人脐带间充质干细胞外泌体miR-100对多囊卵巢综合征患者颗粒细胞炎症的影响

doi:10.6040/j.issn.1671-7554.0.2022.0891

摘要/Abstract

摘要： 目的探究人脐带间充质干细胞(hUC-MSCs)来源的外泌体miR-100对多囊卵巢综合征(PCOS)患者颗粒细胞炎症的影响。方法选取2021年3月至2021年12月于石家庄市第四医院生殖医学中心就诊的70例PCOS患者为研究对象(PCOS组),同期70例因输卵管因素助孕治疗的非PCOS患者为对照(对照组),比较两组患者的临床基线资料及IVF妊娠结局相关参数。采用实时荧光定量PCR(RT-qPCR)检测患者颗粒细胞肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)的表达。采用二元Logistic回归分析影响PCOS的危险因素。RT-qPCR验证hUC-MSCs外泌体中miR-100的表达水平。合成miR-100模拟物及其对照转染至人卵巢颗粒细胞KGN中,RT-qPCR检测miR-100的表达水平。在脂多糖(LPS)诱导的KGN炎症细胞模型中转染miR-100模拟物及其对照,RT-qPCR检测TNF-α、IFN-γ的mRNA表达水平。采用双荧光素酶报告基因系统检测miR-100对靶基因的调控作用。miR-100过表达后,Western blotting检测KGN炎症模型中Toll样受体4(TLR4)、 p-IκBa、IκBa、p-p65、 p65蛋白水平。结果与对照组比较,PCOS组患者体质量指数(BMI)、黄体生成素(LH)、雌二醇(E₂)、睾酮(T)、胰岛素抵抗指数(HOMA-IR)、窦卵泡数(AFC)、获卵数升高(P值分别为<0.001、<0.001、0.020、<0.001、<0.001、<0.001、<0.001);但MII卵率和2PN受精率降低,差异有统计学意义(P均<0.001)。与对照组患者比较,PCOS组患者TNF-α、IFN-γ的mRNA水平升高,差异有统计学意义(P均<0.001)。二元Logistic回归分析结果显示,LH、HOMA-IR、TNF-α、IFN-γ是影响PCOS的危险因素(P值分别为0.031、0.001、0.030、0.005)。RT-qPCR结果显示,miR-100在hUC-MSCs-exos中富集,miR-100过表达可抑制LPS诱导的颗粒细胞炎症因子TNF-α、IFN-γ的mRNA表达水平(P值分别为0.041、<0.001)。双荧光素酶报告基因结果显示,miR-100能结合TLR4 mRNA 3’UTR并有效抑制其表达(P<0.001)。Western blotting结果显示,与miR-NC组比较,过表达miR-100后KGN炎症模型中TLR4、 p-IκBa和p-p65蛋白表达降低(P值分别为<0.001、<0.001、0.049),IκBa蛋白水平升高(P<0.001)。结论 PCOS患者颗粒细胞TNF-α、IFN-γ表达升高,是影响PCOS的危险因素。hUC-MSCs来源的外泌体miR-100可通过靶向TLR4/NF-κB信号通路抑制PCOS颗粒细胞的炎症反应。

关键词: 多囊卵巢综合征, 人脐带间充质干细胞, 外泌体, 微小RNAs, 颗粒细胞, 炎症

Abstract: Objective To explore the effects of exosomal miR-100 derived from human umbilical cord mesenchymal stem cells(hUC-MSCs)on the inflammation of granulosa cells(GCs)in polycystic ovary syndrome(PCOS). Methods A total of 70 PCOS patients treated during Mar. 2021 and Dec. 2021 were enrolled as the PCOS group, and another 70 patients treated due to tubal factors were selected as the control group. The clinical characteristics and in vitro fertilization(IVF)outcomes were compared between the two groups. The expressions of pro-inflammatory cytokines TNF-α and IFN-γ in patients’ GCs were detected with real-time quantitative PCR(RT-qPCR). The risk factors influencing the occurrence of PCOS were analyzed with binary Logistic regression analysis. The expression of miR-100 in hUC-MSCs exosomes(exos)was verified with RT-qPCR. After the miR-100 mimics and miR-NC were synthesized and transfected into human ovarian granulosa cells(KGN), the expression of miR-100 was determined with RT-qPCR. After the miR-100 mimics and miR-NC were transfected into lipopolysaccharide(LPS)-induced KGN cells, the mRNA expressions of TNF-α and IFN-γ were detected with RT-qPCR. The target gene of miR-100 was verified with dual-luciferase reporter gene assay. After miR-100 was overexpressed in LPS-induced KGN cells, the effector molecular of Toll-like receptor-4/nuclear factor kappa B(TLR4/NF-κB)signaling pathways, including TLR4, p-IκBa, IκBa, p-p65 and p65, were detected with Western blotting. Results PCOS group had higher body mass index(BMI), luteinizing hormone(LH), estradiol(E₂), testosterone(T), homeostasis model assessment for insulin resistance index(HOMA-IR), antral follicle count(AFC), and number of retrieved oocytes than the control group, whereas lower MII oocyte rate and 2PN fertilization rate(P<0.001, P<0.001, P=0.020, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001). The PCOS group had significantly higher expressions of TNF-α and IFN-γ than the control group(P<0.001). Binary Logistic regression analysis revealed that LH, HOMA-IR, TNF-α and IFN-γ were risk factors influencing the occurrence of PCOS(P=0.031, 0.001, 0.030, 0.005). RT-qPCR showed that miR-100 was enriched in hUC-MSCs-exos. Overexpression of miR-100 significantly inhibited LPS-induced increase of the mRNA expressions of TNF-α and IFN-γ(P=0.041, P<0.001). Dual-luciferase reporter gene assay showed that miR-100 directly targeted TLR4 mRNA 3’UTR and inhibited its expression(P<0.001). Western blotting showed that miR-100 efficiently inhibited LPS-induced elevation of the protein expressions of TLR4, p-IκB and p-p65(P<0.001, P<0.001, P=0.049), but the protein expression of IκB significantly increased(P<0.001). Conclusion The increased expressions of TNF-α and IFN-γ are risk factors influencing the occurrence of PCOS. Exosomal miR-100 derived from hUC-MSCs inhibits the inflammation of granulosa cells via TLR4/NF-κB signaling pathway in PCOS.

Key words: Polycystic ovary syndrome, Human umbilical cord mesenchymal stem cell, Exosomes, MicroRNAs, Granulosa cells, Inflammation

中图分类号:

R711.75

赵元元,路军涛,吴小华. 人脐带间充质干细胞外泌体miR-100对多囊卵巢综合征患者颗粒细胞炎症的影响[J]. 山东大学学报 (医学版), 2023, 61(5): 51-58.

ZHAO Yuanyuan, LU Juntao, WU Xiaohua. Effects of human umbilical cord mesenchymal stem cell-derived exosomal miR-100 on inflammation of ovarian granulosa cells in polycystic ovary syndrome[J]. Journal of Shandong University (Health Sciences), 2023, 61(5): 51-58.

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