角质细胞生长因子促进人胚神经干细胞增殖与分化的实验研究  

RESEARCH OF HUMAN KERATINOCYTE GROWTH FACTOR PROMOTING PROLIFERATION AND DIFFERENTIATION OF HUMAN NEURAL STEM CELLS

作　　者：阳运康[1,2] 蒋电明[1] 阳明明[1] 

机构地区：[1]重庆医科大学附属第一医院骨科,重庆400016 [2]泸州医学院附属医院骨与关节外科

出　　处：《中国修复重建外科杂志》2013年第10期1246-1251,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：重庆市卫生局中医药科技项目(渝中医2009-1-10);四川省卫生厅课题资助项目(07018)~~

摘　　要：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�ObjectiveTo study the effects of the human keratinocyte growth factor 2 （hKGF-2） on the survival and differentiation of human neural stem cells （hNSCs）. MethodsThe 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. ResultsReanimated 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 amon

关 键 词：人源性角质细胞生长因子2 人胚神经干细胞 增殖 分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]