BHA和β-ME对表观修饰后的NIH/3T3细胞向神经方向诱导的实验研究

Beta hydroxy acid and β-mercaptoethanol promote differentiation of epigenetic modified NIH/3T3 fibroblasts into neural stem cell-like cell

目的通过联合应用表观修饰药物5-氮-2'-脱氧胞苷(5-aza-2'-deoxycytidine,5-aza-dC)和曲古抑菌素A(TrichstatinA,TSA)对NIH/3T3细胞进行重编程,应用β-羟基酸(betahydroxyacid,BHA)和β-巯基乙醇(β-mercaptoethanol,β-ME)进一步诱导,以期表达与神经细胞密切相关的基因。方法应用流式细胞技术检测实验组(4.5μM5-aza-dC+0.35μMTSA+1mMβ-ME+200μMBHA作用后的NIH/3T3细胞)和对照组中NIH/3T3细胞的DNA甲基化水平。应用RT-PCR的方法检测Oct4,Sox2,c-Myc和Klf4的表达,免疫细胞化学染色检测巢蛋白(nestin)和神经丝轻链(neurofilamentlightchain,NF-L)的表达情况。结果实验组NIH/3T3细胞DNA甲基化水平较对照组明显降低,细胞均呈现出Oct4,Sox2,c-Myc和Klf4基因的阳性表达。经过BHA和β-ME诱导后,NIH/3T3细胞呈巢蛋白和神经丝轻链阳性。结论表观修饰后的细胞经诱导后可以呈现nestin和NF-L的阳性表达。

Objective We reprogrammed NIH/3T3 fibroblasts by combination of 5-aza-2＇-deoxycytidine（5-aza-dC） with trichstatin A（TSA）,then the modified cells were induced by beta hydroxy acid（BHA） andβ-mercaptoethanol（β-ME） to exhibit the positive expression of neural markers in the cells.Methods The total levels of cellular DNA methylation were measured by flow cytometry,the expressions of Sox2,klf4,c-Myc and Oct4 were analyzed by RT-PCR,the expressions of nestin and neurofilament light chain（NF-L） were detected by immunohistochemistry.Results The total DNA methylation level was significant decreased after the treatment of combination of 5-aza-dC with TSA,Sox2,klf4,c-Myc and Oct4 were expressed positively in pharmacological agents modified NIH/3T3 fibroblasts.Nestin and NF-L immunopositive cluster of cells were found in β-ME＋BHA-treated NIH/3T3 cells.Conclusion Nestin and NF-L expressed positively in reprogrammed NIH/3T3 fibroblasts using pharmacological epigenetic modifiers.