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.

累积数据表明幽门螺杆菌感染参与胃癌的发展。在这项研究中，为了阐明幽门螺杆菌感染在胃癌发生中的作用，我们试图在胸膜癌和肾上腺小鼠中伴有h..pylbyri感染的胸腺/c小鼠在胸膜癌和肾上腺小鼠中伴随抑制性的结果，并在以下结果中获得了3天，并且（1）在;（1）bal/c鼠标（1）;（1）;（1）在;（1）Bal/c Miles; H.Pylori在6-8周时。大约50％的小鼠在金融腺发生了胃腺瘤。（2）在感染后9个月中，从腺瘤和非金融腺体的非腺瘤部分提取RNA。在构造单链cDNA之后，通过使用未感染对照小鼠的mRNA进行减法，并用剩余的cDNA制成cDNA库。（3）表达质粒库是通过插入CMV启动子（转换为COS细胞）构建的。 48H孵育后，将COS细胞的培养基施用至GSMO6细胞，并从COS细胞中提取质粒DNA，其上清液的上清液对GSMO6细胞具有显着的生长促进作用。通过重复此过程，最终克隆了cDNA。我们从COS细胞中提取了HGF，TGF-α，Reg，TNF-α和IL-1β基因。（4）cDNA，增强其生长能力，并将cDNA转换为GSMO6细胞。选择后，从增殖细胞中提取基因组，并测序cDNA。（5）（5）如上所述的过程，涉及NF-KB信号传导途径（如NF-KB，TRAF-6，NIK-6，NIK和SRC和SRC和SRC和CELCLIN D1）等基因。参与胃癌发生。