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目的本实验通过联合应用甲基转移酶抑制剂5-氮-2’-脱氧胞苷(5-aza-2’-deoxycytidine,5-aza-dC)和去乙酰化酶抑制剂曲古抑菌素A(Trichstatin A,TSA)对NIH/3T3胎鼠成纤维细胞进行DNA去甲基化重编程,以期使其表达与多向分化潜能性密切相关的基因。方法药物处理组与正常对照组NIH/3T3细胞的形态学观察。流式细胞技术检测药物处理前后NIH/3T3细胞的DNA甲基化水平。应用RT-PCR的方法检测多能性基因Oct4,Sox2,c-Myc和Klf4的表达情况。结果经过药物处理后的NIH/3T3细胞与对照组的细胞比较,从细胞形态来观察,没有明显的区别,均呈现成纤维细胞的外观。药物处理组的DNA甲基化水平较对照组明显降低。药物处理后细胞均呈现出Oct4,Sox2,c-Myc和Klf4基因的阳性表达。结论 5-aza-dC和TSA对NIH/3T3细胞进行表观重编程,可以使重编程后的体细胞中呈现与多向分化潜能基因的阳性表达。
OBJECTIVE: The purpose of this study was to investigate the protective effects of 5-aza-2’-deoxycytidine (5-aza-dC) and strytost A (Trichstatin A, TSA) DNA demethylation reprogramming NIH / 3T3 fetal rat fibroblasts in order to make it express genes closely related to the potential of multi-directional differentiation. Methods The morphology of NIH / 3T3 cells in drug-treated group and normal control group was observed. Flow cytometry was used to detect DNA methylation levels in NIH / 3T3 cells before and after drug treatment. The expression of pluripotency genes Oct4, Sox2, c-Myc and Klf4 were detected by RT-PCR. Results After treatment with NIH / 3T3 cells, the appearance of fibroblasts showed no significant difference when compared with that of the control group. The DNA methylation level of the drug-treated group was significantly lower than that of the control group. After drug treatment, the cells showed positive expression of Oct4, Sox2, c-Myc and Klf4 genes. Conclusions Apparent reprogramming of NIH / 3T3 cells by 5-aza-dC and TSA can make the reprogrammed somatic cells show the positive expression of multidirectional differentiation potential genes.