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目的在体外检测Nrf3基因对肝癌SMMC7721的生长影响作用。方法构建其融合蛋白真核表达载体,脂质体介导该真核表达载体转染肝癌SMMC7721细胞系,FCM观察其在体外对肝癌SMMC7721细胞系细胞周期和凋亡的影响;荧光显微镜观察候选基因亚细胞定位。结果Nrf3在肝癌SMMC7721细胞中表达,荧光显微镜下观察Nrf3定位于细胞核内。FCM分析显示Nrf3影响下肝癌SMMC7721G2/M+S期细胞所占比例较对照组明显减少,G0/G1细胞所占比例明显增加。证实Nrf3可抑制肝癌SMMC7721的DNA合成和有丝分裂,促使细胞阻滞于G0/G1期,抑制肝癌SMMC7721细胞的体外生长。FCM分析,未观察到明显的“亚G1”峰(即凋亡峰),提示Nrf3在体外对肝癌SMMC7721细胞的凋亡无影响。结论Nrf3在体外具有抑制肝癌细胞增殖的功能,对肝癌细胞的凋亡无影响,为肿瘤抑制基因。
Objective To detect the effect of Nrf3 gene on the growth of hepatoma SMMC7721 in vitro. Methods The fusion protein eukaryotic expression vector was constructed. The eukaryotic expression vector was transfected into hepatoma SMMC7721 cell line by lipofectamine. FCM was used to observe the effect of FCM on the cell cycle and apoptosis of hepatocellular carcinoma SMMC7721 cell line. Fluorescence microscope was used to observe the expression of the candidate gene Subcellular localization. Results Nrf3 was expressed in hepatocellular carcinoma SMMC7721 cells. Nrf3 was localized in the nucleus under fluorescence microscope. FCM analysis showed that the proportion of SMMC7721G2 / M + S cells in NCCF-treated hepatocellular carcinoma cells was significantly decreased compared with that of control cells, and the proportion of G0 / G1 cells was significantly increased. Confirmed that Nrf3 can inhibit DNA synthesis and mitosis in hepatoma SMMC7721, promote cell arrest in G0 / G1 phase, inhibit the growth of hepatoma SMMC7721 cells in vitro. FCM analysis showed no significant “sub G1” peak (ie, apoptosis peak), suggesting that Nrf3 had no effect on the apoptosis of SMMC7721 cells in vitro. Conclusion Nrf3 has the function of inhibiting the proliferation of hepatocellular carcinoma cells in vitro and has no effect on the apoptosis of hepatocellular carcinoma cells and is a tumor suppressor gene.