下调NF-κB信号通路促进人羊膜间充质干细胞上皮化  

Down-regulation of N FkB signaling pathway promotes epithelialization of human amniotic mesenchymal stem cells

作　　者：刘拟舟 毛艳华 张文文 张应凤 王佳 李长江 陈秋宏 吴莎莎 王婷婷 黄玺玥 谢乐乐 孙聪聪 LIU Nihou;MAO Yanhua;ZHANG Wenwen;ZHANG Yingfeng;WANG Jia;II Changiang;CHEN Qiuhong;WU Shasha;WANG Tinging;HUANG Xiyue;XIE Lele;SUN Congcong(Department of Obstetrics and Gynecology,the University-Town Hospital Afiliated to Chongqing Medical University,Chongqing,401331,China)

机构地区：[1]重庆医科大学附属大学城医院妇产科,重庆401331

出　　处：《陆军军医大学学报》2023年第16期1712-1721,共10页Journal of Army Medical University

基　　金：重庆市英才计划“包干制”项目(cstc2021ycjh-bgzxm0014);重庆医科大学智慧医学项目(YJSZHYX202107)。

摘　　要：目的探讨NF-κB信号通路在诱导人羊膜间充质干细胞(human amniotic mesenchymal stem cells,hAMSCs)上皮化的过程中发挥的作用。方法分离和培养hAMSCs,将hAMSCs分为对照组和诱导组(n=3),采用RT-qPCR检测上皮化标志物(CK-7、CK-19、E-cadherin)、间质标志物(Vimentin)、NF-κB信号通路关键因子(IκBα、p65)和NF-κB信号通路靶基因cyclin D1的mRNA表达水平;采用免疫荧光检测CK-7、E-cadherin、Vimentin、IκBα、p65和p-p65的蛋白表达水平;为确认NF-κB信号通路在hAMSCs上皮化中的作用,将hAMSCs分为诱导组、抑制剂组、Ad-GFP组(空白腺病毒)、Ad-IκBα组(腺病毒过表达IκBα)、LV-GFP组(空白慢病毒)、LV-RELA-RNAi组(慢病毒沉默p65)(n=3)。RT-qPCR检测CK-7、CK-19、E-cadherin、Vimentin、IκBα和p65的mRNA表达水平,免疫荧光检测CK-7、E-cadherin、Vimentin和IκBα、p65、p-p65的蛋白表达水平。结果与对照组比较,诱导组中CK-7、CK-19、E-cadherin的mRNA表达升高(P<0.01),CK-7、E-cadherin蛋白表达升高(P<0.05),Vimentin的mRNA表达降低(P<0.01),蛋白表达降低(P<0.05),IκBα、p65、cyclin D1的mRNA表达降低(P<0.001),IκBα、p65、p-p65蛋白表达降低(P<0.05);抑制剂组、Ad-IκBα组、LV-RELA-RNAi组中CK-7、CK19、E-cadherin的mRNA表达较诱导组明显升高(P<0.01),CK-7、E-cadherin蛋白表达较诱导组升高(P<0.05),Vimentin的mRNA表达明显下降(P<0.01),蛋白表达降低(P<0.05)。结论NF-κB信号通路参与hAMSCs上皮化的调控,下调NF-κB信号通路能有效促进hAMSCs上皮化。Objective To explore the role of NF-KB signaling pathway in inducing the epithelialization of human amniotic mesenchymal stem cells(hAMSCs).Methods hAMSCs were extracted and divided into control group and induced group(n=3).RT-qPCR was used to detect the mRNA levels of epithelial markers(CK-7,CK-19,E-cadherin),mesenchymal marker(Vimentin),key factors of NF-KB signaling pathway(IkBc,p65)and target gene of NF-KB pathway(cycin D1).The protein expression of CK-7,E-cadherin,Vimentin,IkBc,p65 and p-p65 were detected by immunofluorescence assay.To confirm the role of NF-KB signaling pathway in the epithelialization of hAMSCs,hAMSCs were divided into induced group,inhibitor group,Ad-GFP group(control adenovirus),Ad-IKBo group(a recombinant adenovirus overexpressing IkBa),LV-GFP group(control lentivirus)and LV-RELA-RNAi group(a lentivirus silencing p65)(n=3).The mRNA levels of CK-7,CK-19,E-cadherin,Vimentin,IkBcu and p65 were detected by RT-qPCR.The protein levels of CK-7,E-cadherin,Vimentin,IkBa,p65 and p-p65 were determined by immunofluorescence assay.Results Compared with the control group,the induced group showed the increased mRNA expression of CK-7,CK-19 and E-cadherin(P<0.01),increased protein expression of CK-7 and E-cadherin(P<0.05),decreased mRNA expression of Vimentin(P<0.01),decreased protein expression of Vimentin(P<0.05),decreased mRNA expression of IkBa,p65 and cyclin DI(P<0.001)and protein expression of IkBa,p65 and p-p65(P<0.05).Compared with the induced group,the mRNA expression levels of CK-7,CK-19 and E-cadherin were increased(P<0.01)in inhibitor group,Ad-IkBc group and LV-RELA-RNAi group,and the protein expression of CK-7 and E-cadherin were inereased(P<0.05),while the mRNA expression of Vimentin was decreased(P<0.01)and protein expression of V imentin was decreased(P<0.05).Conclusion NF-KB signaling pathway is involved in the regulation of epithelialization of hAMSCs,and down-regulation of NF-KB signaling pathway could effectively promote the epithelialization of hAMSCs.

关 键 词：NF-ΚB信号通路 人羊膜间充质干细胞 宫腔粘连 上皮化 

分 类 号：R322.65[医药卫生—人体解剖和组织胚胎学] R329.21[医药卫生—基础医学] R329.26