无血清培养条件下诱导胚胎干细胞向多巴胺能神经元定向分化的实验研究

THE STUDY OF DIRECT DIFFERENTIATION OF EMBRYONIC STEM CELL INTO DOPAMINERGIC NEURON IN THE SERUM-FREE MEDIUM

目的 寻求无血清、无饲养层细胞存在的情况下 ,胚胎干细胞向多巴胺能神经元定向诱导分化的最佳条件。 方法 采用阶段诱导的方法 ,在不同阶段加入不同的生长因子 ,对新近分离的胚胎干细胞进行分化培养。首先向无血清培养液中分别加入bFGF和LIF ,实现由胚胎干细胞向神经前体细胞的定向分化 ,通过巢蛋白(nestin)免疫细胞化学染色对神经前体细胞进行鉴定 ;在此基础上 ,撤除bFGF和LIF ,加入B2 7无血清培养基、IL 1后继续培养 ,实现由神经前体细胞向多巴胺能神经元的定向诱导分化 ;通过酪氨酸羟化酶 (TH)免疫细胞化学染色对多巴胺能神经元进行鉴定。 结果 有 85 %的细胞团呈nestin免疫阳性着色 ,多巴胺能神经元的分化比率为13% ,较多巴胺能神经元的自然分化比率 (4 % )有明显提高。 结论 在无血清、无饲养细胞的情况下 ,我们采用阶段诱导的方法 ,在不同的阶段加入不同的生长因子 ,可有效诱导多巴胺能神经元的分化 ,使胚胎干细胞的诱导分化过程更易于操作。

Objective To explore the optimal condition of direct differentiation into dopaminergic neurons of embryonic stem cells in serum-free and feeder layer cell free medium. Methods We used the method of phase induction to culture embryonic stem cells. At first, embryonic stem cells were cultured in the serum-free medium with bFGF and LIF so as to realize the direct differentiation from embryonic stem cells to neural precursors. Differentiated cells were determined by nestin immunocytochemical staining. On this basis, we transferred embryonic stem cells to the B27 serum-free medium with IL-1 so as to realize the direct differentiation from neural precursors to dopaminergic neurons. Differentiated cells were determined by TH immunocytochemical staining. Results Approximately 85 percent of cell masses were nestin immuno-positive. The differentiation ratio of dopaminergic neurons was 13%, which increased significantly in comparison with natural differentiation ratio of dopaminergic neurons.Conclusion Without serum and feeder layer cell, we can induce embryonic stem cells to differentiate into dopaminergic neurons effectively by adding different growth factors at different phases, which makes the inductive processes more easily.