解毒化瘀健脾方对胃黏膜异型增生模型大鼠Thbs1、E-cad基因去甲基化和蛋白诱导表达的影响

Effect of “Jiedu Huayu Jianpi Decoction”on demethylation and inducible expression of Thbs1 and E-cad gene in gastric mucosa dysplasia rats cells

目的观察解毒化瘀健脾方对胃黏膜异型增生模型大鼠血小板反应蛋白1(thrombospondin 1,Thbs1)和E-钙黏蛋白(E-cadherin,E-cad)基因甲基化状态和蛋白表达的影响,探讨解毒化瘀健脾方治疗胃黏膜异型增生的作用机制。方法采用低浓度MNNG(N-甲基-N'-硝基-N-亚硝基胍)的综合造模方法,建立实验性胃黏膜异型增生大鼠模型;分模型对照组、西药维甲酸治疗组、解毒化瘀健脾方治疗组,并选择正常大鼠作为阳性对照组;应用甲基化特异PCR技术检测大鼠胃黏膜THBS1、E-cad基因甲基化状态;Realtime PCR、免疫组化技术检测Thbs1和E-cad在mRNA和蛋白表达水平的变化。结果模型组胃黏膜异型增生细胞Thbs1的甲基化阳性检出率为25.00%,显著高于正常对照组的0.00%(P<0.05);模型组E-cad基因的甲基化阳性检出率(33.33%)高于正常对照组(16.67%),但差异不显著(P>0.05);解毒化瘀健脾方组Thbs1及E-cad甲基化阳性检出率相比,模型组显著降低为0.00%(P<0.05);解毒化瘀健脾方组Thbs1和E-cad mRNA(P<0.05,P<0.01)和蛋白表达(P<0.05,P<0.01)水平均显著高于模型组。结论解毒化瘀健脾方可能通过对异型增生胃黏膜细胞Thbs1、E-cad基因的去甲基化和诱导蛋白表达量的增加,实现对胃黏膜异型增生的治疗作用。

Objective To observe the effect of ＂Jiedu Huayu Jianpi Decoction＂on the methylation status and the expression of thrombospondin 1（ Thbs1）,E-Cadherin（E-cad） in rats with gastric mucosal dysplasia（GMD）,and to explore its possible molecular mechanism. Methods We use a low concentration of MNNG（N- methyl-N＇- nitro-N-nitrosoguanidine） integrated modeling method,established the experimental rat model with GMD; the animal model rats were randomly divided into： model control group,western medicine retinoic acid treatment group,＂Jiedu Huayu Jianpi Decoction＂treatment group,and the normal rats were selected as the positive control group; the methylation specific PCR technique was used to detect the methylation status of Thbs1 and E-cad gene in gastric mucosal cells of rats; the Realtime PCR,immunohistochemistry were used to detect the expression of Thbs1 and E-cad in mRNA and protein level.Results Thbs1 gene＇s positive methylation rate in the model group was 25. 00 %,which was significantly higher than that of the normal control group（0. 00 %）（P〈0.05）; the E-cad gene＇s positive methylation rate in the model group（33.33 %） was higher than that of the normal control group（16.67 %）,but no significant difference was found between them; the Thbs1 and E-cad＇s positive methylation rate in ＂Jiedu Huayu Jianpi Decoction＂group（ 0. 00 %） was significantly decreased than that of the model group（ P〈0. 05）; The expression of Thbs1,E-cad in mRNA（ P〈0. 05,P〈0. 01） and protein（ P〈0. 01,P〈0. 01） were significantly higher than that of the model groups. Conclusion ＂Jiedu Huayu Jianpi Decoction＂may achieve the therapeutic effect on GMD by the way of prompted Thbs1,E-cad gene demethylation and increased their protein expression.