化瘀理气中药组方对大鼠胃黏膜异型增生p16基因甲基化与蛋白表达的影响

The effect of traditional Chinese herbs Huayu Liqi compound on the methylation and expression of p16 in gastric mucosal dysplasia rats cells

目的观察解毒化瘀健脾中药治疗胃黏膜异型增生过程中其化瘀与理气组方对模型大鼠p16基因甲基化状态和蛋白表达的影响。方法将胃黏膜异型增生大鼠分模型对照组、维甲酸治疗组(西药对照组)、化瘀组方、理气组方治疗组,并以正常大鼠作为阳性对照组进行相应药物干预;应用甲基化特异PCR技术检测大鼠胃黏膜p16基因甲基化状态;Real-time PCR、免疫组化技术、Western blot检测p16在m RNA和蛋白表达水平的变化。结果模型组胃黏膜异型增生细胞p16基因的甲基化阳性检出率均为33.33%(6/18),高于正常对照组(20.00%),但差异无统计学意义(χ2=1.023,P=0.312);化瘀和理气治疗组p16基因甲基化检出率均为0.00%(0/15);m RNA(P<0.001,P<0.001)和蛋白表达(P<0.05,P<0.01)水平两组的表达均明显高于模型组。结论解毒化瘀健脾中药治疗胃黏膜异型增生过程中其化瘀与理气组方中药均对异型增生胃黏膜细胞p16基因具有一定的去甲基化作用,并能显著增加其转录与蛋白翻译水平的表达量。

【Objective】To observe the effect of traditional Chinese herbs Huayu Liqi compound on the methylation and the expression of cyclin-dependent kinase inhibitor 2A（p16） in rats with gastric mucosal dysplasia（GMD）.【Methods】The GMD model rats were randomly divided into： model control group, western medicine retinoic acid treatment group, Huayu Liqi prescription treatment group, and the normal rats were used as positive control group of experimental treatment; The methylation specific PCR technique was used to detect the methylation status of p16 gene in gastric mucosal cells of rats, the Real-time PCR, Western blot and immunohistochemistry were used to detect the expression of p16 in mRNA and protein level. 【Results】The p16 gene＇s positive methylation rate in the GMD model group was 33.33%（6/18）, which was higher than that in the normal control group（20.00%）, but the difference was not significant（χ^2=1.023, P =0.312）; Both in Huayu Liqi prescription treatment group, p16 gene＇s positive methylation rate was 0.00%（0/15）, and lower than that in the model group in GMD cells; And in Huayu and Liqi herbs treatment group, the expression of p16 in mRNA（P〈0.001, P〈0.001） and protein（P〈0.05, P〈0.01） level were significantly higher than that in the model groups. 【Conclusion】Huayu Liqi herbs had a certain degree of demethylation of p16 genes in gastric mucosal cells with GMD, and then induced the increased expression of p16 genes.