集落刺激因子1在活化星形胶质细胞条件培养基诱导神经干细胞分化过程中的表达及功能

Expression and function of colony-stimulating factor 1 in differentiation of neural stem cells induced by activated astrocyte conditioned medium

背景:颅脑外伤后形成的反应性星形胶质细胞可能对神经干细胞向神经元分化产生不利影响,因此研究颅脑外伤后反应性星形胶质细胞对神经干细胞分化的调节作用及其机制对促进神经功能恢复具有很大临床应用前景。目的:探讨反应性星形胶质细胞对神经干细胞向神经元分化的影响。方法:体外培养SD大鼠星形胶质细胞,分为对照组和脂多糖刺激组,干预3 d后,采用PCR、ELISA、Western blot检测星形胶质细胞中集落刺激因子1的表达;体外培养SD大鼠原代神经干细胞,在成神经元诱导培养基中加入星形胶质细胞条件培养基共培养3 d,通过免疫荧光和流式细胞术检测β微管蛋白Ⅲ、胶质纤维酸性蛋白的表达;体外培养原代神经干细胞,在成神经元诱导培养基中加入集落刺激因子1重组蛋白干预3 d,应用流式细胞技术和免疫荧光检测集落刺激因子1对神经干细胞向神经元分化的作用。结果与结论:①脂多糖刺激后的星形胶质细胞及条件培养基中集落刺激因子1表达极低;②脂多糖刺激的星形胶质细胞条件培养基与神经干细胞共培养后,抑制了神经干细胞向神经元分化;③神经干细胞加入集落刺激因子1重组蛋白干预后,促进了神经干细胞向神经元分化;④结果表明,脂多糖刺激后星形胶质细胞中集落刺激因子1分泌水平下调,抑制神经干细胞向神经元分化,而集落刺激因子1过表达可促进神经干细胞向神经元分化,对大鼠脑损伤具有修复作用。

BACKGROUND:The excessive proliferation of reactive astrocytes after traumatic brain injury may have an impact on the differentiation of neural stem cells into neurons.Therefore,studying the regulation of reactive astrocytes on neural stem cell differentiation after traumatic brain injury and its regulation mechanism may have great clinical application prospect for promoting the recovery of neural function.OBJECTIVE:To investigate the effect of reactive astrocytes on the differentiation of neural stem cells into neurons.METHODS:Astrocytes of SD rats were cultured in vitro and divided into control group and lipopolysaccharide stimulation group.After 3 days of intervention,the expression of colony-stimulating factor 1 in astrocytes was detected by PCR,ELISA and western blot assay.Primary neural stem cells of SD rats were cultured in vitro,and astrocyte conditioned medium was added to the neuron induction medium for 3 days.Immunofluorescence and flow cytometry were used to detect the expression of β-tubulin III and glial fibrillary acidic protein.Primary neural stem cells were cultured in vitro,and the recombinant protein of colony-stimulating factor-1 was added to the neuron induction medium for 3 days.The effect of colony-stimulating factor-1 on the differentiation of neural stem cells into neurons was detected by flow cytometry and immunofluorescence.RESULTS AND CONCLUSION:(1)The expression of colony-stimulating factor-1 in astrocytes stimulated by lipopolysaccharide and in conditioned medium was very low.(2)The coculture of neural stem cells and astrocyte conditioned medium stimulated by lipopolysaccharide inhibited the differentiation of neural stem cells into neurons.(3)Neural stem cells promoted the differentiation of neural stem cells into neurons by adding colony stimulating factor 1 recombinant protein.(4)The results showed that colony-stimulating factor 1 secretion of astrocytes was down-regulated after stimulation with lipopolysaccharide,and inhibited the differentiation of neural stem cells into neurons.The overexpression of colony-stimulating factor 1 can promote the differentiation of neural stem cells into neurons,which can repair brain injury in rats.