白细胞介素12联合粒细胞巨噬细胞集落刺激因子对H22肝癌细胞凋亡的影响

Effects of IL-12 combined with GM-CSF on apoptosis of H22 hepatoma cells

目的 研究基因粒细胞巨噬细胞集落刺激因子（GM-CSF）和IL-12基因联合作用对H22肝癌细胞凋亡的影响.方法 体外培养肝癌细胞株,分别设置GM-CSF转染组、IL-12转染组、GM-CSF和IL-12共染组、阴性对照组（空载组）.构建PIB-CMV3-GM-CSF和PIB-CMV3-IL-12真核表达载体,转染肝癌细胞36 h后,采用荧光显微镜检测转染效果;RT-PCR 检测IL-12、GM-CSF、p53、p38及（C-JUN mRNA表达水平;Western blot 法检测IL-12、GM-CSF、p53、p38及C-JUN蛋白表达水平;流式细胞术检测细胞凋亡情况.结果 经荧光显微镜观察发现,转染GM-CSF组、转染IL-12组及共转染组均有较强的绿色荧光,说明质粒成功转入细胞.另外,空载组、GM-CSF转染组、IL-12转染组、GM-CSF和IL-12基因联合组p53 mRNA的表达量分别为1.2±0.10、4.3±0.98、4.2±0.34、9.2±0.87,蛋白表达量分别为1.0±0.10、3.6±0.34、3.8±0.30、5.0±0.60,与空载组比较,3组质粒转染组p53 mRNA和蛋白表达量均显著上调（P均〈0.01）;而与转染GM-CSF组、转染IL-12组比较发现,联合组能更加明显上调p53 mRNA和蛋白的表达量（P均〈0.01）.与此同时,空载组、GM-CSF转染组、IL-12转染组、GM-CSF和IL-12基因联合组p38 mRNA表达量分别为7.5±0.9、3.5±0.45、3.7±0.25、1.0±0.11,p38 蛋白表达量分别为10.1±1.03、6.1±0.67、7.1±0.61、1.0±0.12,C-JUN mRNA分别为11.2±1.20、4.1±0.19、3.3±0.30、1.0±0.01,C-JUN 蛋白分别为2.25±0.2、1.8±0.13、1.4±0.12、1.0±0.09,与空载组相比,3组质粒转染组p38 和C-JUN mRNA和蛋白的表达量均显著下调（P均〈0.01）;与转染GM-CSF组、转染IL-12组比较发现,联合组能更加明显的下调p38和C-JUN mRNA和蛋白的表达量（P均〈0.01）.流式细胞术检测结果显示,空载组、GM-CSF转染组、IL-12转染组、GM-CSF和IL-12基因联合组细胞早期凋亡率分别为（3.43±0.9）%、（5.87±1.02）%、（7.32±1.1）%、（17.47±2.11）%,与空载组比较,3组质粒转染组细胞早期凋亡率均显著的上调（P均〈0.01）;并且基因联合组细胞早期凋亡率与其他质粒组相比上调趋势更加明显（P均〈0.01）.结论 GM-CSF和IL-12基因联合作用可显著促进肝癌细胞凋亡,其作用机制可能是通过上调p53的表达、抑制p38和C-JUN的表达,进而促进肝癌细胞凋亡.

Objective To investigate the effects of granulocyte macrophage colony-stimulating factor （GM-CSF） combined with interleukin-12 （IL-12） genes on apoptosis of hepatoma cells.Methods The hepatoma cell lines were cultured in vitro and were divided into four groups： GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group,negative control group （empty load group）,respectively.The PIB-CMV3-GM-CSF and PIB-CMV3-IL-12 eukayotic expression vector was built,and 36 h after transfection,fluorescence microscope was used to detect the transfection effect;the expression level of IL-12,GM-CSF,p53,p38 and C-JUN mRNA were detected by RT-PCR,and Western blot was used to examine the expression level of IL-12,GM-CSF,p53,p38 and C-JUN protein.In addition,the flow cytometry was applied to detect cell apoptosis.Results Through fluorescence microscope,green fluorescence was observed in cells of GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group,indicating that the plasmid has successfully transferred into cells.In addition,the expression of p53mRNA in empty load group,GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group were 1.2±0.10,4.3±0.98,4.2±0.34,9.2±0.87,and the protein expression were 1.0±0.10,3.6±0.34,3.8±0.30,5.0±0.60.Compared with the empty load group,the expression level of p53 mRNA and protein were significantly increased in the three plasmid transfection groups （P〈0.01）.The expression of p53 mRNA and protein were significantly increased in co-transfection group than GM-CSF group and IL-12 group （P〈0.01）,while in the comparison with GM-CSF transfection group and IL-12 transfection group,the expression level of p53mRNA and protein in the co-transfection group could be improved to a higher degree（P〈0.01）.Meanwhile,p38 C-JUN mRNA expression levels in empty load group,GM-CSF transfection group,IL-12 transfection group,GM-CSF and IL-12 co-transfection group were as follows： 7.5± 0.9,3.5±0.45,3.7±0.25,1.0±0.11,while p38protein expression levels were 10.1±1.03,6.1± 0.67,7.1 ± 0.61,1.0 ± 0.12,respectively,C-JUN mRNA expression levels were 11.2 ± 1.20,4.1 ± 0.19,3.3 ± 0.30,1.0 ± 0.01,separately,C-JUN protein expression levels were 2.25 ± 0.2,1.8 ± 0.13,1.4 ± 0.12,1.0 ± 0.09.P38, C-JUN mRNA and protein levels were significantly reduced in the three plasmid transfection groups compared with the empty load group （P〈0.01）.The expression of p38,C-JUN mRNA and protein were reduced to a lower degree in co-transfection group than in GM-CSF transfection group and IL-12 transfection group （P〈0.01）.Flow cytometer showed that the hepatoma cell apoptosis rate of the empty load group,GM-CSF transfection group,IL-12 transfection group,co-transfection group were （3.43±0.9）%,（5.87±1.02）%,（7.32±1.1）%,（17.47±2.11）%,the rates of the three plasmid transfection groups were significantly higher than that of the empty load group （P〈0.01）.And the apoptosis rate was significantly increased in the co-transfected group compared with other plasmid groups （P〈0.01）. Conclusion The combination of GM-CSF and IL-12 could significantly accelerate the apoptosis of hepatoma cells by up-regulating the expression of p53,and down-regulating the expression of p38 and C-JUN.