CNTF 联合RA 诱导成肌细胞类神经分化及与REST 的相关性

CNTF combined with RA induces differentiation of myoblasts into neural like cells and its correlation with REST

摘要:

目的 探讨睫状神经营养因子（CNTF）联合视黄酸（RA）诱导成肌细胞类神经分化的方法以 及该诱导过程与神经元限制性沉默因子（REST）相关性。方法 以CNTF 诱导C2C12 去分化，再以RA 诱导 C2C12 类神经分化。通过细胞形态学、细胞免疫荧光、流式细胞术、Western blot 等技术对去分化及类神经 分化的细胞进行鉴定及相关分析，比较细胞类神经分化前后REST 表达变化。结果 诱导后细胞聚集生长，呈 典型的神经球样。细胞免疫荧光染色显示神经球能够被神经特异性标志物NSE、Sox1、GFAP 标记，神经球 离散为单个细胞接种后生长状态良好，神经干细胞标志物（nestin）呈阳性表达。Western blot 结果显示CNTF 去分化诱导后成肌细胞分化相关蛋白Myogenin、P21 表达降低，类神经分化后NSE、Sox1、GFAP 表达增多， 且成肌细胞特异性标志物Desmin 及REST 表达下降。流式细胞仪分析表面标志物NSE 显示诱导组与对照组 比较细胞有差异，且诱导组特异性阳性细胞率达84% 左右。结论 CNTF 联合RA 可诱导成肌细胞类神经分化， 且该诱导过程可能与REST 表达降低相关。

Abstract:

Objective To investigate methodological application of ciliary neurotrophic factor (CNTF) plus retinoic acid (RA) in neural like differentiation of myoblasts and its potential correlation with neuron restrictive silencing factor (REST). Methods Differentiation of C2C12 was induced by CNTF followed by neural like differentiation induced by RA. Cell morphology, immunofluorescence, flow cytometry analysis and Western blot were utilized to identify cellular type. Expression of REST pre and post neuronal differentiation was measured. Results Cells gathered as “neurospheres” after induction. Immunofluorescence staining showed expression of neural markers such as NSE, Sox1 and GFAP. Neural like cells grew well after dispersed from “neurospheres” with high expression of nestin which is a neural stem cell biomarker. Western blot showed decreased expression of myoblast related markers such as Myogenin, P21, Desmin and REST post CNTF induction.Expression of neural associated markers including NSE, Sox1, GFAP was significantly increased. Flow cytometry data supported the upregulated expression of NSE in differentiated cells and NSE positive cell in differentiation group was about 84%. Conclusion CNTF combined with RA can induce differentiation of myoblasts into neural like cells, which is probably mediated by expression of REST.