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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 investigate the effect of CLU on activation of astrocytes and the differentiation of hippocampal neural stem cells into neurons induced by CLU in vitro. Methods The astrocytes were isolated from the hippocampus of hippocampus and normal hippocampus by DMEM / F12 culture, and the conditioned medium of 12h, 24h, 48h, normal group and control group were prepared. ELISA assay The concentration of CLU protein in different culture media was detected in vitro. CLU-induced differentiation of neural stem cells was induced in vitro by inducing the hippocampal neurons by inducing the MAP-2 immunofluorescence and Hoechst labeling Differentiation of stem cells into neurons. Results The concentrations of CLU in the conditioned medium of the normal group and the control group were lower (0.1037 ± 0.0119) ng / L and (0.1170 ± 0.0078) ng / L, respectively. The concentration of CLU in the conditioned medium of the cutting group was significantly higher at 12h, 24h and 48h (0.1940 ± 0.0364) ng / L, (0.4250 ± 0.0724) ng / L and (0.2903 ± 0.0472) ng / L respectively, which was the highest in 24h group, which was about 4 times of that in normal group and control group. The number of MAP-2 positive cells in CLU induction group was significantly higher than that in blank control group (P <0.05), and the percentage of total cell number in CLU induction group was (11.67 ± 2.57)% and (4.52 ± 1.54)%, -2 positive cells have longer, more abundant processes. Conclusion CLU can excise more CLU in hippocampus of hippocampus after excising hippocampus, and CLU can promote differentiation of hippocampal neural stem cells into mature neurons in vitro.