miR-145-3p对大鼠肠神经干细胞增殖与分化的影响

Effects of MiR-145-3p on Proliferation and Differentiation of Enteric Neural Stem Cells

【目的】探讨微小核糖核酸145-3p(miR-145-3p)对SD大鼠肠神经干细胞(ENSCs)增殖及分化的影响。【方法】原代培养法提取孕18 d的SD大鼠肠神经干细胞(ENSCs),观察其细胞学特性,采用巢蛋白(Nestin)及神经胶质纤维蛋白(GFAP)免疫荧光进行鉴定。分别采用脂质体及慢病毒载体过表达ENSCs的miR-145-3p进行转染,采用荧光显微镜及实时定量聚合酶链式反应(qRT-PCR)检测转染效果。Edu细胞增殖实验及诱导分化实验明确miR-145-3p对ENSCs增殖及分化的影响。【结果】原代培养ENSCs可见大量神经球样结构形成,免疫荧光证实细胞Nestin和GFAP均阳性,具有干细胞特性;在慢病毒转染组内,miR-145-3p过表达组的miR-145-3p相对表达量为3.52±0.24,显著高于阴性对照组的0.01±0.01,其差异具有统计学意义(P<0.05);miR-145-3p过表达组的Edu阳性率为(15.23±1.17)%,显著低于阴性对照组的(22.38±1.52)%;采用分化培养基处理后,miR-145-3p慢病毒载体转染组的GFAP阳性细胞比例为(21.47±1.98)%,显著低于阴性对照组的(33.86±2.52)%。【结论】对于ENSCs而言,慢病毒载体转染效率较高,miR-145-3p可抑制ENSCs的增殖与分化,其可能在肠神经系统发育过程中发挥重要作用。

【Objective】To establish the method for culturing and purifying enteric neural stem cells(ENSCs)derived from rat and to investigate the effect of miR-145-3p on proliferation and differentiation of ENSCs.【Methods】ENSCs which primarily extracted from 18-days SD rats embryos,were observed by cytological characteristics and identified by Nestin and GFAP immunofluorescence staining.The liposome transfection and lentivirus transfection were used to transfect the ENSCs.Overexpression of miR-145-3p in ENSCs was transfected through liposome and infected with lentivirus with miR-145-3p,which followed by fluorescence microscope and qRT-PCR measurement.Edu cell proliferation assay was used to analyze proliferating cells by recording the proportion of Edu/DAPI in each group.The neurospheres was differentiated by differentiation medium and the GFAP/DAPI ratio of differentiated cells was recorded.【Results】Primary culture of ENSCs showed the formation of a large number of neurosphere-like structures(undifferentiated neurospheres).The immunofluorescence showed that Nestin and GFAP were positive in these cells.The qRT-PCR showed that the relative expression level of miR-145-3p in overexpression group was 3.52±0.24 significantly higher than the negative control group 0.01±0.01,the difference was statistically significant(P<0.01).It indicated that lentivirus was successfully transfected into ENSCs.Edu cell infiltration showed that the positive rate of Edu in the miR-145-3p overexpression group was(15.23±1.17)%,while the negative control group was(22.38±1.52)%(P<0.05).After NESCs cells were exposed to the differentiation medium,the proportion of GFAP positive cells in the miR-145-3p overexpression group was(21.47±1.98)%,which was lower than that in the negative cell control group(33.86±2.52)%(P<0.05).【Conclusions】Lentivirus transfection is a high efficient transfection method in ENSCs.Overexpression of miR-145-3p inhibits the proliferation and differentiation of ENSCs,which may play an important role in the development of enteric nervous system.