羧甲基壳聚糖对氧化应激诱导雪旺细胞凋亡及BDNF与GDNF表达的影响

EFFECT OF CARBOXYMETHYLATED CHITOSAN ON APOPTOSIS AND EXPRESSION OF BRAIN DERIVED NEUROTROPHIC FACTOR AND GLIAL CELL LINE DERIVED NEUROTROPHIC FACTOR IN OXIDATIVE STRESS INDUCED Schwann CELLS IN VITRO

目的研究羧甲基壳聚糖（carboxymethylated chitosan,CMCS）对氧化应激诱导大鼠雪旺细胞（Schwann cells,SCs）凋亡的保护作用及对SCs内脑源性神经营养因子（brain derived neurotrophic factor,BDNF）和胶质细胞源性神经营养因子（glial cell line derived neurotrophic factor,GDNF）表达的影响。方法取24只3-5日龄SD大鼠（体重25-30 g,雌雄不限）双侧坐骨神经,体外分离培养SCs,S-100免疫荧光染色鉴定。于SCs培养基中加入0.01、0.10、1.00 mmol/L H2O2溶液进行诱导,作用3、6、12、24 h后采用CCK-8检测细胞增殖以及流式细胞术检测细胞凋亡,筛选H2O2诱导氧化应激SCs凋亡模型的最佳作用浓度及时间。取第2代SCs,根据不同处理方法分成5组：空白对照组（PBS处理）、1.00 mmol/L H2O2诱导组、1.00 mmol/L H2O2＋50μg/m L CMCS处理组、1.00 mmol/L H2O2＋100μg/m L CMCS处理组、1.00 mmol/L H2O2＋200μg/m L CMCS处理组。培养24 h后,采用CCK-8检测SCs增殖,流式细胞术检测SCs早期凋亡,实时定量PCR及Western blot检测SCs内BDNF、GDNF m RNA及蛋白表达。结果分离培养的细胞经S-100免疫荧光染色鉴定阳性率达95%以上。CCK-8及流式细胞术检测示,H2O2可明显抑制SCs增殖并诱发早期凋亡,且呈浓度依赖性,1.00 mmol/L H2O2溶液作用24 h效果最明显,为模型制备最佳作用浓度及时间。当加入50-200μg/m L CMCS后,SCs增殖活性增加、细胞凋亡率降低,且随CMCS浓度增加上述效应增强。实时定量PCR及Western blot检测示,与空白对照组比较,1.00 mmol/L H2O2诱导组BDNF、GDNF m RNA及蛋白表达明显降低,差异有统计学意义（P〈0.05）;加入50、100、200μg/m L CMCS作用后,随CMCS浓度增加,BDNF、GDNF m RNA及蛋白表达逐渐增加,与空白对照组及1.00 mmol/L H2O2诱导组比较以及各CMCS处理组间比较,差异均有统计学意义（P〈0.05）。结论 CMCS对氧化应激诱导大鼠SCs凋亡具有保护作用,并促进凋亡SCs分泌BDNF与GDNF。

Objective To investigate the protective effects of carboxymethylated chitosan（CMCS） on oxidative stress induced apoptosis of Schwann cells（SCs）,and the expressions of brain derived neurotrophic factor（BDNF） and glial cell line derived neurotrophic factor（GDNF） in oxidative stress induced SCs.Methods Twenty-four 3-5 days old Sprague Dawley rats（weighing 25-30 g,male or female） were involved in this study.The bilateral sciatic nerves of rats were harvested and SCs were isolated and cultured in vitro.The purity of SCs was identified by immunofluorescence staining of S-100.SCs were treated with different concentrations of hydrogen peroxide（H_2O_2,0.01,0.10,and 1.00 mmol/L） for 3,6,12,and 24 hours to establish the apoptotic model.The cell counting kit 8（CCK-8） and flow cytometry analysis were used to detect the cell viability and apoptosis induced by H_2O_2,and the optimal concentration and time for the apoptotic model of SCs were determined.The 2nd passage SCs were divided into 5 groups and were treated with PBS（control）,with 1.00 mmol/L H_2O_2,with 1.00 mmol/L H_2O2 ＋ 50 μg/mL CMCS,with 1.00 mmol/L H_2O_2＋ 100 μg/mL CMCS,and with 1.00 mmol/L H_2O_2 ＋ 200 μg/mL CMCS,respectively.After cultured for 24 hours,the cell viability was assessed by CCK-8,cell apoptosis was detected by flow cytometry analysis,the expressions of mRNA and protein of BDNF and GDNF were detected by real-time quantitative PCR and Western blot.Results The immunofluorescence staining of S-100 indicated the positive rate was more than 95%.CCK-8 and flow cytometry results showed that H_2O_2 can inhibit the proliferation of SCs and induce the SCs apoptosis with dose dependent manner,the effect was the most significant at 1.00 mmol/L H_2O_2 for 24 hours;after addition of CMCS,SCs exhibited the increased proliferation and decreased apoptosis in a dose dependent manner.Real-time quantitative PCR and Western blot analysis showed that 1.00 mmol/L H_2O_2 can significantly inhibit BDNF and GDNF expression in SCs when compared with control group（P〈0.05）,50-200 μg/mL CMCS can reverse the oxidative stress-induced BDNF and GDNF expression in SCs in a dose dependent manner,showing significant difference compared with control group and 1.00 mmol/L H_2O_2 induced group（P〈0.05）.There were significant differences among different CMCS treated groups（P〈0.05）.Conclusion CMCS has the protective stress on oxidative stress induced apoptosis of SCs,and may promote the BDNF and GDNF expressions of neurotrophic factors in oxidative stress induced SCs.