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为比较皮质和中脑来源的神经干细胞(NSCs)定向分化为多巴胺神经元的情况,应用无血清和单克隆培养技术,分别从皮质和中脑组织中分离克隆、扩增NSCs,然后分成3组诱导分化:①对照组用10%胎牛血清诱导分化;②IL-1α组在10%胎牛血清的基础上加IL-1α诱导分化;③联合因子组在10%胎牛血清的基础上加IL-1α、IL-11、LIF及GDNF进行诱导分化。用酪氨酸羟化酶(TH)免疫组化染色检测多巴胺神经元。在100倍镜下随机取5个视野,计数每个视野中的TH阳性神经元数;并用图像分析软件处理TH阳性神经元的胞体面积和细胞周长;用STATA7.0统计软件进行统计处理。结果显示,皮质对照组中仅见极少的TH阳性神经元(0.25±0.163),中脑对照组中见少量TH阳性神经元(2±0.378);而皮质IL-1α组有较多的TH阳性神经元(15.5±0.866),中脑IL-1α组中的TH阳性神经元则为皮质IL-1α组的150%(22.875±1.517);皮质联合因子组中的TH阳性神经元数(25.75±0.940)与中脑联合因子组中的TH阳性神经元数(28.875±2.125)接近,无显著差异。胞体面积和细胞周长的结果显示出IL-1α组和联合因子组大于对照组、而联合因子组又优于IL-1α组的趋势。上述结果提示,中脑来源的NSCs本身具有一定的分化为多巴胺神经元的区域特异性,而皮质来源的NSCs在IL-1α、IL-11、LIF及GDNF等细胞因子的诱导下可改变其区域特异性而分化为较多较为成熟的多巴胺神经元。
To compare the directional differentiation of cortical and midbrain-derived neural stem cells (NSCs) into dopaminergic neurons, serum-free and monoclonal culture techniques were used to separate and clone NSCs from cortex and midbrain tissue, respectively, and then divided into three groups Induction of differentiation: ① control group with 10% fetal bovine serum induced differentiation; ②IL-1α group in 10% fetal bovine serum plus IL-1α-induced differentiation; ③ combination factor group in 10% fetal bovine serum on the basis of IL -1α, IL-11, LIF and GDNF. Dopamine neurons were detected by tyrosine hydroxylase (TH) immunohistochemistry. The number of TH-positive neurons in each visual field was counted in five fields at 100 times magnification. The cell body area and cell perimeter of TH-positive neurons were analyzed by image analysis software. Statistical analysis was performed with STATA7.0 statistical software. The results showed that there were only a few TH-positive neurons (0.25 ± 0.163) in the cortical control group and a small number of TH-positive neurons in the control group (2 ± 0.378), while the cortical IL-1α group had more TH-positive neurons (15.5 ± 0.866) neurons, and TH positive neurons in midbrain IL-1α group were 150% (22.875 ± 1.517) of the cortical IL-1α group; the number of TH positive neurons in the cortical combination group (25.75 ± 0.940) was similar to that of TH-positive neurons (28.875 ± 2.125) in the midbrain combined factor group, with no significant difference. Cell body area and cell cycle results showed that the IL-1α group and the combination factor group than the control group, and the combination factor group and IL-1α group trend. These results suggest that mesencephalic NSCs themselves have a regional specificity for differentiating into dopamine neurons, whereas cortical NSCs can change their regions under the induction of cytokines such as IL-1α, IL-11, LIF and GDNF Specificity and differentiation into more mature dopamine neurons.