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激活的星形胶质细胞分泌聚集素蛋白及其对海马神经干细胞向神经元分化的促进作用
邹琳清 金国华* 李浩明 秦建兵 田美玲 朱培培 王海琴
解剖学报 ›› 2013, Vol. 44 ›› Issue (6) : 761-766.
激活的星形胶质细胞分泌聚集素蛋白及其对海马神经干细胞向神经元分化的促进作用
Activated astrocyte secrete clusterin that enhances neuronal differentiation from rat hippocampal neural stem cells
目的 探讨切割穹隆海马伞的侧海马提取液“激活”的星形胶质细胞分泌聚集素蛋白（CLU）及体外CLU诱导海马神经干细胞分化为神经元的情况。
方法 分别用切割穹隆海马伞的海马提取液、正常海马提取液、DMEM/F12 培养基培养星形胶质细胞，制备切割12h、24h、48h组，正常组和对照组条件培养基；ELISA检测比较不同条件培养基中CLU蛋白的浓度；体外细胞培养采用CLU诱导海马神经干细胞分化，设诱导组和空白对照组，7d后通过微管相关蛋白2（MAP-2）免疫荧光及Hoechst标记观察海马神经干细胞向神经元的分化情况。 结果 正常组和对照组条件培养基中CLU浓度较低，约为（0.1037±0.0119）ng/L和（0.1170±0.0078）ng/L；切割组12h、24h、48h条件培养基中其浓度明显增高，分别为（0.1940±0.0364）ng/L、（0.4250±0.0724）ng/L和（0.2903±0.0472）ng/L，以24h组最高，约为正常组和对照组的4倍。CLU诱导组中MAP-2阳性细胞数明显高于空白对照组（P<0.05），其占总细胞数的百分比分别为(11.67±2.57)%和（4.52±1.54）%，且诱导组中MAP-2阳性细胞突起更长、更丰富。 结论 切割穹隆海马伞侧海马提取液“激活”的星形胶质细胞可分泌更多的CLU，体外细胞培养中CLU可促进海马神经干细胞向成熟的神经元分化。
Objective To detect the secretion of clusterin in "reactive astrocytes" which are activated by fimbria-fornix transected hippocampal extracts, and to investigate the neuronal differentaition of hippocampal neural stem cells (NSCs) by clusterinin vitro. Methods The astrocytes were cultured in fimbria-fornix transected hippocampal extracts, normal hippocampal extracts and DMEM/F12 medium, the conditional medium contained normal hippocampal extracts and transected 12hours, 24hours, 48hours hippocampal extracts. The concentrations of clusterin was detected by ELISA assay. To investigate the neuronal differentaition of hippocampal neural stem cells (NSCs) by clusterin, the cells were divided into the induced group and control group. Seven days later, the neuronal differentiation of hippocampal NSCs was investigated by MAP-2 immunofluorescence and labeled by Hoechst. Results The concentrations of clusterin in normal group and control group were lower than the transected 12hours, 24hours, 48hours groups, especially the 24hours group which concentration was about 4-folds higher than normal and control group，about (0.1037±0.0119)ng/L,(0.1170±0.0078) ng/L, (0.1940±0.0364) ng/L, (0.4250±0.0724) ng/L and (0.2903±0.0472) ng/L, respectively. The number of MAP-2 positive cells in clusterin induced group was higher than the control group (P<0.05), and the percentages of the total number of cells of these two groups were (11.67± 2.57)% and (4.52±1.54)%, respectively. The processes in the induced group was longer and more than the control group. Conclusion Reactive astrocytes activated by fimbria-fornix transected hippocampal extracts secrete more clusterin. The clusterin in culture cells may promote hippocampal NSCs differentiation into mature neurons in vitro.
星形胶质细胞 / 聚集素蛋白 / 海马 / 神经干细胞 / 神经元 / 免疫荧光 / 大鼠
Astrtocyte / Clusterin / Hippocampus / Neural stem cell / Neuron / Immunofluorescence / Rat
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