胚胎大鼠脊髓神经干细胞体外培养向胆碱能神经元的分化  

Differentiation of choline neurons from embryonic rat spinal cord neural stem cells in vitro

作　　者：张潇 张金枝 刘真真 林艳 ZHANG Xiao;ZHANG Jin-zhi;LIU Zhen-zhen(Department of Neurology,Sichuan People's Hospital,Sichuan Academy of Medical Sciences,Chengdu Sichuan 610000,China)

机构地区：[1]四川省医学科学院四川省人民医院神经内科,四川成都610000

出　　处：《临床和实验医学杂志》2019年第7期676-680,共5页Journal of Clinical and Experimental Medicine

基　　金：四川省卫生厅科研计划项目(编号:120063)

摘　　要：目的在一定条件下对胚胎大鼠脊髓神经干细胞进行体外培养与诱导,观察其向胆碱能神经元的分化情况,并进行鉴定。方法选取孕龄17 d的Wistar大鼠5只,利用胚胎大鼠进行脊髓源神经干细胞分离与培养,并进行神经上皮干细胞蛋白(Nestin)的细胞免疫荧光鉴定。对神经干细胞进行胆碱能神经元定向诱导分化,对照组为单纯诱导培养基,其余各组以诱导培养基为基础,添加生长因子,分为维甲酸(RA)组、音猬因子(SHH)组、SHH+神经营养因子-3(NT-3)组、SHH+RA组,每组1只。细胞诱导分化14 d后,进行细胞免疫荧光染色,观察各组胆碱乙酰转移酶(ChAT)阳性细胞比率与细胞分化情况。结果经诱导分化后,神经干细胞向胆碱能神经元进行分化。培养14 d,大部分神经细胞从神经干细胞球中央迁出,神经细胞间突起发生交错,呈现出网状结构。经免疫荧光检测,可观察到细胞胞质呈现出红色荧光。对照组未发现Ch AT阳性细胞,SHH、RA诱导组可观察到少量的Ch AT阳性细胞,SHH+NT-3组和SHH+RA组则可以观察到较多的Ch AT阳性细胞。经统计学比较,SHH+NT-3组和SHH+RA组Ch AT阳性细胞比率均显著高于对照组、RA组、SHH组,差异均具有统计学意义(P <0.05),且SHH+NT-3组和SHH+RA组组间比较,对照组、RA组、SHH组两两比较,差异均无统计学意义(P> 0.05)。结论在添加一定生长因子的条件下对胚胎大鼠脊髓神经干细胞进行体外培养,可以诱导其向胆碱能神经元分化。Objective The neural stem cells of embryonic rat spinal cord were cultured and induced in vitro under certain conditions to observe the differentiation of neural stem cells into cholinergic neurons and identify them.Methods A total of 5 Wistar rats with gestational age of 17 D were selected.The embryonic rat spinal cord neural stem cell was isolated and cultured,and neuroepithelial stem cell protein(neuroepithelial stem proteins,Nestin)cell was identified by immunofluorescence.The differentiation of neural stem cells was directed by cholinergic neurons.The control group was simply induced culture medium,and the rest groups were divided into retinoic acid(RA)group,hedgehog factor(SHH)group,SHH+neurotrophic factor-3(NT-3)group and SHH+RA group based on induction culture medium.After 14 d of cell differentiation,cell immunofluorescence staining was performed to observe the proportion of Choline acetyltransferase(ChAT)positive cells and cell differentiation in each group,each group had one.Results Neural stem cells differentiated into cholinergic neurons by induction.After cultured for 14 days,most nerve cells migrated out from the center of the neural stem cell ball,and the processes between nerve cells were interlaced,showing a reticular structure.The red fluorescence can be observed in cytoplasm by immunofluorescence detection.No ChAT positive cell was found in the control group,a small number of ChAT positive cells were observed in the SHH and RA induction groups,while a large number of ChAT positive cells were observed in the SHH+nt-3 group and the SHH+RA group.After statistical comparison,the proportion of ChAT positive cells in SHH+nt-3 group and SHH+RA group were significantly higher than those in the control group,RA group and SHH group(P<0.05),and the comparison between SHH+NT-3 group and SHH+RA group showed no statistically significant difference(P>0.05).Conclusion Experimental results show that in vitro culture of embryonic rat spinal cord neural stem cells with certain growth factors can induce different

关 键 词：胚胎大鼠 脊髓神经干细胞 体外培养 胆碱能神经元 细胞诱导 细胞分化 鉴定 

分 类 号：R741[医药卫生—神经病学与精神病学] R-332[医药卫生—临床医学]