Yang Yunkang, Jiang Dianming, Yang Mingming
Department of Orthopaedics, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2013 Oct;27(10):1246-51.
To study the effects of the human keratinocyte growth factor 2 (hKGF-2) on the survival and differentiation of human neural stem cells (hNSCs).
The hNSCs at 17 passages preserved in liquid nitrogen were resuscitated and cultured for 7 days with normal methods to form neural spheres. The specific Nestin antigen and differentiated cells antigen were identified using immunohistochemistry technology. Some concentrated hNSCs were incubated in 12-well culture plate with 1 mL basic medium [(DMEM/F12 + N2 (1 : 100) + epidermal growth factor (EGF) (20 ng/mL)] and divided into 7 groups, 6 wells each group. hKGF-2 (0, 10, 30, 60, 90, and 120 ng/mL) and bFGF (10 ng/mL) were added in groups A (control), B, C, D, E, F, and G, respectively. The neurospheres and the cell number were recorded for analyzing growth and multiplication of neural spheres. Some concentrated hNSCs were incubated in 6-well culture plate (cover glass coated with polylysine) with 3 mL DMEM/F12 medium and divided into 4 groups, 6 wells each group. N2 (1 : 100), N2 (1 : 100) + hKGF-2 (90 ng/mL), FBS (1 : 20), and FBS (1 : 20) + hKGF-2 (90 ng/mL) were added in groups A1, B1, C1, and D1, respectively. Then, the growth and multiplication of neural spheres were observed during culture; the separated neural spheres was identified and analyzed with indirect immunofluorescence and flow cytometry.
Reanimated hNSCs could form neural spheres containing a lot of Nestin antigen; differentiated cells by induction expressed the specific antigens of neurofilament 200 (NF-200) and glial fibrillary acidic protein (GFAP). At 7 days after culture, enlarged neural spheres were observed in each group. The neurospheres and the cell number of hNSCs increased with increased concentration of hKGF-2, showing a gradually increasing tendency; they were significantly higher in groups E, F, and G than that in groups A, B, C, and D (P < 0.05); significant differences were found among groups B, C, and D (P < 0.05), but no significant difference between groups A and B, and among groups E, F, and G (P > 0.05). After induction in vitro, the cell growth showed a progressive increase, significant difference was found among groups (P < 0.05); the percentage of NF-200 positive cells in group B1 was significantly higher than that in the other 3 groups (P < 0.05); the percentage of GFAP positive cells in group B1 was significantly lower than that in the other 3 groups (P < 0.05), but no significant difference among groups A1, C1, and D1 (P > 0.05). At 14 days after culture, cell growth reached the peak, which were mainly astero-cells.
The hNSCs are pure after incubated to 17 passages in vitro. hKGF-2 can promote the clone and the growth of differentiated cells, and increase the proportion of neuron.
研究人角质形成细胞生长因子2（hKGF-2）对人神经干细胞（hNSCs）存活及分化的影响。
复苏液氮保存的第17代hNSCs，常规培养7天形成神经球。采用免疫组化技术鉴定特异性巢蛋白抗原及分化细胞抗原。将部分浓缩的hNSCs接种于12孔培养板，每孔加入1 mL基础培养基[DMEM/F12 + N2（1∶100）+表皮生长因子（EGF）（20 ng/mL）]，分为7组，每组6孔。A组（对照组）不添加hKGF-2，B、C、D、E、F、G组分别添加hKGF-2（0、10、30、60、90、120 ng/mL）和碱性成纤维细胞生长因子（bFGF）（10 ng/mL）。记录神经球数量及细胞数，分析神经球的生长增殖情况。将部分浓缩的hNSCs接种于6孔培养板（包被多聚赖氨酸的盖玻片），每孔加入3 mL DMEM/F12培养基，分为4组，每组6孔。A1组添加N2（1∶100），B1组添加N2（1∶100）+ hKGF-2（90 ng/mL），C1组添加胎牛血清（FBS）（1∶20），D1组添加FBS（1∶20）+ hKGF-2（90 ng/mL）。培养过程中观察神经球的生长增殖情况；采用间接免疫荧光和流式细胞术对分离的神经球进行鉴定分析。
复苏的hNSCs可形成含大量巢蛋白抗原的神经球；诱导分化的细胞表达神经丝200（NF-200）和胶质纤维酸性蛋白（GFAP）特异性抗原。培养7天后，各组神经球均增大。hNSCs的神经球数量及细胞数随hKGF-2浓度升高而增加，呈逐渐上升趋势；E、F、G组明显高于A、B、C、D组（P < 0.05）；B、C、D组间差异有统计学意义（P < 0.05），但A与B组间及E、F、G组间差异无统计学意义（P > 0.05）。体外诱导后，细胞生长呈逐渐增加趋势，各组间差异有统计学意义（P < 0.05）；B1组NF-200阳性细胞百分比明显高于其他3组（P < 0.05）；B1组GFAP阳性细胞百分比明显低于其他3组（P < 0.05），但A1、C1、D1组间差异无统计学意义（P > 0.05）。培养14天时，细胞生长达高峰，主要为星形细胞。
体外培养至第17代的hNSCs纯度较高。hKGF-2可促进hNSCs的克隆及分化细胞生长，增加神经元比例。
