羧甲基壳聚糖抗氧化损伤诱导雪旺细胞凋亡的作用及机制研究

Anti-apoptotic effect of carboxymethylated chitosan on oxidatively injured Schwann cells

[目的]探讨羧甲基壳聚糖（carboxymethylated chitosan,CMCS）对氧化应激诱导雪旺细胞（schwann cells,SCs）凋亡的保护作用及作用机制。[方法]体外培养SCs,S-100免疫荧光染色鉴定。将SCs分为空白对照组、H2O2诱导组、H2O2加CMCS处理组。通过CCK-8检测不同组SCs增殖情况,流式细胞仪计数检测SCs早期凋亡率,检测各组细胞内超氧化物歧化酶（SOD）及丙二醛（MDA）的含量,Real-time PCR技术检测Bcl-2及Bax mRNA表达水平,Western blot法检测Bcl-2,Bax,Caspase-3、-9的表达水平。[结果]H2O2诱导SCs后其细胞增殖活性明显降低,加入不同浓度CMCS后增殖活性增加。流式细胞技术检测结果表明,经H2O2诱导SCs早期凋亡率增加,加入50-200μg/ml CMCS后凋亡率逐渐降低。经H2O2诱导组SCs与空白对照组比较SOD含量明显减少,MDA含量明显增加（P〈0.05）;加入CMCS后SOD含量增加,MDA含量减少（P〈0.05）。Real-time PCR及Western blot检测结果表明经H2O2诱导SCs内Bcl-2表达降低,Bax,Caspase-3、-9表达增高,加入CMCS后Bcl-2表达恢复,Bax,Caspase-3、9表达抑制。[结论]CMCS对H2O2诱导SCs凋亡具有保护作用。

[Objective]To investigate the effect of carboxymethylated chitosan（ CMCS） on oxidative stress- induced apoptosis in Schwann cells（ SCs） and its underlying mechanism. [Method]SCs were cultured in vitro and purified using S- 100 staining. Purified SCs were divided into control,H2O2- induced,and H2O2＋ CMCS treatment groups. Cell proliferation of SCs was evaluated using a CCK- 8 assay and flow cytometry was used to detect the early SC apoptosis rate. Intracellular superoxide dismutase（ SOD） and malondialdehyde（ MDA） levels were measured in SCs. Real- time PCR was used to detect Bcl- 2 and Bax mRNA levels and western blotting was performed to detect Bcl- 2,Bax,Caspase- 3,and- 9 levels. [Result]CCK- 8 assay results indicated that the percentage of proliferating H2O2- induced SCs was significantly reduced by the addition of CMCS,and the proliferating activity was significantly lower than in the H2O2- treated group. Flow cytometry results showed that the apoptotic rate in the 0. 01- 1. 0 m M H2O2- induced group was increased,and the addition of 50- 200 μg / ml CMCS decreased this rate. Compared to the control group,H2O2- induced SCs had reduced SOD content and MDA levels. After the addition of CMCS,SOD content increased and MDA content decreased significantly（ P〈0. 05）. Real- time PCR and western blot analyses showed that the level of Bcl- 2 was reduced in H2O2- induced SCs,whereas Bax,Caspase- 3,and- 9 were increased. Bcl- 2expression was restored by the addition of CMCS,and Bax,Caspase- 3,and- 9 levels were reduced in response to CMCS treatment. [Conclusion]CMCS can protect SCs from oxidative stress- induced apoptosis.