干扰CLDN6基因表达通过调控MAPK/ERK信号通路对多囊卵巢综合征大鼠卵巢颗粒细胞凋亡的影响  

The effect of interference with CLDN6 gene expression on the apoptosis of ovarian granular cells in rats with polycystic ovary syndrome by regulating the MAPK/ERK signaling pathway

作　　者：孙彩萍[1] 张丹[2] 张珂[1] Sun Caiping;Zhang Dan;Zhang Ke(Department of Obstetrics,The Second Affiliated Hospital of Zhengzhou University,Zhengzhou 450014;Reproductive Center,The Second Affiliated Hospital of Zhengzhou University,Zhengzhou 450014)

机构地区：[1]郑州大学第二附属医院产科,郑州450014 [2]郑州大学第二附属医院生殖中心,郑州450014

出　　处：《现代妇产科进展》2022年第2期96-101,106,共7页Progress in Obstetrics and Gynecology

基　　金：2019年度河南省医学科技攻关计划(联合共建)项目(No:LHGJ20190319)。

摘　　要：目的探讨Claudin-6(CLDN6)在多囊卵巢综合征(PCOS)大鼠中的表达及其对卵巢颗粒细胞(GC)增殖与凋亡的影响和可能作用机制。方法SD雌性大鼠皮下注射脱氢表雄酮(DHEA)诱导建立PCOS模型,采用实时荧光定量PCR(RT-qPCR)和Western blot法检测卵巢组织中CLDN6表达。分离培养原代PCOS大鼠卵巢GC,采用小分子干扰RNA(siRNA)技术和pcDNA3.1质粒转染下调/上调卵巢GC中CLDN6表达水平,RT-qPCR检测转染效果;CCK-8法和流式细胞术检测细胞活力和凋亡;Western blot法检测细胞CLDN6、丝裂原活化蛋白激酶(MAPK)/细胞外信号调节激酶(ERK)通路和增殖、凋亡相关蛋白的表达。结果CLDN6在PCOS大鼠卵巢组织和GC中呈高表达。转染后,与空白组相比,对照组GC中CLDN6 mRNA和蛋白表达水平、GC活性、Cyclin D1、Bcl-2、p-ERK1/2蛋白表达显著升高,凋亡率、Cleaved Caspase-3、Bax蛋白表达显著降低(P<0.05);与对照组相比,si-CLDN6组GC中CLDN6 mRNA和蛋白表达水平、GC活性、Cyclin D1、Bcl-2、p-ERK1/2蛋白表达显著降低,凋亡率、Cleaved Caspase-3、Bax蛋白表达显著升高(P<0.05),pcDNA3.1-CLDN6组GC中CLDN6 mRNA和蛋白表达水平、GC活性、Cyclin D1、Bcl-2、p-ERK1/2蛋白表达显著升高,凋亡率、Cleaved Caspase-3、Bax蛋白表达显著降低(P<0.05);上调CLDN6表达基础上,ERK1/2抑制剂PD98059可明显减弱CLDN6高表达对GC增殖的促进作用和GC凋亡的抑制作用。结论CLDN6在PCOS大鼠卵巢组织和GC中呈高表达,下调CLDN6可抑制PCOS大鼠卵巢GC增殖并诱导GC凋亡,其作用机制可能与抑制MAPK/ERK信号通路有关。Objective:To investigate the expression of Claudin-6(CLDN6)in polycystic ovary syndrome(PCOS)rats,its effects on the proliferation and apoptosis of ovarian granular cells(GC)and its possible mechanism.Methods:SD female rats were injected subcutaneously with dehydroepiandrosterone(DHEA)to induce the establishment of a PCOS model.Real-time fluorescent quantitative PCR(RT-qPCR)and Western blot were used to detect the expression of CLDN6 in ovarian tissue.The primary PCOS rat ovarian GC was isolated and cultured,the small-molecule interfering RNA(siRNA)technology was used to transfect the pcDNA3.1 plasmid to down-regulate/up-regulate the expression level of CLDN6 in ovarian GC,RT-qPCR was used to detect the transfection effect;CCK-8 method and flow cytometry were used to detect cell viability and apoptosis;Western blot was used to detect the expression of CLDN6,mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase(ERK)pathway,proliferation and apoptosis-related proteins.Results:CLDN6 was highly expressed in ovarian tissue and GC of PCOS rats.After transfection,compared with the blank group,the expression level of CLDN6 mRNA and protein,GC activity,protein expression of Cyclin D1,Bcl-2 and p-ERK1/2 in GC in the control group increased significantly,and the apoptosis rate and protein expression of Cleaved Caspase-3,Bax reduced significantly(P<0.05);compared with the control group,the expression level of CLDN6 mRNA and protein,GC activity,protein expression of Cyclin D1,Bcl-2 and p-ERK1/2 in GC in the si-CLDN6 group reduced significantly,and the apoptosis rate and protein expression of Cleaved Caspase-3,Bax increased significantly(P<0.05),the expression level of CLDN6 mRNA and protein,GC activity,protein expressions of Cyclin D1,Bcl-2,and p-ERK1/2 in GC in the pcDNA3.1-CLDN6 group increased significantly,and the apoptosis rate and protein expression of Cleaved Caspase-3,Bax reduced significantly(P<0.05);on the basis of up-regulating the expression of CLDN6,the use of ERK1/2 inhibitor PD98059 could

关 键 词：多囊卵巢综合征 Claudin-6 卵巢颗粒细胞 丝裂原活化蛋白激酶/细胞外信号调节激酶通路 凋亡 增殖 

分 类 号：R71[医药卫生—妇产科学]