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目的探讨人源性角质细胞生长因子2(human keratinocyte growth factor 2,hKGF-2)对体外长期培养人胚神经干细胞(human neural stem cells,hNSCs)存活和分化的影响。方法将液氮冻存17代hNSCs复苏常规培养7 d形成神经球后,取少许行免疫细胞化学染色鉴定干细胞及分化细胞特异抗原。一部分神经球浓缩后种植至12孔培养板中,分为7组,每组6孔,均添加1 mL基础培养液[含N2(1∶100)、20 ng/mL EGF的DMEM/F12培养基]后,B、C、D、E、F组分别添加10、30、60、90、120 ng/mL KGF-2,G组添加10 ng/mL bFGF,A组为空白对照组;培养7、14 d观察并计数神经球与hNSCs,了解神经球生长与增殖情况。另一部分浓缩后种植至置有多聚赖氨酸包被玻片的6孔培养板内,分为4组,每组6孔,均添加3 mL DMEM/F12培养基后,A1、B1、C1、D1组对应加入N2(1∶100)、N2(1∶100)+90 ng/mL hKGF-2、FBS(1∶20)、FBS(1∶20)+90 ng/mL hKGF-2;培养14 d内观察神经球生长与分化情况。7 d时各取5孔玻片行免疫荧光细胞化学鉴定和流式细胞仪检测,分析神经球的生长与分化状况。结果复苏后培养形成的神经球含大量巢蛋白阳性细胞,充满整个神经球;诱导分化后表达分化细胞特异性蛋白神经丝200(neurofilament200,NF-200)和胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)。各组神经球培养7 d后均有不同程度增大;随hKGF-2浓度增加,神经球数量和hNSCs数目均依次增多,呈递增趋势,E、F、G组显著高于A、B、C、D组(P<0.05);B、C、D组间两两比较差异亦有统计学意义(P<0.05);但A、B组间以及E、F、G组间比较差异无统计学意义(P>0.05)。体外诱导过程中,A1、B1、C1、D1组分化细胞生长旺盛程度呈递增趋势,组间比较差异均有统计学意义(P<0.05);B1组NF-200阳性率显著高于其余3组(P<0.05),GFAP阳性率显著低于其余3组(P<0.05);A1、C1、D1组间比较差异均无统计学意义(P>0.05)。培养14 d后各组生长均达峰值,以星形细胞为主。结论体外培养17代的hNSCs系纯化hNSCs,hKGF-2能促进其增殖和诱导分化为神经元细胞。
Objective To investigate the effect of human keratinocyte growth factor 2 (hKGF-2) on the survival and differentiation of human neural stem cells (hNSCs) cultured in vitro. Methods Seventeen passages of hNSCs cryopreserved in liquid nitrogen were cultured for 7 days to form neurospheres. Then immunocytochemical staining was used to identify stem cells and differentiated cells specific antigens. Part of the neurospheres were seeded in 12-well plates and divided into 7 groups of 6 wells each. 1 mL of basal medium [DMEM / F12 medium containing N2 (1: 100) and 20 ng / mL EGF ], 10, 30, 60, 90 and 120 ng / mL KGF-2 in group B, C, D, E and F, 10 ng / mL bFGF in group G, blank control group A, 14 d observed and counted neurospheres and hNSCs, to understand the growth and proliferation of neurospheres. The other part was concentrated to a 6-well plate containing polylysine coated glass slides and divided into 4 groups with 6 wells in each group. After adding 3 mL of DMEM / F12 medium, A1, B1 and C1 , N2 (1: 100) +90 ng / mL hKGF-2, FBS (1:20), FBS (1:20) +90 ng / mL hKGF- Observation of neurosphere growth and differentiation within 14 days. Immunofluorescence cytochemistry and flow cytometry were used to detect the growth and differentiation of neurospheres at 5 days after 7 days. Results The neurospheres formed after resuscitation contained a large amount of nestin positive cells, which filled the entire neurospheres. After differentiation, neurofilament 200 (NF-200) and glial fibrillary acidic protein , GFAP). The number of neurospheres and the number of hNSCs increased gradually with the increase of hKGF-2 concentration, and showed an increasing trend, and the number of neurospheres in groups E, F and G was significantly higher than that of A, B and C (P <0.05). There was also a significant difference between groups B, C and D (P <0.05), but there was no significant difference between groups A, B and groups E, F and G Significance (P> 0.05). In vitro induction, the proliferation of A1, B1, C1 and D1 cells showed an increasing trend with significant difference between the two groups (P <0.05). The positive rate of NF-200 in B1 group was significantly higher than that in the other three groups (P <0.05). The positive rate of GFAP was significantly lower than the other three groups (P <0.05). There was no significant difference between A1, C1 and D1 groups (P> 0.05). After 14 days of culture, the growth of each group reached the peak, mainly astrocytes. Conclusion hNSCs cultured in vitro for 17 passages can be purified hNSCs, hKGF-2 can promote their proliferation and differentiation into neurons.