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目的探讨经载体介导的RNA干涉法建立周期蛋白E1表达稳定抑制细胞系的可行性。方法将源自pSSC-9质粒的neo基因亚克隆至干涉载体pSilencer 1.0-U6以构建稳定干涉载体pSineo,再将其与合成的靶向周期蛋白E1基因特定的RNA干涉模板片段连接,重组载体经脂质体LipofectamineTM2000介导法转染肝癌细胞株BEL-7402;转染细胞经G418筛选,常规抽提G418抗性克隆细胞的基因组DNA并行PCR法鉴定外源导入载体的稳定整合情况。结果酶切及测序鉴定结果均表明,重组载体pSineo-cyc E1符合预期设计;PCR鉴定结果显示,稳定筛选出的6个克隆均整合有靶向周期蛋白E1基因的干涉载体。结论本研究建立了6个稳定整合有靶向细胞周期蛋白E1基因RNA干涉载体的肝癌细胞系,为肿瘤的癌基因靶向干涉治疗作了有益探索。
OBJECTIVE: To investigate the feasibility of establishing a stably inhibitory cell line expressing cyclin E1 by vector-mediated RNA interference. Methods The neo gene from pSSC-9 plasmid was subcloned into the interference vector pSilencer 1.0-U6 to construct the stable interference vector pSineo, which was then ligated with the specific RNA interference template fragment targeting the cyclin E1 gene. The transfected cells were selected by G418 and the genomic DNA of G418 resistant clones were routinely extracted. The stability of exogenous vector was identified by PCR. Results The results of restriction enzyme digestion and sequencing showed that the recombinant vector pSineo-cyc E1 was in accordance with the expected design. The PCR results showed that the stable clones were all integrated with the interfering vector targeting cyclin E1 gene. Conclusion Six hepatocellular carcinoma cell lines stably integrated with the RNA interference vector targeting the cyclin E1 gene have been established in this study.