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CLONING OF GROWTH FACTORS AND TYROSINE KINASES INVOLVED IN H.pylori-INDUCED GASTRIC CARCINOGENESIS

幽门螺杆菌诱发胃癌相关生长因子和酪氨酸激酶的克隆

基本信息

批准号：
11470130
负责人：
CHIBA Tsutomu
金额：
$ 8.51万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Accumulating data suggest involvement of H.pylori infection in the development of gastric cancer. In this study, in order to elucidate roles of H.pylori infection in gastric carcinogenesis, we tried to clone growth factors and tyrosine kinases involved in the development of gastric cancer and adenomas occurred in thymectomised balb/c mice with H.pylori infection, and the following results were obtained ;(1) Balb/c mice were thymectomised 3 days after birth, and infected with H.pylori at 6-8 weeks. Approximately 50% of the mice developed gastric adenoma in the fundic gland.(2) At 9 months after infection RNAs were extracted from adenoma and non-adenoma portions of the fundic glands. After construction of single strand cDNAs, subtraction was performed by using mRNAs from non-infected control mice, and cDNA library was made from the remaining cDNAs.(3) Expression plasmid library was constructed by inserting CMV promotor, transfected to COS cells. Culture medium of the COS cells after 48h incubation was administered to GSMO6 cells, and the plasmid DNA was extracted from the COS cells, the supernatant of which had a significant growth promoting effect on GSMO6 cells. By repeating this procedure, cDNA was finally cloned. We obtained HGF, TGF-α, reg, TNF-α, and IL-1β genes.(4) cDNA was extracted from the COS cells, the growth ability of which was enhanced, and the cDNA was transfected to GSMO6 cells. After selection, genomes were extracted from the proliferating cells, and the cDNA was sequenced.(5) From the procedure as mentioned above, genes involved in NF-kB signaling pathway such as NF-kB, traf-6, NIK, and src and cyclin D1 were obtained.(6) From these data, it is suggested that anti-apoptotic action by various inflammatory cytokines with resulting NF-kB activation is involved in gastric carcinogenesis.

越来越多的资料表明幽门螺杆菌感染与胃癌的发生有关。本研究为探讨幽门螺杆菌（H.pylori）感染在胃癌发生过程中的作用，对H.pylori感染的Balb/c小鼠胸腺切除后发生胃癌和腺瘤的生长因子和酪氨酸激酶进行了克隆，获得了以下结果：（1）Balb/c小鼠出生后3d切除胸腺，6-8周感染H.pylori。大约50%的小鼠在胃底腺中发展为胃腺瘤。(2)感染后9个月，从胃底腺的腺瘤和非腺瘤部分提取RNA。在构建单链cDNA后，通过使用来自未感染对照小鼠的mRNA进行消减，并从剩余的cDNA制备cDNA文库。(3)插入CMV启动子构建表达质粒文库，转染COS细胞。将孵育48小时后的COS细胞培养基给予GSMO 6细胞，并从COS细胞中提取质粒DNA，其上清液对GSMO 6细胞具有显著的生长促进作用。通过重复该过程，最终克隆cDNA。我们获得了HGF、TGF-α、reg、TNF-α和IL-1β基因。(4)从生长能力增强的COS细胞中提取cDNA，并将cDNA转染到GSMO 6细胞中。选择后，从增殖细胞中提取基因组，并对cDNA进行测序。(5)通过上述方法，获得了参与NF-κ B信号通路的基因，如NF-κ B、traf-6、NIK、src和cyclin D1。(6)从这些数据中，它表明，抗凋亡作用的各种炎性细胞因子所产生的NF-κ B活化参与胃癌的发生。