重组人Neurturin对体外培养的胚胎大鼠多巴胺能神经元的保护作用

目的建立大鼠胚胎多巴胺能神经元原代培养方法 ,研究重组人Neurturin(rhNTN)体外对黑质多巴胺能神经元的营养和保护作用。方法孕 14dWister大鼠 ,取出胎鼠中脑腹侧区组织 ,经胰酶消化、分散 ,接种至涂有多聚左旋赖氨酸基质的 6孔板中 ,37℃培养 2 4h后将培养液更换为B2 7无血清培养基 ,实验组同时加入纯化的rhNTN。继续培养 ,于培养第 7、14、2 1天直接观察及TH免疫细胞化学染色观察结果。结果培养 14、2 1d ,对照组中多数神经元已经萎缩、死亡 ,少量存活的多巴胺能神经元胞体小 ,突起细短 ;实验组中存活的多巴胺能神经元数目较多 ,神经元胞体粗壮 ,突起长 ,生长状况良好。结论rhNTN能促进中脑黑质多巴胺能神经元的存活及突起生长 ,对多巴胺能神经元具营养和保护作用。

Protective effect of recombinant human Neurturin on dopaminergic neurons of rat embryos in vitro

PurposeTo establish the primary culture of neurons of rat fetus and to study the nutritive and protective effects of recombinant human Neurturin(rhNTN)on cultured nigro substantial dopaminergic neurons in vitro.MethodsThe dopaminergic neurons were obtained from the ventral mesencephalon of Wister rat embryos (E14) after iced Hanks washing, trypsin digestion and dispersion. Cells were plated in 6-well culture plates coated with poly-L-lysine (PLL). After incubation at 37℃ for 24h in 2ml of DMEM containing 10% FCS and 10% horse serum in 5% CO 2, the medium was exchanged with serum free B27 medium containing 1% glutamine. Meanwhile, rhNTN expressed by E.coli was added to the experimental group and the same amount of PBS was added to the control group. After culturing for 7 days, the growth of cells was observed and TH immunocytochemical staining was performed. ResultsAfter culturing for 14 days and 21 days, most of the dopamine (DA) neurons in control group died and the surviving cells were small and the neurites was slender and short. However, in experiment group more DA neurons survived, the cells were big and the neurites thick and long.ConclusionrhNTN could promote the survival and neurite outgrowth of mesencephalon dopaminergic neurons in vitro and had nutritive and protective function on DA neurons.