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

doi:10.12280/gjfckx.20240599

摘要/Abstract

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 pellucida 3 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，E₂）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E₂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）。④pZP3组体内Treg细胞数量降低（P<0.01），hUC-MSCs干预后小鼠Treg细胞数量较pZP3组升高（P<0.000 1）。pZP3组卵巢组织中BMP15、AMH、WNT以及FSHR的mRNA表达水平较对照组降低，hUC-MSCs干预后小鼠的表达水平较pZP3组升高（均P<0.05）。⑤pZP3组FOXO3a磷酸化水平高于对照组（P<0.000 1），hUC-MSCs干预后，FOXO3a磷酸化水平较pZP3组下降（P<0.000 1）。测序结果提示，pZP3组GDF-15表达上调， hUC-MSCs组中GDF-15表达较pZP3组下调。结论： POI小鼠体内GDF-15和p-FOXO3a表达上调，hUC-MSCs通过下调GDF-15的表达和降低FOXO3a的磷酸化水平，改善POI小鼠的卵巢组织形态，实现对POI小鼠的治疗作用。

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

Abstract:

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 (E₂) 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: ①After 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 E₂ level was decreased, indicating that the model construction was successful.②Compared 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). ③Compared 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 Caspase-3 in the mice in the pZP3 group were not significantly different from that of the control group (P>0.05). pZP3 group inflammatory factors IL-1β and TNF-α expression were up-regulated, and their expression was significantly down-regulated after hUC-MSCs intervention (both P<0.05). ④The number of Treg cells in mice in the pZP3 group was significantly lower (P<0.01) and significantly higher after hUC-MSCs intervention (P<0.000 1). mRNA expression levels of BMP15, AMH, WNT and FSHR in ovarian tissues of mice in the pZP3 group were lower than those in the control group. Their expression levels were elevated compared with those in the pZP3 group after the intervention of hUC-MSCs (all P<0.05). ⑤The phosphorylation level of FOXO3a was increased in the pZP3 group compared with the control group (P<0.000 1) and decreased after hUC-MSCs intervention (P<0.000 1). The sequencing results suggested that GDF-15 expression was up-regulated in POI mice and down-regulated in the hUC-MSCs group. Conclusions: The expression of GDF-15 and p-FOXO3a was up-regulated in POI mice, hUC-MSCs improved the morphology of ovarian tissues in POI mice and achieved the therapeutic effect on POI mice by down-regulating the expression of GDF-15 and reducing the phosphorylation level of FOXO3a.

Key words: Human umbilical cord mesenchymal stem cell, Primary ovarian insufficiency, Growth differentiation factor 15, FOXO3, Ovarian reserve

司潇洒, 王建波, 翁平, 史朋飞, 王长亮, 陈晨, 王勇. 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[J]. 国际妇产科学杂志, 2024, 51(4): 424-432.

SI Xiao-sa, WANG Jian-bo, WENG Ping, SHI Peng-fei, WANG Chang-liang, CHEN Chen, WANG Yong. Human Umbilical Cord Mesenchymal Stem Cells Treat Mouse Premature Ovarian Insufficiency through GDF-15/FOXO3a[J]. Journal of International Obstetrics and Gynecology, 2024, 51(4): 424-432.

图/表 10

表1 引物序列

基因名称	序列
BMP15	上游：5'-TCCTTGCTGACGACCCTACAT-3'
	下游：5'-TACCTCAGGGGATAGCCTTGG-3'
AMH	上游：5'-GGGAGCAAGCCCTGTTAGTG-3'
	下游：5'-AGCGGGAATCAGAGCCAAA-3'
WNT	上游：5'-AGACGTGCGAGAAACTCAAAG-3'
	下游：5'-GGAACTGGTATTGGCACTCCT-3'
FSHR	上游：5'-AGTGTTTAATGCCTGTGTTGGA-3'
	下游：5'-ACCCTGAGGCCTTCCAGA-3'
TNF-α	上游：5'-ATACACTGGCCCGAGGGAAC-3'
	下游：5'-CCACATCTCGGATCATGCTTTC-3'
IL-6	上游：5'-ACAAAGCCAGAGTCCTTCAGAG-3'
	下游：5'-GGCAGAGGGGTTGACTT-3'
IL-1β	上游：5'-TCCAGGATGAGGACATGAGCA-3'
	下游：5'-GAACGTCACACACCAGCAGGT-3'
GAPDH	上游：5'-GTGAAGGTCGGTGTGAACGG-3'
	下游：5'-GTGATGGCATGGACTGTGGTC-3'

图1 造模期间小鼠体质量监测

图2 小鼠动情周期检测（甲苯胺蓝染色×100）注：A动情前期，B动情期，C动情后期，D 动情间期。NEC有核上皮细胞，CC无核上皮细胞，L白细胞；标尺=100 μm。

图3 各组脾脏、卵巢对比注：A 各组脾脏对比图，B脾脏指数量化图，C 卵巢指数量化图，D 各组卵巢对比图。组间比较，**P<0.01，****P<0.000 1。

图4 POI小鼠模型注：A 各组小鼠动情周期各阶段示图，其中纵坐标4代表动情前期，3代表动情期，2代表动情后期，1代表动情间期；B 各组小鼠血清FSH水平；C 各组小鼠血清E2水平。组间比较，*P<0.05，**P<0.01。。

图5 小鼠卵巢病理组织学变化注：A 各组卵巢形态学检测（HE染色×200），标尺=250 μm；B 不同阶段卵泡数量统计分析。组间比较，*P<0.05，**P<0.01，***P<0.001。

图6 hUC-MSCs调节POI诱导的凋亡和炎症反应注：A 卵巢组织中p53蛋白表达，B 卵巢组织中Bax蛋白表达，C 卵巢组织中Caspase-3蛋白表达，D 卵巢组织中IL-6、IL-1β、TNF-α的mRNA水平。组间比较，*P<0.05，**P<0.01，***P<0.001，****P<0.000 1。

图7 hUC-MSCs调节POI诱导免疫反应和炎症反应注：A 流式实验检测各组脾脏组织中Treg细胞水平，B Treg细胞水平量化图，C~F q-PCR法检测卵巢组织中BMP-15、AMH、WNT、FSHR的mRNA水平。组间比较，*P<0.05，**P<0.01，***P<0.001，****P<0.000 1。

图8 hUC-MSCs抑制GDF-15和磷酸化FOXO3a表达注：A 各组卵巢中p-FOXO3a、FOXO3a蛋白表达，B 各组卵巢中p-FOXO3a、FOXO3a蛋白表达量化图，C 对照组与pZP3组的转录组对比结果， D hUC-MSCs组与pZP3组转录组对比结果。组间比较，*P<0.05，**P<0.01，***P<0.001，****P<0.000 1。

图9 hUC-MSCs缓解POI引起的颗粒细胞损伤注：A 颗粒细胞中p-FOXO3a、FOXO3a蛋白表达，B 颗粒细胞中p53蛋白表达，C 颗粒细胞中FOXO3a蛋白表达量化图，D 颗粒细胞中p-FOXO3a蛋白表达量化图，E 颗粒细胞中p53蛋白表达量化图，F 颗粒细胞中IL-6、IL-1β、TNF-α的mRNA水平。组间比较，*P<0.05，**P<0.01，***P<0.001，***P<0.000 1。

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