X-染色体相关凋亡抑制蛋白相关因子1抑制肝癌细胞增殖和诱导凋亡的实验研究

Experimental study on related factors 1 gene of X-chromosome linked inhibitor of apoptosis on inhibiting cell proliferation and inducing apoptosis in hepatocellular cell line

目的研究X-染色体相关凋亡抑制蛋白(XIAP)相关因子1(XAF1)对肝癌细胞增殖和凋亡的影响。方法构建正义pcDNA3～XAF1和反义pcDNA3-XAF1-AS质粒,通过脂质体转染技术将质粒DNA转入SMMC7721肝癌细胞株并建立稳定高表达XAF1的肝癌细胞克隆。应用MTT、流式细胞仪、TUNEL法检测肝癌细胞的增殖和凋亡；应用RT-PCR和Western印迹法检测基因在mRNA和蛋白水平的表达。结果XAF1在SMMC7721肝癌细胞株中有表达,但低于正常肝脏细胞水平。稳定高表达XAF1基因可抑制肝癌细胞的增殖和诱导凋亡,并能增加其对化疗药物5-氟尿嘧啶和羟基喜树碱的敏感性。结论XAF1能抑制肝癌细胞增殖和诱导其凋亡。

Objective To investigate the expression and the effects of X-chromosome linked inhibi tor of apoptosis （XIAP） associated factor 1 （XAF1） on apoptosis and cell proliferation in SMMC7721 hepatocellur carcinoma （HCC） cell line. Methods The expression of XAF1 mRNA and protein in hu man SMMC7721 cell line were detected by semi-quantitative, RT PCR and Western blot. Plasmid constructs expressing sense and antisense XAF1 were generated and transfected into SMMC7721 cell line to establish stable transfectants. Cell proliferation and apoptosis were detected by MTT, flow cytometry and TUNEL. Results XAF1 mRNA and protein were detectable in SMMC7721 cell line but lower than that in normal liver cell. Stable expression of XAF1 significantly inhibited cell proliferation and increased spontaneous apoptosis in SMMC7721 cell （P〈0.05）. Over-expression of XAF1 in stable transfactants increased the sensitivity of SMMC7721 cell to chemotherapeutic drugs such as 5-fluorouracial and hydroxycamptothecin. Conclusions Over expression of XAF1 induces apoptosis and inhibits SMMC7721 cell growth. XAF1 may be a promising candidate for HCC gene therapy.