EGF对BDNF诱导成年大鼠海马结构神经干细胞向神经元分化的影响

发布时间：2018-05-30 06:40

本文选题：神经干细胞 + 大鼠海马结构　；参考：《中国医科大学》2008年硕士论文

【摘要】： 目的探讨表皮生长因子(epidermal growth factor,EGF)对脑源性神经生长因子(brain-derived neurotrophic factor,BDNF)促进成年大鼠海马结构神经干细胞(neural stem cells,NSCs)向神经元分化的影响及EGF浓度为20ng/mL时,BDNF促进成年大鼠海马结构神经干细胞向神经元分化的最佳浓度。材料和方法用含碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)、EGF、B27的DMEM/F12体外培养成年大鼠海马结构神经干细胞,并进行传代培养。当第4代细胞大多数克隆球直径约为2μm时,利用有限稀释法进行单细胞克隆培养。单细胞克隆培养形成的克隆球直径约2μm时进行传代培养,进行以下2组实验: 1、神经干细胞的鉴定部分克隆球行巢蛋白(Nestin)免疫细胞化学染色;部分克隆球用含10%胎牛血清的DMEM/F12进行诱导分化,1w后分别行神经元特异性烯醇化酶(neuronespecific enolase,NSE)、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)免疫细胞化学染色; 2、将单细胞克隆再传代而来的神经干细胞分为2部分进行诱导分化实验 (1)根据培养液中BDNF浓度的不同将细胞分为5组,0ng/mL组、50ng/mL组、100ng/mL组、150ng/mL组和200ng/mL组,0ng/mL组所用培养液为含10%胎牛血清、20ng/mLEGF的DMEM/F12,其它各组所用的培养液在0ng/mL组的基础上加入相应浓度的BDNF。 (2)按培养液中添加诱导因素的不同分为对照组、EGF组、BDNF组和EGF+BDNF组进行诱导分化培养,对照组的培养液为含10%胎牛血清的DMEM/F12,另外3组的培养液在对照组所用培养液的基础上分别加入EGF、BDNF、EGF+BDNF,其中EGF浓度为20ng/mL,BDNF浓度为50ng/mL。上述2部分细胞接种于六孔板中,每组6孔,培养1w行NSE免疫细胞化学染色,计数阳性细胞比例后进行统计学分析。实验结果 1、神经干细胞的鉴定结果单细胞克隆培养形成的克隆球表达Nestin,诱导分化1w后细胞表达NSE、GFAP。 2、诱导因素对神经干细胞分化影响的结果 (1)0ng/mL组、50ng/mL组、100ng/mL组、150ng/mL组和200ng/mL组大鼠海马结构神经干细胞向神经元分化的比例分别为13.33±3.44%、23.17±2.93%、20.67±2.80%、16.67±2.73%、14.17±3.43%,经t检验发现,50ng/mL组、100ng/mL组神经干细胞分化为神经元的比例明显增高(P＜0.05)。 (2)对照组、EGF组、BDNF组、EGF+BDNF组细胞分化为神经元的比例分别为17.67±3.27、13.33±3.44、19.67±2.07、23.17±2.93,经过t检验发现:BDNF组神经干细胞分化为神经元的比例明显高于对照组和EGF组,EGF+BDNF组神经干细胞分化为神经元的比例明显高于BDNF组(P＜0.05)。结论 1、EGF可以提高BDNF促进成年大鼠海马结构神经干细胞向神经元分化的比例。 2、EGF浓度为20ng/mL时,BDNF促进成年大鼠海马结构神经干细胞向神经元分化的最佳浓度为50ng/mL。
[Abstract]:objective
The effect of epidermal growth factor (epidermal growth factor, EGF) on the differentiation of hippocampal neural stem cells (neural stem cells, NSCs) to neuron differentiation in adult rat hippocampal neural stem cells (brain-derived neurotrophic factor, BDNF) and promoting the development of neural stem cells in the hippocampus of adult rats The optimal concentration of neuron differentiation.
Materials and methods
The adult rat hippocampal neural stem cells were cultured with basic fibroblast growth factor (bFGF), EGF, B27 DMEM/F12 in vitro and carried out in vitro. When most of the cloned spheres in the fourth generation cells were about 2 micron in diameter, the single cell clone culture was carried out by the finite dilution method. Single cell clone culture was formed. The diameter of the cloned sphere was about 2 m, and the following 2 groups of experiments were carried out.
1, identification of neural stem cells
Some cloned spherical nestin (Nestin) immunocytochemical staining was cloned, and some cloned spheres were induced by DMEM/F12 containing 10% fetal bovine serum. After 1W, neuron specific enolase (neuronespecific enolase, NSE), glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP) were immunocytochemical staining, respectively.
2, the neural stem cells from single cell clone were divided into 2 parts to induce differentiation experiments.
(1) according to the different concentration of BDNF in the medium, the cells were divided into 5 groups, group 0ng/mL, group 50ng/mL, group 100ng/mL, 150ng/mL group and 200ng/mL group. The medium used for 0ng/mL group was 10% fetal bovine serum, 20ng/mLEGF DMEM/F12, and the culture solution of other groups was added to the 0ng/mL group based on the BDNF. concentration.
(2) the control group was divided into control group according to the induction factors added in the culture solution, the EGF group, BDNF group and EGF+BDNF group were induced and cultured. The culture solution of the control group was DMEM/F12 containing 10% fetal bovine serum, and the other 3 groups were added to EGF, BDNF, EGF+BDNF respectively on the basis of the culture solution in the control group. The concentration of EGF was 20ng/mL, and the BDNF concentration was 5. 0ng/mL.
The above 2 parts of cells were inoculated in six hole plates, 6 holes in each group, cultured 1W, NSE immunocytochemical staining, counting the proportion of positive cells, statistical analysis.
experimental result
1, identification of neural stem cells
The cloned spheres of single cell clone culture expressed Nestin, and after induction of 1W, the cells expressed NSE, GFAP.
2, the effect of inducing factors on the differentiation of neural stem cells.
(1) the proportion of neural stem cells differentiated into neurons in the hippocampus of 0ng/mL group, group 50ng/mL, group 100ng/mL, group 150ng/mL and 200ng/mL group was 13.33 + 3.44%, 23.17 + 2.93%, 20.67 + 2.80%, 16.67 + 2.73%, 14.17 + 3.43%. The proportion of neural stem cells differentiated into neurons in group 50ng/mL was significantly higher (P < 0.05).
(2) the proportion of cells differentiated into neurons in group EGF, group BDNF and EGF+BDNF group was 17.67 + 3.27,13.33 + 3.44,19.67 + 2.07,23.17 + 2.93 respectively. After t test, it was found that the proportion of neural stem cells differentiated into neurons in BDNF group was significantly higher than that of control group and EGF group, and the proportion of neural stem cells differentiated into neurons in EGF+BDNF group was significantly higher than that of BDNF. Group (P < 0.05).
conclusion
1, EGF can increase the ratio of BDNF to the differentiation of neural stem cells into neurons in adult rats.
2, when EGF concentration is 20ng/mL, BDNF promotes the differentiation of adult rat hippocampal formation neural stem cells into neurons, the best concentration is 50ng/mL.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329.28

【参考文献】