稳定整合靶向细胞周期蛋白E1基因RNA干涉载体的肝癌细胞系的建立

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。

Establishment of hepatocellular carcinoma cell line with stable RNA interference vector targeting cyclin E1 gene

Objective To explore the establishment of hepatocellular carcinoma cell line with stable RNA interference vector targeting cyclin E1 gene.Methods A neo gene from pSSC-9 was subcloned into pSilencer 1.0-U6 to construct a stable RNAi vector pSineo.Then,it was ligased with a synthesized RNA interference DNA template targeting cyclin E1 gene.The recombinant was introduced into BEL-7402 cells with Lipofectamine~(TM)2000.After G418 selection,the genomic DNAs of anti-G418 clones were isolated and PCR experiments were carried out for the identification of integration of RNAi vectors.Results The results of digestion of recombinants with restriction enzymes and sequencing indicated that the recombinant pSineo-cycE 1 was meet the design.Our PCR indetification also demonstrated that all 6 cell clones sorted out with G418 selection had stably integrated RNAi vector targeting cyclin E1 gene.Conclusion Six cell lines with stable RNA interference vector targeting cyclin E1 gene are established in this study.This work will be helpful in investigating oncogene targeting RNAi therapy of cancer.