大鼠胚胎中脑前体细胞培养的研究

Dopamine Neurons Obtained Through Primary Culture of Mesencephalic Progenitor cells of E12 Rats

目的 :通过胚胎MPC体外培养获取PD患者CRT治疗所需的DA能神经元。方法 :取自E12鼠胚中脑腹侧的MPC悬液在添加bFGF的DMEM/F12 /N2培养液中原代培养 ,鉴定培养细胞的增殖和分化能力。结果 :在添加了bFGF 10ng·mol-1的DMEM/F12 /N2培养液中MPC增殖良好 ,体外培养 5～ 7d后细胞数量扩增到培养前的 9 782± 0 0 47倍 ;培养液中撤去bFGF后细胞可分化成星形胶质细胞和神经元 ,其中多数Tuj1抗原标记阳性的神经元同时呈TH抗原标记阳性 ,培养细胞分化为DA能神经元的比例约为2 4 3 4% (10 2 / 419)。结论 :E12鼠胚MPC原代培养是获取DA能神经元的可靠途径 ,类似的技术应用于临床可能缓解PD患者CRT治疗供体不足的矛盾。

Aim:To obtain dopamine neurons for cell replacenment therapy in Parkinson's disease through primary culture of mesencephalic progenitor cells of E12 rats.Methods:Mesencephalic progenitor cells harvested from ventral mesencephalon of E12 rats were cultured on poly-D-lysine-coated dishes in DMEM/F12 medium supplemented with N2 and bFGF (10 ng·mL -1).The ability of cell expansion and differentiation after primary culture were evaluated by cell-counting and immunohistochemical analysis.Result:The MPC expanded well under the medium supplemented with bFGF.Within five to seven days in vitro,the cell number multiplied with about a 9.782±0.047-fold.Cultured MPC differentiated into astrocytes and neurons after removal of bFGF form the culture medium.About 24.34%(102/419)of the total cell population differentiated into dopamine neurons,an a majority of neurons which immunoreactive for neuron specific antigen-Tuj1 were immunoreactive-TH for dopamine neuron specific marker-TH too.Conclusion:In vitro culture of MPC is a reliable way to gain more dopmine neurons.Such technique may alleviate the technical and ethical difficulties in obatining sufficient and appropriate donor cells which needed in the cell replacement therapy of Parkinson's disease.