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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 effects of carboxymethyl chitosan (CMCS) on the proliferation and secretion of neurotrophic factor in Schwann cells (SCs) and its mechanism. Methods Twenty SDF rats of 3 to 5 days old were randomly divided into male and female, weight ranged from 25 g to 30 g. SCs were cultured in vitro and sciatic nerve was identified by S-100 immunofluorescence staining. The second-generation SCs in logarithmic growth phase were taken and the cell counting kit 8 was used to detect the effect of CMCS at different concentrations on the proliferation of SCs at different times. The SCs were divided into four groups, and the final concentration of CMCs was adjusted to 50 (group B), 100 (group C) and 200μg / mL (group D) by adding CMCS, and the control group (group A) (S%) and proliferation index (PI) of S phase cells were measured. The contents of NGF and ciliary neurotrophic factor (CNTF) in SCs were detected by real-time quantitative PCR and Western blot. Results S-100 immunofluorescence labeling showed that the positive rate of cells was over 90%. CMCS promoted the proliferation of SCs in the concentration range of 10 ~ 1 000μg / mL, and the effect of proliferation was most obvious in 200 and 500μg / mL group (P <0.01). There was no significant difference between 200μg / mL and 500μg / mL groups Significance (P> 0.05). At 200 μg / mL CMCS cultured SCs at different times, the effect was most obvious at 24 h, which was significantly different from that of control group (200 μg / mL CMCS for 0 h) (P <0.01). The percentages of S% and PI in group B, C and D were significantly higher than those in group A (P <0.05), while those in group C and D were significantly higher than those in group B (P <0.05). There was no significant difference between group C and D Statistical significance (P> 0.05). Real-time quantitative PCR and Western blot showed that the mRNA and protein expressions of NGF and CNTF in B, C and D groups were significantly higher than those in A group (P <0.05), and the effect of D group was the most obvious. Conclusion CMCS can promote the proliferation and cell cycle progression of SCs and the secretion of neurotrophic factor.