肝癌乙型肝炎病毒X抗原相关基因的克隆  

Identification of differentially expressed genes in HBxAg transfected HepG2 cells by suppression subtractive hybridization

作　　者：刘杰[1] LIAN Zhaorui PAN Jinbo Clayton M 胡家露[1] 朱明华[3] 窦科峰[1] 丁杰[1] 吴开春[1] 樊代明[1] Feitelson M 

机构地区：[1]西安第四军医大学西京医院消化疾病研究所,710032 [2]美国Thomas Jefferson University病理及细胞生物学实验室 [3]上海第二军医大学病理教研室

出　　处：《中华消化杂志》2000年第3期159-162,共4页Chinese Journal of Digestion

基　　金：国家自然科学基金!高技术探索资助项目 ( 3 9980 0 41)

摘　　要：目的　观察HepG2细胞因乙型肝炎病毒X抗原 (HBx)转染导致的基因表达差异。 方法①用逆转录病毒感染的方法建立表达乙型肝炎病毒X抗原 (HepG2X)及氯霉素乙酰转移酶 (HepG2Cat)的细胞模型。②用抑制差减杂交的方法做HepG2X和对照细胞 (HepG2Cat)的cDNA文库差示筛选。③用3 2 P随机引物标记法标记探针做NorthernBlot杂交及地高辛缺口翻译标记法做原位分子杂交以筛选基因探针。结果　经cDNA文库差减杂交及抑制性PCR扩增共得到 10个cDNA探针 ,其中 8个被HBx所启动 ,2个因HBx而表达下降。经检测 ,这些基因在HepG2X和对照细胞中的表达均有差异。在被HBx所抑制的 2个差示表达基因中 ,1个与人的蛋白翻译起始因子同源 ,同时发现 ,此基因在正常组织中表达较强 ,而在肿瘤组织中表达被抑制。结论　HBx能够改变宿主细胞的基因表达 。Objective To evaluate differentially expressed genes between HepG2X and HepG2Cat cells. Methods 1. Recombinant retroviruses encoding the X antigen or bacterial chloramphenicol acetyltransferase were constructed and used to infect HepG2 cells. 2. The differences in gene expression between HepG2X and HepG2Cat cells were then evaluated by suppression subtractive hybridization and PCR. 3. In Situ hybridization and northern blot analysis were carried out to screen these differentially expressed genes. Results PCR select cDNA subtraction generated 8 differentially expressed genes from HBx transfected HepG2 cells (turned on by HBx). All these probes distinguished HepG2X cells from HepG2Cat cells. Two bands (turned off by HBx) cloned from control cells, were detected in HepG2Cat cells but not in HepG2X. One differentially expressed gene C2, the human homology of hu suil, encodes a translation initiation factor whose expression was suppressed by X antigen in HepG2 cells. This gene was also found in most normal liver tissues, not in tumor tissues. Conclusion HBx can regulate the expression of genes whose products may be positive or negative regulators of cell growth. These changes may be in part of the mechanism contributes to hepatocellular transformation.

关 键 词：肝肿瘤 抑制差减杂交 基因克隆 HBXAG 乙型肝炎 

分 类 号：R735.7[医药卫生—肿瘤] R512.62[医药卫生—临床医学]