C6细胞中诱导体外培养大鼠神经干细胞迁移蛋白的初步分析

Preliminary study on C6 cell-isolated proteins inducing the migration of in vitro cultured rat neural stem cells

目的:许多体内实验表明神经干细胞具有向胶质瘤细胞迁移的特性,该特性使神经干细胞有可能成为基因治疗脑胶质瘤潜在的基因携带者。实验拟观察大鼠星形胶质瘤C6细胞诱导体外培养的大鼠神经干细胞的迁移作用,并初步分离C6细胞中诱导神经干细胞迁移的蛋白。方法:实验于2005-01/2006-05在南通大学江苏省神经再生重点实验室完成。实验材料:C6细胞用含10%小牛血清的DMEM/F12培养基进行培养,使用时细胞处于对数生长期。清洁级的新生1周内的SD大鼠由南通大学实验动物中心提供,实验过程中对动物处置符合动物伦理学标准。实验方法:从新生5～7dSD大鼠大脑皮质分离培养、纯化神经干细胞。从SD新生红皮鼠大脑皮质分离、培养、纯化星形胶质细胞。采用"TranswellInserts"细胞培养室培养系统共培养36h,比较C6细胞和大鼠星形胶质细胞对体外培养的大鼠神经干细胞的迁移作用;自然系统聚丙烯酰胺凝胶蛋白电泳分离C6细胞和星形胶质细胞差异蛋白,观察各蛋白凝胶条带对神经干细胞的迁移作用。结果:体外培养C6细胞能诱导神经干细胞迁移,迁移细胞数目与星形胶质细胞相比,差异有显著意义(P<0.001)。将C6细胞中差异蛋白条带与神经干细胞共培养,与对应的星形胶质细胞蛋白条带相比,可观察到与相对分子质量约67000蛋白胶条共培养的神经细胞球在接近蛋白胶条的一侧细胞向胶条迁移生长。结论:C6细胞可以诱导体外培养的神经干细胞迁移,其总蛋白在自然系统聚丙烯酰胺凝胶电泳分离实验中获得的相对分子质量为67000的蛋白组分具有诱导神经干细胞迁移的活性。

AIM： Various in vivo studies demonstrate a migration tendency of neural stem cells （NSCs） toward glioma cells, indicating NSCs can be served as a potential carrier for delivery of therapeutic genes to disseminated glioma cells. This present study was to investigate the effect of astroglioma C6 cells inducing the migration of the rat NSCs cultured in vitro and primarily screen the proteins with activity of inducing migration from C6 cells. METHODS： The experiment was carried out in the Jiangsu Provincial Key Laboratory of Neural Regeneration, Nantong University from January 2005 to May 2006. C6 cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum, and were used in logarithmic phase. SD neonatal rats within one week, which were cleaning grade, were offered by the Experimental Animal Center of Nantong University. The NSCs were separated, cultured and purified from the cerebral cortex of the 5-7 days old neonatal SD rats. The astrocytes were separated, cultured and purified from the cerebral cortex of newborn SD rats. The ＂Transwell Inserts＂ chamber migration assay was used to investigate the activity of inducing NSCs migration in C6 cells or the rat astrocytes. After 36-hour co-incubation with NSCs, the total proteins of C6 cells and astrocytes were separated by native polyacrylamide gel electrophoresis, and the differential protein bands between C6 cells and astrocytes were respectively co-cultured with NSCs to observe the migration of NSCs by protein gel straps. RESULTS： Contrasted with astrocytes, the C6 cells apparently induced migration of NSCs （P 〈 0.001）. The differential protein bands of C6 cells were co-cultured with NSCs, and it was observed that the proteins with 67 000 relative molecular mass could induce migration of neural spheres. CONCLUSION： The C6 cells can induce the migration of NSCs cultured in vitro, and the protein components with 67 000 relative molecular mass isolated in native polyacrylamide gel electrophoresis show the activity of inducing NSC migration.