激活态雪旺细胞对神经干细胞分化作用的研究  

Effect of activated Schwann cells in inducing differentiation of neural stem cells

作　　者：张睿[1] 张军[1] 赵承斌[1] 

机构地区：[1]哈尔滨医科大学附属第四医院骨科,黑龙江哈尔滨150086

出　　处：《中国医药导报》2014年第7期19-22,共4页China Medical Herald

基　　金：黑龙江省自然科学基金项目(编号D200901);黑龙江省教育厅科技研究项目(编号11531160)

摘　　要：目的探讨激活态雪旺细胞(SCs)对神经干细胞(NSCs)的分化作用。方法取出生24 h内的SD乳鼠脊髓NSCs,分离培养并传至4代。取体重(100±5)g的SD大鼠,结扎右侧坐骨神经,1周后取双侧坐骨神经,左侧提取正常坐骨神经分离培养SCs(普通SCs),右侧提取结扎变性的坐骨神经分离培养SCs(激活态SCs),均采用双酶消化加差速贴壁法。将SCs与NSCs应用Transwell培养皿联合培养,分为三组:A组为激活态SCs与NSCs;B组为普通SCs与NSCs;C组仅为NSCs。于培养的第2、4、6天检测各组NSCs活性。1周后,Western blot检测各组SCs表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF)的表达水平,免疫荧光检测各组NSCs分化比例。结果传至4代的NSCs生长稳定,分离的SCs 7 d后大量增殖呈旋涡状。联合培养后MTT法检测细胞活性,第2、4天三组细胞的活性差异无统计学意义(P>0.05),第6天C组活细胞比例开始降低,A、B组与C组比较差异有统计学意义(P<0.05),A组与B组比较差异无统计学意义(P>0.05)。Western blot检测A组细胞表皮生长因子(EGF)表达水平(0.486±0.028)、碱性成纤维细胞生长因子(bFGF)表达水平(0.385±0.023)均高于B组,差异有统计学意义(P<0.05)。MAP-2荧光染色,每高倍视野阳性细胞比例A组[(35.26±2.53)%]高于B组[(27.63±3.45)%],差异有统计学意义(P<0.05);GFAP荧光染色,每高倍视野阳性细胞比例A组[(62.42±3.78)%]低于B组[(70.18±1.26)%],差异有统计学意义(P<0.05)。结论激活态SCs能够促进NSCs向神经元的分化进程,提高神经元的分化比例。Objective To discuss the effect of activated Schwann cells（SCs） in the differentiation of neural stem cells（NSCs）. Methods The spinal cord of SD rat was taken in the 24 hours after birth. The NSCs from spinal cord were separated and cultured until the fourth passage.（100±5） g SD rat was used to ligature the right sciatic nerve. After one week, both the right and left sciatic nerve were isolated to culture SCs by double enzymes digestion and differential attachment. The SCs from the right sciatic nerve were named activated SCs, and the ones from the left side called common SCs. Then the SCs and NSCs were co-culture by Transwell culture dish, and they were divided into group A,group B and group C. group A was activated SCs and NSCs, group B was common SCs and NSCs, and group C was just NSCs as control. The cytoactive was detected at the second, fourth and sixth days. One week later, the EGF and bFGF secreted by SCs were detected in Western blot, and the differentiation ratio of NSCs was detected by immunofluorescence. Results The fourth passage NSCs grew stably, and the SCs proliferated as vortex after 7 days. The difference of cytoactive between the second and fourth day was not statistically significant（P 0.05） according to the MTT detection. However, the cytoactive began to reduce at the sixth day by MTT detection, and the cytoactive of group A and group B were superior than those of group C, the differences were statistically significant（P 0.05）. The difference of cytoactive between group A and group B, the difference was not statistically significant（P 0.05）. The expression of EGF and bFGF in group A [（0.486 ±0.028）,（0.385 ±0.023）] were both higher than those in group B,the differences were statistically significant（P 0.05）.The positive cells ratio of each high power field of group A [（35.26±2.53）%] was superior than that of group B [（27.63±3.45）%] according to the MAP-2 staining, the difference was statistically significant（P 0.05）. The positive cells ratio

关 键 词：雪旺细胞 神经干细胞 细胞分化 联合培养 神经元 

分 类 号：R741.02[医药卫生—神经病学与精神病学]