目的：研究逆转录病毒介导的Notch1（ICN）基因转染对人肝癌细胞生长的影响，并对其作用机制进行了探讨。方法： 采用磷酸钙沉淀法质粒共转染293T细胞，制备出表达组成性活化形式的Notch1（ICN）和/或绿色荧光蛋白（GFP）的逆转录病毒载体MSCV-ICN/GFP及对照逆转录病毒载体MSCV-GFP，并检测病毒滴度。用逆转录病毒感染人肝癌细胞Hep3B，观察病毒的感染效率，MTT比色法测定瞬时表达Notch1（ICN）对Hep3B人肝癌细胞生长的影响，利用流式细胞仪分析细胞周期分布的变化，Western blot方法检测细胞周期调控蛋白的变化。结果：通过质粒共转染可获得具有较高滴度的重组逆转录病毒。MTT比色法显示瞬时表达Notch1（ICN）基因可以抑制人肝癌细胞Hep3B的生长，瞬时表达Notch1（ICN）基因可使Hep3B细胞周期停滞在G0/G1期并上调细胞周期调控蛋白P53的表达水平。结论：Notch1基因可通过影响细胞周期分布和P53蛋白的表达水平而抑制人肝癌细胞生长。

Objective: To investigate the effects of Notch1 gene transfection on the growth of human hepatocarcinoma cells and to explore the mechanisms of Notch signaling. Methods: Recombinant retrovirus MSCV-ICN/GFP and MSCV-GFP were prepared by calcium phosphate precipitation mediated gene transduction of 293T package cells. The recombinant retroviruses were used to infect human hepatocarcinoma cells Hep3B. After infected, proliferation of Hep3B cells was observed by MTT assay. Cell cycle distribution was analyzed by FACS and p53 expression was detected by Western blot. Results: After infection of MSCV-ICN/GFP recombinant retrovirus, the growth of Hep3B cells was inhibited significantly and cell cycle distribution analysis showed that Hep3B cells in G0/G1 phase were increased. Western immunoblotting showed that Notch1 signaling up regulated p53 expression. Conclusion: Notch1 activation could inhibit the growth of hepatocarcinoma cells through G0/G1 cell cycle arrest and p53 induction.

国家自然科学基金（30121002）资助