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)和教育部长江学者奖励计划资助项目.
This work was supported by grants from The National Natural Science Foundation of China (30330240, 30125013, 30371772), Natural Science Foundation of Guangdong Province (37047) and Cheung King Scholars Programme to TM Gao.
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 (bFGF), 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 β-tubulin Ⅲ(Tuj1) and Glial fibrillary acidic protein (GFAP), respectively. Western blot analysis showed an expression of Cav1.2α1C subunits in NSCs, but no Cav1.3α1D subunits. 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.
冯锦丽,胡德辉,高天明. L-型钙通道对大鼠胚胎海马神经干细胞增殖和分化的调控[J].生物化学与生物物理进展,2007,34(3):275-282
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