羧甲基壳聚糖对体外培养雪旺细胞增殖周期及其分泌神经营养因子的影响

EFFECT OF CARBOXYMETHYLATED CHITOSAN ON PROLIFERATION AND SYNTHESIS OF NEUROTROPHIC FACTORS IN Schwann CELLS IN VITRO

目的探讨羧甲基壳聚糖(carboxymethylated chitosan,CMCS)对雪旺细胞(Schwann cells,SCs)增殖周期及分泌神经营养因子的影响及其作用机制。方法取3～5日龄SPF级SD大鼠20只,雌雄不限,体重25～30 g,取双侧坐骨神经体外培养SCs,经S-100免疫荧光标记鉴定。取处于对数生长期的第2代SCs,细胞计数检测试剂盒8检测不同浓度CMCS及作用不同时间对SCs增殖的影响。将SCs分为4组,加入CMCS调整终浓度为50(B组)、100(C组)、200μg/mL(D组),对照组(A组)加入等量PBS,流式细胞技术检测处于S期细胞的百分比(S%)和增殖指数(proliferation index,PI)分析细胞周期;实时定量PCR与Western blot检测SCs合成NGF和睫状神经营养因子(ciliary neurotrophic factor,CNTF)的含量。结果 S-100免疫荧光标记鉴定示细胞阳性率达90%以上。CMCS在10～1 000μg/mL浓度范围内对SCs增殖均有促进作用,200、500μg/mL组促增殖效应最明显(P<0.01),200μg/mL与500μg/mL组间比较差异无统计学意义(P>0.05)。采用200μg/mL CMCS培养SCs不同时间,发现作用24 h时其促增殖效果最明显,与对照组(200μg/mL CMCS作用0 h)比较差异有统计学意义(P<0.01)。B、C、D组S%及PI均显著高于A组,差异有统计学意义(P<0.05);C、D组显著高于B组(P<0.05),C、D组间差异无统计学意义(P>0.05)。实时定量PCR和Western blot检测均显示,B、C、D组NGF与CNTF mRNA和蛋白表达均显著高于A组(P<0.05);其中D组作用最明显。结论 CMCS可以促进SCs增殖及细胞周期进程以及分泌神经营养因子。

Objective To investigate the effect of carboxymethylated chitosan（CMCS） on the proliferation,cell cycle,and secretion of neurotrophic factors in cultured Schwann cells（SCs）. Methods SCs were obtained from sciatic nerves of 20 Sprague Dawley rats（3-5 days old;male or female;weighing,25-30 g） and cultured in vitro,SCs were identified and purified by immunofluorescence against S-100.The cell counting kit 8（CCK-8） assay was used to determine the proliferation of SCs.The SCs were divided into 4 groups： 50 μg/mL CMCS（group B）,100 μg/mL CMCS（group C）,200 μg/mL CMCS（group D）,and the same amount of PBS（group A） were added.The flow cytometry was used to analyze the cell cycle of SCs;the real-time quantitative PCR and Western blot analysis were used to detect the levels of never growth factor（NGF） and ciliary neurotrophic factor（CNTF） in cultured SCs induced by CMCS. Results The purity of cultured SCs was more than 90% by immunofluorescence against S-100;the CCK-8 results indicated that CMCS in concentrations of 10-1 000 μg/mL could promote the proliferation of SCs,especially in concentrations of 200 and 500 μg/mL（P 0.01）,but no significant difference was found between 200 and 500 μg/mL（P 0.05）.CMCS at a concentration of 200 μg/mL for 24 hours induced the highest proliferation,showing significant difference when compared with that at 0 hour（P 0.01）.The percentage of cells in phase S and the proliferation index were significantly higher in groups B,C,and D than in group A（P 0.05）,in groups C and D than in group B（P 0.05）;and there was no significant difference between group C and group D（P 0.05）.Real-time quantitative PCR and Western blot results showed that the levels of NGF and CNTF in groups B,C,and D were significantly higher than those in group A（P 0.05）,especially in group D. Conclusion CMCS can stimulate the proliferation,and induce the synthesis of neurotrophic factors in cultured SCs.