miR-146调控TLR4/NF-κB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  

Study on the Mechanism of miR-146 in Reducing Follicular Granulosa Cell Apoptosis and Intervening Premature Ovarian Failure by Regulating TLR4/NF-κB

作　　者：何凤屏 刘彦慧 熊符[3] 唐莉 马秋林 郭义红 刘玉兰 HE Feng-ping;LIU Yan-hui;XIONG Fu;TANG Li;MA Qiu-lin;GUO Yi-hong;LIU Yu-lan(Dongguan Institute of Reproduction and Genetics,Dongguan Maternal and Child Health Hospital Affiliated to Southern Medical University,Guangdong Dongguan 523125,China;Molecular Biology Laboratory of Yuebei People’s Hospital Affiliated to Medical College of Shantou University,Guangdong Shantou 512026,China;Key Laboratory of Genetics,School of Basic Medicine,Southern Medical University,Guangzhou 510515,China)

机构地区：[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

出　　处：《现代检验医学杂志》2023年第1期77-82,共6页Journal of Modern Laboratory Medicine

基　　金：广东省基础与应用基础研究基金项目(2020B 1515120009)。

摘　　要：目的探究miR-146通过调控Toll样受体4(TLR4)/核因子-κB(NF-κB)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-κB,肿瘤坏死因子α(TNF-α)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-κB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(0.51±0.14 vs 1.52±0.21),差异具有统计学意义(t=7.338,P<0.01);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(9.43±2.03,6.43±1.60 vs 16.82±2.11)、初级卵泡(6.15±1.11,5.01±1.10 vs 8.88±1.12)、次级卵泡(5.11±1.71,4.01±1.26 vs 7.11±1.34)均降低,闭锁卵泡(10.17±1.41,11.46±1.96 vs 7.18±1.64)升高,差异具有统计学意义(F=7.787,8.214,9.726,7.811,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TLR4(68.18±5.92pg/ml,91.11±16.34 pg/ml vs 24.81±2.81 pg/ml),NF-κB(74.19±8.11 pg/ml,88.11±16.71pg/ml vs 68.18±5.92 pg/ml),TNF-α(72.81±2.10 pg/ml,94.31±2.26 pg/ml vs 28.07±3.67 pg/ml)和IL-6(69.19±7.11,81.11±16.34 vs 19.43±10.81 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(1.18±0.19.0.61±0.14 vs1.8Objective To investigate the expression of miR-146 in premature ovarian failure(premature ovarian failure,POF)model mice and its role and mechanism in premature ovarian failure and follicular apoptosis.Methods Wild 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-κB(NF-κB),Tumor necrosis factor(TNF-α)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-κB expression level were detected by Western blot.Results Compared with the control group,the expression level of miR-146 in follicles of POF group(0.51±0.14 vs 1.52±0.21)was down-regulated,and the difference was statistically significant(t=7.338,P<0.01).The knockout mice showed that:in the control group,the primordial follicles of POF and miR-146 were 9.43±2.03,6.43±1.60 vs 16.82±2.11,the primary follicles were 6.15±1.11,5.01±1.10 vs 8.88±1.12,the secondary follicles were 5.11±1.71,4.01±1.26 vs 7.11±1.34,and the atretic follicles were 10.17±1.41,11.46±1.96 vs 7.18±1.64,respectively,and the differences were statistically significant(F=7.787,8.214,9.726,7.811,all P<0.01).Compared with the control group,TLR4(68.18±5.92,91.11±16.34 vs 24.81±2.81 pg/ml),NF-κB(74.19±8.11,88.11±16.71 vs 68.18±5.92 pg/ml),TNF-α(72.81±2.10,94.31±2.26 v

关 键 词：miR-146 卵巢早衰 Toll样受体4(TLR4)/核因子-κB(NF-κB)信号通路 

分 类 号：R711.75[医药卫生—妇产科学] R392.11[医药卫生—临床医学]