嗅鞘细胞在大鼠坐骨神经缺损修复中的炎性调节作用研究  

REGULATORY EFFECT OF OLFACTORY ENSHEATHING CELLS ON INFLAMMATORY CYTOKINES IN REPAIR OF RAT SCIATIC NERVE DEFECT

作　　者：姚东东[1,2] 张洁元[3] 李兵仓[3] 周婵[4] 刘彬[1,2] 

机构地区：[1]西南大学生命科学学院,重庆400715 [2]淡水鱼类资源与生殖发育教育部重点实验室 [3]第三军医大学大坪医院野战外科研究所,创伤,烧伤与复合伤国家重点实验室 [4]重庆市畜牧科学院

出　　处：《中国修复重建外科杂志》2016年第12期1538-1544,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(31271036;81371341);国家重点基础研究发展计划(973)资助项目(2012CB518106);全军后勤科研计划重大专项资助项目(AWS14C003-5);创伤;烧伤与复合伤国家重点实验室Ⅰ类研究课题(SKLZZ201003);第三军医大学第三附属医院院所青年创新基金(2014YQN07)~~

摘　　要：目的探讨嗅鞘细胞（olfactory ensheathingcell，OEC）移植修复大鼠坐骨神经缺损后，其在修复过程中对坐骨神经局部微环境内炎性因子表达的调节作用。方法取绿色荧光蛋白（green fluorescent protein, GFP ）转基因SD大鼠嗅球，原代培养GFP—OEC并鉴定。取100只SD大鼠随机分为两组，制备右侧坐骨神经缺损（10mm长）模型后，采用聚乳酸一聚羟基乙酸共聚物[poly （lactic acid-co-glycolic acid）, PLGA]神经导管修复缺损处，实验组（n=55）于导管两端注入GFP—OEC与细胞外基质（extrace Uularmatrix，ECM）的混合液，对照组（n=45）注入等量DMEM与ECM的混合液。术后观察大鼠一般情况，于6h、1d、3d以及1、2、3、4、6周Westernblot检测IL-4、IL-6、IL-13和TNF—α的表达；术后2、4、6周实验组取材观察GFP—OEC存活情况；9周两组取材行HE染色观察神经组织形态，并行坐骨神经感觉及运动功能检测、神经电生理检测。结果经免疫荧光染色鉴定，原代培养细胞为GFP—OEC。Westernblot检测显示，与对照组比较，2～6周实验组IL-4相对表达量明显升高，3d、1周时IL-6及TNF—α相对表达量降低，1d以及3～6周时IL-13相对表达量升高，比较差异有统计学意义（P〈0．05）。术后2、4、6周荧光显微镜下观察，实验组神经移植体内的神经再生端均有GFP—OEC存活；术后9周，HE染色示实验组再生神经组织较松散，细胞形态不规则；对照组再生神经组织成泡状空隙，细胞明显减少。术后9周，实验组大鼠坐骨神经功能恢复程度优于对照组，后足回缩时间、坐骨神经功能指数、肌肉动作电位潜伏期及复合肌肉动作电位波幅比较，差异均有统计学意义（P〈0．05）。结论坐骨神经缺损后植入OEC可促进抗炎因子IL-4和IL-13的表达，同时抑制促炎因子IL-6和TNF-α的表达，改善了坐骨神经局部的炎症微环境，进而有效地促进坐骨神Objective To investigate the expression regulation of inflammation cytokines interleulon 4 （IL-4）, IL- 6, IL-13, and tumor necrosis factor u （TNF-α） in rats with sciatic nerve defect following olfactory ensheathing cell （OEC） transplantation. Methods The primary OEC for cell culture and identification was dissociated from the olfactory bulb of the green fluorescent protein-Sprague Dawley （GFP-SD） rat. One hundred SD rats were randomly divided into 2 groups, and the right sciatic nerve defect （10 mm in length） model was made, then repaired with poly （lactic acid-co-glycolic acid） （PLGA）. The mixture of equivalent cultured GFP-OEC and extracellular matrix （ECM） was injected into both ends of PLGA nerve conduit in the experimental group （n=55）, and the mixture of DMEM and ECM in the control group （n=45）. The general situation of rats was observed after operation. At 6 hours, 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, and 6 weeks, the inflammatory cytokines were detected by Western blot. At 2, 4, and 6 weeks, the survival of GFP-OEC was observed in the experimental group. At 9 weeks, HE staining was used to observe the morphology of nerve tissue, and the sensory and motor function and the electrophysiological index were detected. Results The cultured primary cells were GFP-OECs by immunofluorescence staining. Compared with the control group, the experimental group showed significantly increased expression level of IL-4 at 2-6 weeks （P〈0.05）, significantly decreased expression level of IL-6 and TNF-a at 3 days and 1 week （P〈0.05） and significantly increased expression level of IL-13 at 1 day and 3-6 weeks （P〈0.05） by Western blot detection. At 2, 4, and 6 weeks, the surviving GFP-OEC of regenerative nerve end was observed in the experimental group under the fluorescence microscope. At 9 weeks, regenerative nerve tissue was loose, and cell morphology was irregular in the experimental group, while the regenerative nerve tissue had vesicular voids and the

关 键 词：周围神经再生 嗅鞘细胞 抗炎因子 促炎因子 大鼠 

分 类 号：R688[医药卫生—骨科学]