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目的建立肝癌细胞微细胞介导染色体转移方法,为肝癌转移抑制基因的染色体功能定位建立技术平台。方法人单染色体供体细胞通过微核化、出核、融合步骤将随机标记有耐药neo基因的正常人8号染色体导入到大鼠肝癌高转移细胞系C5F中,对微细胞杂交克隆进行药物筛选和单细胞克隆,并用序列标签位点-PCR和全染色体涂染荧光原位杂交方法验证人染色体转移的结果。结果获得具有G418和HAT双重抗性的微细胞杂交细胞,通过单细胞分离克隆方法获得15个具有双重抗性的微细胞杂交克隆,序列标签位点-PCR结果发现导入染色体的随机片段丢失,全染色体涂染荧光原位杂交结果发现导入的人8号染色体与大鼠染色体发生了稳定的重组。结论成功建立微细胞介导的染色体转移技术,为肝癌转移抑制基因的染色体功能定位奠定了技术基础。
Objective To establish a method of microcell-mediated chromosomal transfer of hepatocellular carcinoma cells and to establish a technical platform for the localization of chromosomal function of metastasis suppressor genes of hepatocellular carcinoma. Methods The human chromosome-specific donor cells were introduced into the high-metastatic cell line C5F of rat liver cancer by micronuclei, nucleus and fusion steps. The normal human chromosome 8 was labeled with drug resistant neo gene. Screening and single cell cloning. The results of human chromosome transfer were verified by sequence-tagged site-PCR and whole chromosome smear fluorescence in situ hybridization. Results The results showed that there were 15 pairs of minicell hybrids with double resistance by G418 and HAT, and 15 pairs of double-resistant clones were obtained by single-cell isolation and cloning method. The sequence tag site-PCR results showed that the random fragments introduced into the chromosomes were lost and all Chromosomal staining Fluorescence in situ hybridization revealed that the introduced human chromosome 8 was stably recombined with the rat chromosome. Conclusion The successful establishment of microcell-mediated chromosomal transfer technology laid the technical foundation for the chromosomal location of the metastasis-suppressor gene in hepatocellular carcinoma.