小鼠间充质祖细胞向神经元样细胞诱导分化的实验研究

Study on directed differentiation of mesenchymal progenitor cells into neuron-like cells in mice

目的:体外培养C57小鼠密质骨碎片来源的间充质祖细胞(Mesenchymal progenitor cells,MPC),探讨MPC体外向神经元样细胞定向诱导分化的可行性。方法:取C57小鼠后肢长骨,剪成骨碎片,经Ⅱ型胶原酶消化后体外培养MPC,流式细胞术检测鉴定其免疫学表型。取生长良好的第三代MPC,神经元原代培养上清液定向诱导,对诱导后的MPC,以神经元特异性标志物神经元特异性烯醇化酶(Neuron specific enolase,NSE)及神经丝蛋白(Neurofilament protein,NF)为对照,应用免疫细胞化学染色法,检测神经元标志物的表达。结果:成功培养原代MPC,传代后生长良好,流式细胞术检测CD29、CD44组阳性率与其同型对照组相比有明显差异(P<0.05)。经神经元原代培养上清液诱导后,MPC可表达神经元特异性标志物NSE及NF。结论:从小鼠密质骨碎片分离培养出的间充质祖细胞获得率高,并可诱导分化为神经元样细胞。

Objective：To culture mesenchymal progenitor cells（MPC）from compact bone fragments in C57 mice in vitro and to study the feasibility of inducing directed differentiation of MPC into neuron-like cells in vitro. Methods：Bones of hind limbs of C57 mice were sheared into bone fragments and digested by collagenase type Ⅱ. Then,MPC were cultured in vitro and analyzed by flow cytometry for identification of its immunology phenotype. MPC of P3 in good growth were induced directionally by the supernatant cultured with primary neuron,and then detected the expression of neuronal specific markers neuron specific enolase（NSE）and neurofilament protein（NF）by immunocytochemical staining. Results：The primary MPC were cultured successfully and they grewwell after passage. Compared with that of the control group,the positive rate of CD29 and CD44 of MPC of antibody group had significant difference （P〈0.05）. Induced by supernatant cultured with primary neuron,MPC could express neuronal specific markers NSE and NF-H. Conclusion：The harvest rate of MPC isolated and cultured from compact bone fragments of mice is high and MPC could be induced and differentiated into neuron-like cells.