5-aza-dC和TSA对NIH/3T3细胞多能性基因表达作用的实验研究

The effect of 5-aza-dC and TSA on expressions of pluripotent genes in NIH/3T3 cells

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 To study the effect of 5-aza-dC and TSA on expressions of pluripotent genes in NIH/3T3 cells,detect the gene expression pattern,DNA methylation level of pharmacological agents reprogrammed cells.Methods Morphological observation of pharmacological agents modified NIH/3T3 fibroblasts was completed under microscope.The total level of cellular DNA methylation of pharmacological agents modified NIH/3T3 fibroblasts was measured by flow cytometry.The expressions of embryonic marker Sox2,klf4,c-Myc and Oct4 were analyzed by RT-PCR.Results There was no distinct difference between the pharmacological agents modified cells and the control cells.The embryonic markers Sox2,klf4,c-Myc and Oct4 were expressed in pharmacological agents modified NIH/3T3 fibroblasts.Total DNA methylation level was significant decreased after the treatment.Conclusion The expression of embryonic markers(Sox2,klf4,c-Myc and Oct4) was induced successfully in reprogrammed NIH/3T3 fibroblasts by 5-aza-dC and TSA.