摘要目的 探讨大黄素是否增强5AzA-cdR对胰腺癌裸鼠移植瘤抑癌基因p16、RASSF1A、ppENK的去甲基化作用.方法 建立胰腺癌PANC1细胞株裸鼠皮下移植瘤模型,随机将裸鼠分为对照组、大黄素组、5AzA-cdR组以及大黄素联合5AzA-cdR组(联合用药组).观察各组移植瘤的生长情况,采用甲基化特异性PCR法(MSP)检测各组移植瘤组织p16、RASSF1A、ppENK的甲基化水平,采用荧光定量PCR法和蛋白质印迹法检测上述3个抑癌基因mRNA和蛋白的表达量.结果 对照组、大黄素组、5AzA-cdR组、联合用药组移植瘤重量分别为(0.28 ±0.01)、(0.17 ±0.01)、(0.12 ±0.02)、(0.08±0.01)g;体积分别为(517 ±0.02)、(382 ±0.01)、(232±0.03)、(169 ±0.01)mm3;p16甲基化程度为1.00±0.00、0.89±0.02、0.63 ±0.02、0.19±0.01;RASSF1A甲基化程度为1.00±0.00、0.88 ±0.02、0.51 ±0.01、0.32±0.01;ppENK甲基化程度为1.00±0.00、0.92±0.02、0.77 ±0.02、0.31±0.01;p16mRNA表达量为1.00±0.00、1.71 ±0.02、2.67±0.02、3.81±0.01;RASSF1A mRNA表达量为1.00±0.00、1.92 ±0.02、2.73 ±0.03、3.77±0.01;ppENK mRNA表达量为1.00± 0.00、1.69±0.03、2.17±0.02、4.28 ±0.01;p16蛋白表达量为1.00±0.00、1.71 ±0.02、2.67 ±0.02、3.81±0.01;RASSF1A蛋白表达量为1.00±0.00、1.92 ±0.02、2.73 ±0.03、3.77±0.01;ppENK蛋白表达量为1.00±0.00、1.69±0.03、2.17 ±0.02、4.28 ±0.01.3个治疗组移植瘤的重量及体积均显著小于对照组,3个抑癌基因甲基化程度均低于对照组,抑癌基因mRNA及蛋白表达均显著高于对照组,其中联合用药组又显著强于2个单独用药组,差异均有统计学意义(P值＜0.05或＜0.01).结论 大黄素联合5AzA-cdR用药可增强5AzA-cdR对胰腺癌移植瘤组织抑癌基因p16、RASSF1A、ppENK的去甲基化作用.

abstractsObjective To investigate whether emodin combined with 5AzA-cdR can enhance the demethylation of tumor suppressor genes p16,RASSF1A and ppENK in nude mice with subcutaneously transplanted pancreatic cancer.Methods Pancreatic cancer cells Panc1 burdened subcutaneous xenograft nude mice model was established,which were randomly divided into control group,emodin group,5AzA-cdR group and emodin combined 5AzA-cdR group (combined group).The growth of transplanted tumors wasobserved in each group.Methylation specific PCR (MSP) was used to detect the methylation levels of p16,RASSF1A and ppENK in the xenograft tumor tissue among three groups.The mRNA and protein expression of three tumor suppressor genes were detected by FQ-PCR and Western blotting,respectively.Results The weight of xenografts in the control group,emodin group,5AzA-cdR group,and combination group were (0.28 ±0.01),(0.17 ± 0.01),(0.12 ± 0.02),(0.08 ± 0.01)g,respectively.The tumor volume was (517 ±0.02),(382 ± 0.01),(232 ± 0.03),(169 ± 0.01) mm3.The methylation levels of p16 were 1.00 ± 0.00,0.89 ± 0.02,0.63 ± 0.02,and 0.19 ± 0.01;the methylation levels of RASSF1A were 1.00 ± 0.00,0.88 ± 0.02,0.51 ± 0.01,and 0.32 ± 0.01;the methylation degree of ppENK was 1.00 ± 0.00,0.92 ± 0.02,0.77 ± 0.02 and 0.31 ± 0.01,respectively.The expression of p16 mRNA was 1.00 ± 0.00,1.71 ±0.02,2.67 ± 0.02,3.81 ± 0.01.The expression of RASSF1A mRNA was 1.00 ± 0.00,1.92 ±0.02,2.73 ± 0.03,3.77 ± 0.01.The expression of ppENK mRNA was 1.00 ± 0.00,1.69 ± 0.03,2.17 ± 0.02 and 4.28 ± 0.01.The expression of p16 protein was 1.00 ± 0.00,1.71 ± 0.02,2.67 ± 0.02,3.81 ± 0.01;the expression of RASSF1A protein was 1.00 ± 0.00,1.92 ± 0.02,2.73 ± 0.03.3.77 ± 0.01;ppENK protein expression levels were 1.00 ±0.00,1.69 ±0.03,2.17 ±0.02,4.28 ±0.01.The weight and volume of xenografts in the three treatment groups were significantly smaller than those in the control group.The methylation of three tumor suppressor genes was lower than that of the control group,and the expression of tumor suppressor mRNA and protein was all significantly higher than the control group,which the combination drug group was also significantly stronger than that in emodin group and 5AzA-cdR group,and the differences were statistically significant (P ＜ 0.05 or ＜ 0.01).Conclusions The combination of emodin and 5AzA-cdR can enhance the demethylation effect of 5A6A-cdR on the tumor suppressor genes p16,RASSF1A and ppENK in the tumor tissue of pancreatic cancer xenograft model.