脑源性神经营养因子促进海马神经干细胞的存活、增殖及向神经元分化

Enhancement of Survival,Proliferation and Differentiation into Neurons of Neural Progenitor Cells Isolated from Rats Hippocampus by Brain Derived Neurotrophic Factor

探讨脑源性神经营养因子(brain derived neurotrophic factor,BDNF)对海马神经干细胞(neural progenitor/stem cells,NPCs)的存活、增殖及分化的影响。采用无血清培养基体外分离、纯化、扩增胎鼠海马NPCs。通过细胞形态观察、nestin免疫荧光染色及血清促分化检测NPCs的干细胞特性;采用神经球计数及神经球直径测定观察BDNF对NPCs的促增殖作用,筛选出在适当细胞密度下,促进NPCs增殖的有效浓度;采用Tunel染色及全自动生化分析仪测定细胞培养上清液乳酸脱氢酶(lactic dehydrogenase,LDH)的含量探讨BDNF对海马NPCs存活的影响;采用抗-β-微管蛋白(tubulin)III(Tuj-1)染色检测NPCs分化成神经元的百分率,同时测定分化神经元突起的长度。分离的海马NPCs表现为nestin免疫染色阳性,具有自我增殖能力、且能分化为神经元和星形胶质细胞;当细胞密度为5×105个/ml时,10～200ng/ml BDNF能显著促进NPCs的增殖,其中40ng/ml BDNF促增殖作用最强,40ng/ml BDNF能显著增大神经球直径;40ng/ml BDNF显著减少NPCs的凋亡率(Tunel+/DAPI+),抑制LDH漏出;40ng/ml BDNF能显著促进NPCs分化为Tuj-1免疫染色阳性神经元,且分化后神经元的突起长度显著大于对照组。上述结果提示:BDNF促进海马NPCs的存活、增殖及向神经元方向分化。

The objective of the study is to explore the effect of brain derived neurotrophic factor （BDNF） on the neural progenitor/stem cells （NPCs） of rat hippocampus. NPCs were obtained from Wistar rats at embryonic day 15-16 and cultured in serum-free medium, then the neurospheres were harvested, fixed and immunostained for nestin （the protein marker of stem cells）; NPCs were induced to differentiate with 1% fetal calf serum containing medium; The single NPC was obtained by a mechanical dissociation and grown for 3 days in medium containing BDNF at the concentration of 10-200 ng/ml then the number of neurosphere were counted and the diameter of neurosphere were measured; Tunel staining positive cells and the level of lactic acid dehydrogenase （LDH） in culture medium were used to evaluate the effect of BDNF on the NPCs survival; 13 tubulin III （Tuj-1） immunostaining was used to label the neurons differentiated from NPCs, and the percentage of Tuj-1 positive ceils among DAPI positive cells in the BDNF and control group were compared. Additionally, the length of neurite in Tuj-1 immunostaining positive cells was measured. The results showed that nestin-positive NPCs exhibited the ability of self-proliferation and could be induced to differentiate into neurons and astrocytes. BDNF at the concentration of 10-200 ng/ml enhanced the proliferation of NPCs at the cell density of 5×10^5 cells/ml, while 40 ng/ml BDNF exhibited the strongest proliferation enhancement; Diameter of newly formed neurosphere with 40 ng/ml BDNF was increased obviously while the apoptosis rate （Tunel＋/DAPI＋） and LDH leakage of 40 ng/ml BDNF group was significantly decreased compared with the control group. In 40 ng/ml BDNF group, more neurons （Tuj-1 cells） were observed and total neuritic length in Tuj-1 cells were significantly longer, compared to the control group. Our study suggest that BDNF has a neuroprotective effect, can promote NPCs proliferation and induce them differentiation into neurons.