小分子化合物诱导骨髓间充质干细胞类神经分化的实验研究*

Experimental study of neural differentiation induced by small molecular compounds in bone marrow mesenchymal stem cells*

摘要:

目的 探讨小分子化合物组合（Forskolin、SB431542、DMH1、CHIR99021）诱导骨髓间充质干细胞（BMSCs）类神经分化的可行性。方法 全骨髓贴壁法分离大鼠BMSCs，流式细胞仪鉴定细胞。培养细胞至第3代，以含有Forskolin的神经元培养基预诱导1?d，再以含有Forskolin、SB431542、DMH1、CHIR99021的神经元培养基正式诱导3?d。以神经元培养基为对照组。通过观察细胞形态、免疫荧光检测、Western blotting技术对类神经分化细胞进行鉴定。结果 大鼠BMSCs以梭形细胞为主，可见长短不一的突起；经流式细胞仪鉴定，CD90、CD105表达呈阳性，而CD34和CD45表达呈阴性。诱导后，细胞有神经样形态改变，免疫荧光检测到神经元特异性烯醇化酶（NSE）、巢蛋白（Nestin）阳性表达，诱导3?d后Western blotting显示实验诱导组与对照组比较，NSE、Nestin和SOX1表达增多（P?<0.05）。结论 小分子化合物组合（Forskolin、SB431542、DMH1、CHIR99021）可诱导BMSCs类神经分化。

Abstract:

Objective To investigate the feasibility of differentiation of bone marrow mesenchymal stem cells (BMSCs) into neural cells induced by small molecule compound FSDC (Forskolin, SB431542, DMH1, CHIR99021). Methods Bone marrow mesenchymal stem cells were isolated by whole bone marrow adherence and identified by flow cytometry. Cells were cultured to the third generation, pre-induced with Forskolin-containing neuronal culture medium for 1 day, and formally induced for 3d with neuron culture medium containing Forskolin, SB431542, DMH1 and CHIR99021. The neuron culture medium was used as the control group. The neuron-differentiated cells were identified by observation of cell morphology, immunofluorescence and Western blotting. Results Rat bone marrow-derived mesenchymal stem cells were mainly spindle-shaped cells, showing irregular protrusions. The expression of CD90 and CD105 was positively detected by flow cytometry, while the expression of CD34 and CD45 was negative. After induction, the cells showed obvious neuromorphic changes. The positive expression of NSE, Nestin were detected by immunofluorescence. After 3 days of induction, Western blotting showed that the expression of NSE, Nestin and SOX1 in the experimental group was significantly increased compared with the control group (P?