解毒化瘀健脾方及拆方对胃黏膜异型增生大鼠多基因去甲基化诱导表达的比较

Comparative analysis of each prescription of Jiedu Huayu Jianpi Fang for multiple gene demethylation and expression in mucosal dysplasia in rats

目的:从诱导基因表达角度评估解毒化瘀健脾方及拆方对胃黏膜异型增生模型大鼠治疗的贡献作用.方法:甲基化特异性PCR(methylmion specific PCR,MSP)法检测正常对照组、胃黏膜异型增生组、西药维甲酸治疗组、解毒化瘀健脾全组方治疗组、中药单组方解毒组、化瘀组、益气组、养阴组、理气组抑癌基因及肿瘤相关基因p16、PETN、凝血栓蛋白酶基因1(thrombospondin 1,Thbs1)、E-Cadherin、Runx3的甲基化程度;Real-time PCR及免疫组织化学法检测解毒化瘀健脾中药各组处理因促使各基因去甲基化导致的mRNA及蛋白表达水平表达变化.结果:解毒化瘀健脾方及各拆方组均可一定程度实现p16、PETN、Thbs1、E-Cadherin、Runx3的去甲基化;转录水平各组相比CG组基因表达变化分别为:A组p16极显著升高(P<0.01),PETN、E-Cadherin、Thbs1、B组Thbs1、C组p16、PETN、Thbs1、E组Thbs1、F组p16、Thbs1显著升高(P<0.05);蛋白水平各组相比CG组蛋白表达的变化为:A组p16、PTEN、E-Cadherin、RUNX3、THBS1、B组p16、C组p16、THBS1、D组除RUNX3外的蛋白、E组除PTEN的蛋白、F组除THBS1外的蛋白均显著上调(P<0.05);统计各组在转录和蛋白翻译水平的变化积分,结果表明解毒化瘀健脾中药方贡献分值最为突出,其次为单组方理气组、养阴组、化瘀、益气组及解毒组.结论:解毒化瘀健脾方对胃黏膜异型增生的治疗作用优于各拆方治疗组,对胃黏膜异型增生的治疗应以理气养阴为主.

AIM： To evaluate the contribution of each prescription of Jiedu Huayu Jianpi Fang to the therapeutic effects on gastric mucosal dysplasia from the perspective of induced gene expression. METHODS： We divided gastric mucosal dysplasia rats into a model control （MG） group, a Western medicine （retinoic acid） treatment （PCG） group, a combined prescription （Jiedu Huayu Jianpi） treatment （A） group, and Jiedu （B）, Huayu （C）, Yiqi （D）, Yangying （E） and Liqi （F） single prescription treatment groups. Normal rats were used as controls （CG）. Methylation specific PCR technique was used to detect the methylation status of p16, PETN, Thbsl, E-Cadherin, and Runx3 genes in gastric mucosal cells of rats. Real-time PCR and immunohistochemistry were used to detect the mRNA and protein expression of each genes. RESULTS： All of the Jiedu Huayu Jianpi treatment groups showed a certain degree of demethylation of p16, PETN, Thbsl, E-Cadherin, and Runx3 gene. Compared with group CG, the mRNA expression of p16 （P 〈 0.01）, PETN, E-Cadherin and ThbsI in group A, Thbsl in group B, p16, PETN and Thbsl in group C, Thbsl in group E, p16 and Thbsl in group F increased significantly （P 〈 0.05 for all）; and the protein expression of p16, PTEN, E-cad, RUNX3, and THBS1 in group A, p16 in group B, p16 and THBS1 in group C, p16, PTEN, E-cad and THBS1 in group D, p16, E-cad, RUNX3 and THBS1 in group E, p16, PTEN, E-cad, RUNX3 and THBS1 in group F increased significantly （P 〈 0.05 for all）. The effect of Jiedu Huayu Jianpi Fang on gastric mucosal dysplasia was the most prominent, followed by Liqi, Yangyin, Huayu, Yiqi and Jiedu single prescriptions. CONCLUSION： The effect of Jiedu Huayu Jianpi Fang on gastric mucosal dysplasia is much better than those of each single prescription. Gastricmucosal dysplasia should be treated mainly by means of Liqi and Yangyin.