miR-146调控TLR4...及干预卵巢早衰中的机制研究_何凤屏.pdf

77现代检验医学杂志第 38 卷第 1 期2023 年 1 月J Mod Lab Med,Vol.38,No.1,Jan.2023miR-146 调控 TLR4/NF-B 通路减少卵泡颗粒细胞凋亡及 干预卵巢早衰中的机制研究何凤屏1，2，刘彦慧1，熊符3，唐莉1，马秋林1，郭义红1，刘玉兰2（1.南方医科大学附属东莞妇幼保健院，东莞市生殖与遗传研究所，广东东莞 523125；2.汕头大学医学院附属粤北人民医院分子生物学实验室，广东汕头 512026；3.南方医科大学基础医学院遗传重点实验室，广州 510515）摘要：目的探究 miR-146 通过调控 Toll 样受体 4（TLR4）/核因子-B（NF-B）减少卵泡颗粒细胞凋亡和干预卵巢早衰（premature ovarian failure,，POF）的机制。方法野生小鼠实验：60 只 SPF 级 C57BL/6 小鼠随机分为对照组（n=30）和 POF 组（n=30）。POF 组建立卵巢早衰模型，造模后 15 天 qPCR 检测 2 组小鼠卵巢组织中 miRNA-146表达水平；基因敲除鼠实验：40 只 C57BL/6 小鼠和 20 只 miR-146 敲除小鼠分为三组：对照组（n=20）、野生型 POF组（n=20）和 miR-146 敲除 POF 组（n=20），野生型 POF 组和 miR-146 敲除 POF 组建立 POF 模型，建模后 15 天 HE染色分析各组小鼠卵巢组织病理情况及原始卵泡、初级卵泡、次级卵泡和闭锁卵泡数，ELISA 检测各组小鼠卵巢组织炎症信号通路分子 TLR，NF-B,肿瘤坏死因子（TNF-）和白介素 6（IL-6）的表达水平；Western blot 检测卵巢组织凋亡蛋白 BCL2-Associated X 蛋白（Bax），B 淋巴细胞瘤-2 基因（Bcl2）和 TLR4 信号通路蛋白 TLR4，NF-B表达水平。结果野生鼠实验表明与对照组相比，POF组小鼠卵泡中miR-146表达水平下调（0.510.14 vs 1.520.21），差异具有统计学意义（t=7.338，P0.01）；基因敲除鼠实验表明：与对照组相比，野生型 POF 组和 miR-146 敲除 POF组原始卵泡（9.432.03，6.431.60 vs 16.822.11）、初级卵泡（6.151.11，5.011.10 vs 8.881.12）、次级卵泡（5.111.71，4.011.26 vs 7.111.34）均降低，闭锁卵泡（10.171.41，11.461.96 vs 7.181.64）升高，差异具有统计学意义（F=7.787,8.214,9.726,7.811,均 P0.01）。与对照组相比，野生鼠 POF 组和 miR-146 敲除 POF 组小鼠卵巢组 织 TLR4（68.185.92pg/ml，91.1116.34 pg/ml vs 24.812.81 pg/ml），NF-B（74.198.11 pg/ml，88.1116.71 pg/ml vs 68.185.92 pg/ml），TNF-（72.812.10 pg/ml，94.312.26 pg/ml vs 28.073.67 pg/ml）和 IL-6（69.197.11，81.1116.34 vs 19.4310.81 pg/ml）分泌显著升高，差异均有统计学意义（F=6.281,7.264,8.724,6.817,均 P 0.01）；与对照组相比，野生鼠 POF 组和 miR-146 敲除 POF 组小鼠卵巢组织 Bax 蛋白表达水平降低（1.180.19.0.610.14 vs 1.810.21），Bcl2 蛋白表达升高（0.590.05,0.910.05 vs 0.580.02），TLR4（1.100.12,0.410.04 vs 1.130.11）和 NF-B（0.810.02,0.310.06 vs 0.870.27）降低，差异均有显著性统计学意义（F=7.235,6.714,7.612,7.737,均 P0.01）。结论miR-146 具有减少卵泡颗粒细胞凋亡的作用，其机制可能与 TLR4/NF-B 信号通路的调控和抑制炎症因子的释放相关。关键词：miR-146;卵巢早衰；Toll 样受体 4（TLR4）/核因子-B（NF-B）信号通路中图分类号：R711.75；R392.11文献标识码：A文章编号:1671-7414（2023）01-077-06doi:10.3969/j.issn.1671-7414.2023.01.015Study on the Mechanism of miR-146 in Reducing Follicular Granulosa Cell Apoptosis and Intervening Premature Ovarian Failure by Regulating TLR4/NF-BHE Feng-ping1,2,LIU Yan-hui1,XIONG Fu3,TANG Li1,MA Qiu-lin1,GUO Yi-hong1,LIU Yu-lan2（Dongguan Institute of Reproduction and Genetics,Dongguan Maternal and Child Health Hospital Affiliated to Southern Medical University,Guangdong Dongguan 523125,China;2.Molecular Biology Laboratory of Yuebei Peoples Hospital Affiliated to Medical College of Shantou University,Guangdong Shantou 512026,China;3.Key Laboratory of Genetics,School of Basic Medicine,Southern Medical University,Guangzhou 510515,China）Abstract:ObjectiveTo investigate the expression of miR-146 in premature ovarian failure（premature ovarian failure，基金项目：广东省基础与应用基础研究基金项目（2020B 1515120009）。