exosomes group than that in the control group(P＜0.05).Conclusion The miR-22-3p of extracellular vesicles derived from bone marrow mes-enchymal stem cells can inhibit ovarian granulosa cell injury induced by CTX.
基金项目
河南省医学科技攻关计划(LHGJ20220989)
维普
万方数据