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目的:寻找一种有效分离神经干细胞球并获得大量单个神经干细胞的方法。方法:取20周胎龄人胚胎脑组织分离单细胞,应用DMEM/F12培养基培养形成神经干细胞球,取神经干细胞球,分别以2.50g/L、1.25g/L胰蛋白酶及0.6u/ml、1.2u/ml、1.8u/ml、2.4u/ml酵素-Ⅱ(DispaseⅡ)消化,观察消化过程及细胞形态,对消化后的单个细胞进行培养,观察细胞生长情况,并进行免疫细胞化学染色鉴定。结果:各浓度Dispase-Ⅱ均能较完全地消化神经干细胞球,其中1.2u/ml、1.8u/mlDispase-Ⅱ消化后的细胞生长状况最佳。胰蛋白酶消化后细胞有较大的损伤,成活细胞少。消化后的细胞Nestin抗体染色结果(+),证实为神经干细胞。结论:一定浓度的DispaseⅡ消化神经干细胞球后,可获得大量成活单个神经干细胞。
Objective: To find a way to effectively isolate NSCs and obtain a large number of single neural stem cells. METHODS: Single cells were isolated from the brain tissue of 20-week-old human embryos and cultured in DMEM / F12 medium to form NSCs. The NSCs were harvested at the concentrations of 2.50g / L, 1.25g / L trypsin and 0.6u / ml , 1.2u / ml, 1.8u / ml and 2.4u / ml Dispase Ⅱ respectively. The digestive process and cell morphology were observed. The single cells after digestion were cultured, and the growth of cells was observed. Immunocytochemistry Identification. Results: Dispase-Ⅱ could digest neural stem cells more completely, and the cell growth was the best at 1.2u / ml and 1.8u / ml Diispase-Ⅱ. After trypsin digestion cells have greater damage, fewer surviving cells. After digestion of cells, Nestin antibody staining results (+) were confirmed as neural stem cells. Conclusion: After a certain concentration of Dispase Ⅱ digested neural stem cells, a large number of survived single neural stem cells can be obtained.