作者简介：何凤屏（1968-），女，博士，教授，研究方向：分子生物学，E-mail：。通讯作者：刘彦慧（1971-），男，博士，主任医师，研究方向：生殖与遗传学，E-mail：。78现代检验医学杂志第 38 卷第 1 期2023 年 1 月J Mod Lab Med,Vol.38,No.1,Jan.2023POF）model mice and its role and mechanism in premature ovarian failure and follicular apoptosis.MethodsWild mouse experiment：60 SPF C57BL/6 mice were randomly divided into control group(n=30)and POF group(n=30).POF group established premature ovarian failure model.15 days after modeling,the expression level of miR-146 in the follicles of the two groups was detected by qPCR.Knockout mouse experiment：Forty C57BL/6 mice and 20 miR-146 knockout mice were divided into three groups:control group(n=20)and wild-type POF group(n=20),miR-146 knockout POF group(n=20),wild-type POF group and miR-146 knockout POF to establish POF model.15 days after modeling,HE staining was used to analyze the ovarian pathology and the number of primordial follicles,primary follicles,secondary follicles and atretic follicles,Toll like receptor-4(TLR4)and Nuclear factor-B（NF-B），Tumor necrosis factor（TNF-）and Interleukin-6（IL-6）expression level were detected by ELISA.The apoptotic proteinsBCL2-Associated X protein（Bax）,B-cell lymphoma-2（BCL2）and TLR4 Signal pathway proteins TLR4 and NF-B expression level were detected by Western blot.Results Compared with the control group,the expression level of miR-146 in follicles of POF group(0.510.14 vs 1.520.21)was down-regulated,and the difference was statistically significant(t=7.338,P0.01).The knockout mice showed that:in the control group,the primordial follicles of POF and miR-146 were 9.432.03，6.431.60 vs 16.822.11,the primary follicles were 6.151.11，5.011.10 vs 8.881.12,the secondary follicles were 5.111.71，4.011.26 vs 7.111.34,and the atretic follicles were 10.171.41，11.461.96 vs 7.181.64,respectively,and the differences were statistically significant(F=7.787,8.214,9.726,7.811,all P0.01).Compared with the control group,TLR4(68.185.92,91.1116.34 vs 24.812.81 pg/ml),NF-B(74.198.11,88.1116.71 vs 68.185.92 pg/ml),TNF-(72.812.10,94.312.26 vs 28.073.67 pg/ml)and IL-6(69.197.11,81.1116.34 vs19.4310.81 pg/ml)the secretion of was significantly increased,and the differences were statistical significance(F=6.281,7.264,8.724,6.817,all P 0.01).The expression level of Bax protein in ovarian tis