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《人端粒酶逆转录酶上调cdx2表达促进胃黏膜肠化生》由会员上传分享，免费在线阅读，更多相关内容在工程资料-天天文库。
1、人端粒酶逆转录酶上调Cdx2表达促进胃黏膜肠化生陈柏君*，周源*，肖煜峰，胡长江，谢睿，何兵，汪苏敏，吴玉云，凌贤龙，杨仕明（400037重庆，第三军医大学新桥医院消化内科）[摘要]目的探讨人端粒酶逆转录酶（humantelomerasereversetranscriptase，hTERT）在胃黏膜肠化生的作用及其可能存在的分子机制。方法hTERT真核表达质粒构建成功后，用脂质体转染法转染人胃癌MKN45细胞。应用qPCR技术从RNA水平检测转染前后相关肠化生基因(Cdx1、Cdx2和Cdx4)的改变；进一步采用Westernblot技术从蛋白水平证实其表达变化；继而
2、构建Cdx2基因启动子，采用双荧光素酶实验分析hTERT与Cdx2的作用机制；采用COIP证实hTERT与p65结合情况；加入NF-κB抑制剂后，双荧光素酶实验和Westernblot检测Cdx2表达变化情况。结果转染MKN45细胞hTERT基因后，qPCR证实肠化生相关基因Cdx1、Cdx2mRNA表达明显上调，而Cdx4mRNA则无明显变化；Westernblot证实过表达hTERT后只有Cdx2的蛋白表达上调；双萤光素酶报告实验证实与对照组相比，hTERT组Cdx2的启动子活性明显增加（P<0.05）；免疫共沉淀（CO-IP）实验证实hTERT与NF-κB亚基p
3、65存在蛋白-蛋白相互作用；另外双荧光素酶实验和Westernblot表明NF-κB抑制剂（Bay78-1102）能有效抑制hTERT对Cdx2的启动子的活性，并下调Cdx2蛋白的表达。结论hTERT可以通过活化NF-κB信号通路促进Cdx2的表达，从而促进胃黏膜的肠化生的发生。[关键词]人端粒酶逆转录酶；肠上皮化生；胃黏膜；胃黏膜癌前病变[中图法分类号][文献标志码]AHumantelomerasereversetranscriptaseupregulatestheexpressionofCdx2andpromotesintestinalmetaplasiaofga
4、stricmucosaChenBaijun*,ZhouYuan*,XiaoYufeng,HuChangjiang,XieRui,HeBing,WangSumin,WuYuyun,LingXianlong,YangShiming(InstituteofGastroenterology,XinqiaoHospital,ThirdMilitaryMedicalUniversity,Chongqing,400037,China)[Abstract]ObjectiveToinvestigatetheroleofhumantelomerasereversetranscriptas
5、e(hTERT)inintestinalmetaplasiaofgastricmucosaandtherelevantmolecularmechanism.MethodsAfterthehTERTeukaryoticexpressionplasmidsweresuccessfullyconstructed,hTERTwastransferredintohumangastriccancercelllineMKN45bylipofectamine2000.QuantitativePCRandWesternblotwereappliedtoexaminetheexpress
6、ionoftheintestinalmetaplasiagenes,Cdx1,Cdx2andCdx4.DualluciferasereporterassaywasusedtotesttheactivityoftheCdx2promoter.Furthermore,co-immunoprecipitationwasemployedtodemonstratetheinteractionbetweenhTERTandp65.Atlast,NF-kBinhibitorwasusedtotestthealleviationofthehTERT-inducedCdx2promot
7、erandproteinexpression.ResultsQuantitativePCRresultsshowedthattheexpressionofintestinalmetaplasiagenesCdx1andCdx2wasup-regulatedbyhTERT,whileCdx4genehadnoobviouschange.WesternblotresultsverifiedthatonlyCdx2proteinlevelwasincreased.Dualluciferasereporterassayresultsdemonstratedt
