DETECTION OF DNA ALTERATIONS DURING HEPATOCARCINOGENESIS

大鼠肝上皮（RLE）细胞提供了非常有价值的体外模型， 研究自发和化学诱导的肝癌发生。 因此，本项目的主要目标是表征，使用 变性梯度凝胶电泳（DGGE）和二维 聚丙烯酰胺凝胶电泳（2D-PAGE），早期细胞 由于遗传改变和随后的生物化学事件， 自发和化学介导过程中蛋白质表达的变化 肝癌发生RLE细胞在培养中自发转化， 在裸鼠中由这些转化体产生的肿瘤的表型为 主要是分化良好的肝细胞癌样 肿瘤类似地，在体外用黄曲霉毒素B1处理RLE细胞， 产生了一系列转化体，其在移植到裸 小鼠，导致各种各样的肿瘤类型，其中一些 类似于肝细胞癌。DGGE最初的工作是针对 优化分离程序。利用2D-DGGE生成2D图谱 至少250个来自Hae III和Hinf I消化的正常 大鼠肝脏基因组DNA。与~（32）P标记的人小卫星杂交 探针33.6和33.15证明了使用高度 作为片段的超多态性基因座探针的重复DNA序列 侦测直接克隆选定DNA片段的程序 从2D-DGGE凝胶也建立了未来的DNA功能 特征研究。

Rat liver epithelial (RLE) cells provide a very valuable in vitro model to study both spontaneous and chemically induced hepatocarcinogenesis. Therefore, the main objectives of this project are to characterize, using denaturing gradient gel electrophoresis (DGGE) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), the early cellular biochemical events as a result of genetic alterations and subsequent changes in protein expression during spontaneous and chemically mediated hepatocarcinogenesis. RLE cells spontaneously transform in culture and the phenotype of the tumors produced in nude mice from these transformants was primarily that of a well-differentiated hepatocellular carcinoma-like tumor. Similarly, treatment of RLE cells in vitro with aflatoxin Bl produced a series of transformants which, upon transplantation into nude mice, resulted in a diverse variety of tumors types, some of which resembled hepato-cellular carcinomas. Initial DGGE work was directed toward optimizing separation procedures. 2D-DGGE was utilized to generate 2D maps of at least 250 individual fragments from HaeIII and HinfI digested normal rat liver genomic DNA. Hybridization to 32P-labelled human minisatellite probes 33.6 and 33.15 demonstrated the feasibility of using highly repetitive DNA sequences as hyperpolymorphic loci probes for fragment detection. Procedures for the cloning of selected DNA fragments directly from 2D-DGGE gels were also established for future DNA functional characterization studies.