人脐带间充质干细胞通过GDF-15/FOXO3a治疗小鼠卵巢功能不全的研究  

Human Umbilical Cord Mesenchymal Stem Cells Treat Mouse Premature Ovarian Insufficiency through GDF-15/FOXO3a

作　　者：司潇洒 王建波 翁平 史朋飞 王长亮 陈晨 王勇[1] SI Xiao-sa;WANG Jian-bo;WENG Ping;SHI Peng-fei;WANG Chang-liang;CHEN Chen;WANG Yong(State Key Laboratory of Analytical Chemistry for Life Science,Jiangsu Key Laboratory of Molecular Medicine,Medical School,Nanjing University,Nanjing 210093,China;Jiangsu Unicorn Biotechnology Co.,LTD,Nanjing 210000,China)

机构地区：[1]南京大学医学院生命分析化学国家重点实验室,江苏省医学分子技术重点实验室,210093 [2]江苏育瑞康生物科技有限公司

出　　处：《国际妇产科学杂志》2024年第4期424-432,共9页Journal of International Obstetrics and Gynecology

基　　金：国家自然科学基金(81971346)。

摘　　要：目的:探讨人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)改善卵巢功能不全(premature ovarian insufficiency,POI)小鼠的卵巢损伤及机制。方法:采用足底注射透明带3多肽(zona pellucida3 peptide,pZP3)方法构建POI小鼠模型,将小鼠分为对照组、佐剂对照组、pZP3组和hUC-MSCs组,每组10只。以阴道涂片监测动情周期、以酶联免疫吸附测定检测血清卵泡刺激素(follicle-stimulating hormone,FSH)和雌二醇(estradiol,E_(2))水平,以HE染色观察卵巢组织学,以蛋白质印迹(Western blotting)检测叉头框转录因子O3a(forkhead box O3a,FOXO3a)、p-FOXO3a、p53、胱天蛋白酶-3(Caspase-3)、Bax表达水平,以实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,qRT-PCR)检测POI的标志物骨形态发生蛋白15(bone morp hogenetic protein 15,BMP15)、抗米勒管激素(anti-Müllerian hormone,AMH)、WNT、卵泡刺激素受体(follicle-stimulating hormone receptor,FSHR)以及促炎因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)和IL-1β的表达水平,以RNA-seq检测生长分化因子-15(growth differentiation factor-15,GDF-15)的表达水平。通过环磷酰胺(cyclophosphamide,Cy)构建体外模型。结果:(1)造模6周后,与对照组相比,pZP3组和hUC-MSCs组动情周期出现紊乱,且血清FSH水平升高,血清E_(2)水平降低,表明模型构建成功。(2)相较于对照组,pZP3组闭锁卵泡增加,原始卵泡数目降低,hUC-MSCs干预后小鼠原始卵泡比例和数目均高于pZP3组(P<0.05)。(3)与对照组相比,pZP3组卵巢中凋亡蛋白p53、Bax表达水平上调,hUC-MSCs干预后小鼠凋亡蛋白p53、Bax表达较pZP3组下调(均P<0.05);pZP3组体内Caspase-3变化与对照组差异无统计学意义(P>0.05)。pZP3组炎症因子IL-1β、TNF-α表达上调,hUC-MSCs干预小鼠的IL-1β、TNF-α表达较pZP3组显著下调(均P<0.05)。(4)pZP3组体内Treg细胞数量降低(P<0.01),Objective:To investigate the ovarian damage and mechanism of human umbilical cord mesenchymal stem cells (hUC-MSCs) to improve the ovarian injury in mice with premature ovarian insufficiency (POI).Methods:A mouse model of POI was established by plantar injection of zona pellucida 3 peptide (pZP3),and the mice were divided into control group,adjuvant control group,pZP3 group and hUC-MSCs group,with 10 mice in each group.A vaginal smear was used to monitor the estrous cycle,enzyme-linked immunosorbent assay (ELISA) was used to detect serum follicle-stimulating hormone (FSH) and estradiol (E_2) levels,HE staining was used to observe the histology of the ovary,Western blotting was used to detect the expression levels of FOXO3a,p-FOXO3a,p53,Caspase-3 and Bax,and qRT-PCR was used to detect the markers of POI such as bone morphogenetic protein 15 (BMP15),anti-Müllerian hormone (AMH),WNT,and follicle-stimulating hormone receptor (FSHR).qRT-PCR detected the expression levels of tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6),and IL-1β,and RNA-seq detected the expression level of growth differentiation factor-15 (GDF-15).The in vitro model was established by cyclophosphamide (Cy).Results:(1)After 6 weeks of administration,compared to the control group,the pZP3 group and hUC-MSCs group showed disturbances in the estrous cycle,and the serum FSH level was increased and the E_(2level) was decreased,indicating that the model construction was successful.(2) Compared with the control group,atretic follicles increased and primordial follicle number decreased in the pZP3 group,and the proportion of primordial follicles was significantly increased and the number of primordial follicles was increased after hUC-MSCs intervention (P<0.05).(3) Compared with the control group,the expression levels of apoptotic proteins p53 and Bax were up-regulated in the ovaries of mice in the pZP3 group,and the expression of apoptotic proteins p53 and Bax were down-regulated after the intervention of hUC-MSCs (both P<0.05);the changes of Caspa

关 键 词：人脐带间充质干细胞 原发性卵巢功能不全 生长分化因子15 叉头框转录因子O3 卵巢储备功能 

分 类 号：R711.75[医药卫生—妇产科学]