论文部分内容阅读
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: To investigate the effects of 5-aza-2’-deoxycytidine (5-aza-dC) and trichostatin A (TSA) The cells were reprogrammed and further induced by beta-hydroxy acid (βHA) and β-mercaptoethanol (β-ME) to express genes closely related to nerve cells. Methods The DNA methylation levels of NIH / 3T3 cells in experimental group (NIH / 3T3 cells treated with 4.5μM 5-aza-dC + 0.35μM TSA + 1mMβ-ME + 200μMBHA) and control group were detected by flow cytometry. The expression of Oct4, Sox2, c-Myc and Klf4 were detected by RT-PCR. The expression of nestin and NF-L were detected by immunocytochemistry. Results The DNA methylation level of NIH / 3T3 cells in the experimental group was significantly lower than that in the control group. The positive expression of Oct4, Sox2, c-Myc and Klf4 in the cells were observed. After induced by BHA and β-ME, NIH / 3T3 cells were positive for nestin and neurofilament light chain. Conclusion Apparent expression of nestin and NF-|ÊL can be observed after the apparently modified cells are induced.