基质细胞衍生因子-1α交联层粘连蛋白诱导神经干细胞体外迁移与分化

Stromal cell derived factor-1α-laminin crosstalk induced neural stem cells migration and differentiation in vitro

目的探讨基质细胞衍生因子-1α（SDF-1α）交联层粘连蛋白（LN）在神经干细胞（NSCs）体外迁移及分化中的作用。方法原代提取、培养孕14d胎鼠NSCs，并进行免疫荧光鉴定。选取第3代培养的NSCs，分为多聚赖氨酸（PLL）组、PLL＋SDF-1α组、LN组及LN＋SDF-1α组。PLL组和LN组NSCs分别接种于含0．1mg／mLPLL和1mg／mLLN包被的培养板上；PLL＋SDF-1α组和LN＋SDF-1α组分别于接种后置于1mg／L含SDF-1α的培养基培养。采用Transwell细胞迁移试验检测SDF-1α交联LN对NSCs迁移的影响；采用免疫荧光染色检测NSCs的分化情况。结果成功分离并培养原代NSCs，3～5d即有NSCs克隆球形成，细胞具备典型的NSCs形态，且NSCs特异性抗原巢蛋白呈阳性表达。与PLL组相比，PLL＋SDF-1α组NSC迁移数量无明显变化（个：3．00±0．99比2．30±0．67，P〉0．05）；而与LN组比较，LN＋SDF-1α组NSCs迁移数量明显增多（个：85．33±9．61比31．67±5．86，P〈0．05）。免疫荧光染色显示，PLL组和PLL＋SDF-1α组仅有极少量NSCs分化的神经元前体细胞，分化率几乎为0。LN组可见部分神经元前体细胞，分化率为（12．50±2．56）％；而SDF-1α交联LN后。神经元前体细胞明显增多，分化率为（21．40±3．41）％，明显高于LN组（P〈0．05）。结论SDF-1α交联细胞外基质中的LN能显著并协同增强NSCs的迁移和分化。

Objective To investigate the role of stromal cell derived factor-1α （SDF-1α）-laminin （LN） crosstalk in migration and differentiation of neural stem cells （NSCs）. Methods Original generation of NSCs collected from 14-day pregnant fetal rats were separated and cultivated, and the phenotype characteristics were identified with immunofluorescence. The third generation of NSCs were collected, and they were divided into four groups： poly-L-lysine （PLL） group, PLL＋SDF-1α group, LN group and LN＋SDF-1α group. The NSCs in PLL and LN groups were inoculated into plates coated with 0.1 mg/mL PLL or 1 mg/mL LN, and the NSCs in PLL＋SDF-1α and LN＋SDF-1α groups were inoculated into plates coated with PLL or LN and 1 mg/mL SDF-1α containing medium. The effect of SDF-1α -LN crosstalk on NSCs migrated numbers was determined by using Transwell cell migration test, and the differentiation of NSCs was determined by immunofluorescence staining. Results Primary NSCs were successfully isolated and cultivated, neurospheres formed at 3-5 days with typical NSCs morphology positively expressing Nestin which was the specific antigen of NSCs. Compared with PLL group, the number of NSCs migration in PLL＋SDF-1α group showed no significant change （cells： 3.00 ± 0.99 vs. 2.3 ± 0.67, P 〉 0.05）. Compared with LN group, the number of NSCs migration in LN＋SDF-1α group was significantly increased （cells： 85.33 ± 9.61 vs. 31.67 ± 5.86, P 〈 0.05）. Immunofluorescence staining showed that the differentiation rates of PLL and PLL＋SDF-1α groups were almost zero, and some early differentiation neurons were detected in LN group with the differentiation rates of （12.50 ± 2.56）%. The differentiation of early neuronal ceils was significantly increased after SDF-1α-LN crosstalk, the neuronal differentiation rate was （21.40 ± 3.gl）%, and it was significantly higher than that of LN group （P 〈 0.05）. Conclusion SDF-1αcrosstalk with LN in extracellular matrix can significantly and synergistically enhance the migration and differentiation of NSCs in vitro.