摘要
目的 克隆乙型肝炎病毒X抗原 (HBxAg)相关肝癌差示表达基因并做功能研究。方法 (1)用逆转录病毒方法建立表达HBxAg细胞模型。 (2 )用抑制差减杂交做基因差示表达分析。 (3)做RNA印迹分析 (NorthernBlot)及原位分子杂交筛选基因探针。 (4 )用 5′和 3′迅速末端扩增法 (RACEPCR)进行全长基因序列扩增 ,并进行体外蛋白翻译及制备抗体 ,做蛋白质印迹 (WesternBlot)分析。(5 )进行该基因细胞内转导 ,进行功能研究。结果 共得到 10个cDNA探针 ,其中 8个因HBX表达上调 ,2个表达下降。经筛选及全长基因扩增 ,得到 1个全长为 1.35kb、编码 113个氨基酸的全长基因C2 ,和蛋白翻译起始因子 (Suil)同源 ,其表达被HBX抑制。初步功能研究显示 ,此基因对细胞生长具有抑制作用 ,并且正常肝组织中其mRNA信号明显高于肝癌组织。结论 我们不仅克隆出HBX相关基因C2并进行了功能研究 ,还首次提出DNA病毒 (HBV)
Objective To clone full length differentially expressed genes which are related with HBxAg. Methods HepG2 cells were infected with prepared recombinant retroviruses encoding the X antigen. The differences in gene expression between HepG2×and HepG2Cat cells were evaluated by suppression subtractive hybridization and PCR. In situ hybridization (ISH) and Northern blot analysis were carried out to screen the differentially expressed genes. The full length cDNA clone of the gene was obtained by 5′and 3′rapid amplification of cDNA ends(race) PCR. HepG2 cells transiently transfected with the new full length gene were subjected to fluorescence activated cell sorting(FACS) analysis for DNA content. HepG2 cells stably transfected with the new full length gene were tested for anchorage independent growth in soft agar and for tumorigenicity in nude mice. Results The expression of multiple genes were turned on(8) or off(2) in HepG2X compared to HepG2CAT cells. One differentially expressed gene C2, the human homology of Sui1, encoded a translation initiation factor whose expression was suppressed by X antigen in HepG 2 cells. The full length of this gene was 1.35 kb, which encoded a small protein of 113 amino acids. Introduction of C2 into HepG2 cells could inhibit cell growth in culture, in soft agar, and partially inhibit tumor formation in nude mice. Cells transfected with pcDNA3 HBx showed little or no detectable C2, which was consistent with the suppression of this protein in the presence of HBxAg. C2 was also expressed in nontumor liver, but not in tumor cells from patients with hepatocellular carcinoma. Conclusions HBX can regulate the expression of genes whose products may be positive or negative regulators of cell growth. Our work for the first time demonstrates that the mechanism of DNA virus associated carcinogenesis involves altered patterns of gene expression regulated at the level of translation initiation.
出处
《中华医学杂志》
CSCD
北大核心
2000年第6期456-460,共5页
National Medical Journal of China
基金
国家自然科学基金高技术探索资助项目!(39980041)
关键词
肝肿瘤
互补DNA
乙型肝炎病毒X抗原
Liver neoplasms
Hybridization
DNA
complementary
Hepatitis antigens
