人端粒酶逆转录酶上调Cdx2表达促进胃黏膜肠化生.docVIP

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（human telomerase reverse transcriptase，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Western blot技术从蛋白水平证实其表达变化；继而构建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Western blot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、Cdx2 mRNA表达明显上调，而Cdx4 mRNA则无明显变化；Western blot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p65存在蛋白-蛋白相互作用； 另外双荧光素酶实验和Western blot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] A Human telomerase reverse transcriptase upregulates the expression of Cdx2 and promotes intestinal metaplasia of gastric mucosa Chen Baijun*, Zhou Yuan*, Xiao Yufeng, Hu Changjiang, Xie Rui, He Bing, Wang Sumin, Wu Yuyun, Ling Xianlong, Yang Shiming (Institute of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China) [Abstract] Objective To investigate the role of human telomerase reverse transcriptase (hTERT) in intestinal metaplasia of gastric mucosa and the relevant molecular mechanism. Methods After the hTERT eukaryotic expression plasmids were successfully constructed, hTERT was transferred into human gastric cancer cell line MKN45 by lipofectamine 2000. Quantitative PCR and Western blot were applied to examine the expression of the intestinal metaplasia genes, Cdx1, Cdx2 and Cdx4. Dual luciferase reporter assay was used to test the activity of the Cdx2 promoter. Furthermore, co-immunoprecipitation was employed to demonstrate the interaction between hTERT and p65. At last, NF-kB inhibitor was used to test the alleviation of the hTERT-induced Cdx2 promoter and protein expression. Results Quan