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

发布时间：2018-01-01 23:04

本文关键词：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　出处：《中国修复重建外科杂志》2013年08期 　论文类型：期刊论文

【摘要】：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(P0.01),200μg/mL与500μg/mL组间比较差异无统计学意义(P0.05)。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(P0.01)。B、C、D组S%及PI均显著高于A组,差异有统计学意义(P0.05);C、D组显著高于B组(P0.05),C、D组间差异无统计学意义(P0.05)。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(P0.05);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。
[Abstract]:Objective to investigate the effect of carboxymethylated chitosanine (CMCs) on Schwann cells in Schwann cells. Methods Twenty SD rats of SPF grade at the age of 3 days and 5 days of age were selected, male and female, 2530g and 2530g respectively. The sciatic nerves of both sides were cultured in vitro and identified by S-100 immunofluorescence labeling. The second generation of SCs in logarithmic growth phase was obtained. Cell count assay kit 8 was used to detect the effects of different concentrations of CMCS and time on the proliferation of SCs. SCs was divided into 4 groups, adding CMCS to adjust the final concentration to 50B). The same amount of PBS was added to group C (n = 200 渭 g / mL) and group A (control group). Cell cycle was analyzed by flow cytometry (FCM) and proliferation index (Pi). Real-time quantitative PCR and Western blot were used to detect the synthesis of NGF and ciliary neurotrophic factor by SCs. CNTF). Results S-100 immunofluorescence labeling showed that the positive rate of cells was more than 90%. CMCs could promote the proliferation of SCs in the concentration range of 101000 渭 g / mL. The proliferation effect of 200,500 渭 g / mL group was the most obvious (P0.01). There was no significant difference between the 200 渭 g / mL and 500 渭 g / mL groups (P 0.05). SCs was cultured with 200 渭 g / mL CMCS for different time. It was found that the proliferation effect was the most obvious at 24 h after treatment, and there was a significant difference compared with the control group (200 渭 g / mL CMCS for 0 h). The S% and Pi in group D were significantly higher than those in group A (P 0.05). There was no significant difference between group D and group B (P 0.05). Real time quantitative PCR and Western blot detection showed BMC. The expressions of NGF and CNTF mRNA and protein in group D were significantly higher than those in group A (P 0.05). Conclusion CMCS can promote the proliferation and cell cycle progression of SCs and secrete neurotrophic factors.
【作者单位】：武汉大学人民医院骨科;
【基金】：国家自然科学基金资助项目(30600627、30801166)~~
【分类号】：R329
【正文快照】： 雪旺细胞(Schwann cells,SCs)是周围神经系统的主要胶质细胞,在脊髓及周围神经损伤再生修复中发挥重要作用[1-2]。神经组织工程是目前神经再生领域研究的热点和难点,SCs作为该领域的种子细胞受到广大学者重视。SCs体外增殖缓慢,因此促进体外培养SCs增殖成为研究热点[3]。羧甲

【参考文献】