摘要
为鉴定大鼠胚胎海马神经干细胞(NSCs)是否表达功能性的L-型钙通道,L-型钙通道是否参与了对大鼠胚胎NSCs增殖和分化调控.分离孕15天Wistar大鼠胚胎海马组织,制成单细胞悬液,利用无血清培养技术,在添加bFGF、EGF、N-2和B27 supplement的DMEM/F12培养液中进行培养.采用细胞免疫荧光法对原代至第5代细胞进行鉴定,均有巢蛋白(nestin)的表达,第3代nestin阳性细胞比例达97%.把培养的细胞诱导分化5天后,这些细胞表现为神经元和星形胶质细胞的形态,且分别呈Ⅲ型β-微管蛋白(Tuj1)阳性和胶质纤维酸性蛋白(GFAP)阳性;细胞免疫印迹结果显示,NSCs表达L-型钙通道的Cav1.2α1C亚单位,而无Cav1.3α1D亚单位的表达;利用全细胞膜片钳技术在NSCs上记录到了L-型钙电流,证明了NSCs所表达的L-型钙通道具有功能.进一步对细胞进行药理学干预,发现L-型钙通道的激活不仅可以促进胚胎NSCs的增殖,而且使增殖的NSCs向神经元分化的比例显著增加.以上结果表明,Wistar大鼠胚胎海马NSCs表达功能性的L-型钙通道;L-型钙通道参与了胚胎NSCs增殖和分化的调控.
In order to identify whether functional L-type calcium channels are expressed in neural stem cells （NSCs） from rat embryonic hippocampus, and whether L-type calcium channels participate in the modulation of proliferation and differentiation of NSCs, the rat embryonic hippocampal tissue was dispersed into a single cell suspension, and the dissociated cells were cultured in serum-free DMEM/F12 medium containing epidermal growth factor （EGF）, basic fibroblast growth factor CoFGF）, N-2 and B27 supplement. Immunofluorescent labeling showed an expression of nestin-positive cells. Following five-days culture in differentiation medium, neuron-like and astrocyte-like cells were observed, which expressed 13-tubulin Ⅲ （Tuj 1） and Glial fibrillary acidic protein （GFAP）, respectively. Western blot analysis showed an expression of Cavl.2α1C subunits in NSCs, but no Cavl.3α1D subunita. Moreover, L-type calcium channel currents were recorded in those cells by using whole-cell patch clamp techniques. It was found that activation of L-type calcium channels promotes proliferation and differentiation to neuronal type of NSCs. The results indicated that rat embryonic hippocampal NSCs express functional L-type calcium channels, L-type calcium channels modulate proliferation and differentiation of NSCs.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2007年第3期275-282,共8页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金(30330240
30125013
30371772)资助项目
广东省自然科学基金重点项目(37047)
教育部长江学者奖励计划资助项目~~
关键词
大鼠
胚胎神经干细胞
L-型钙通道
增殖
分化
rat, embryonic neural stem cells, L-type calcium channel, proliferation, differentiation
