小神经胶质细胞去除联合骨髓间质干细胞移植修复小鼠脊髓损伤的实验研究  

Depletion of microglia combined with transplantation of bone marrow mesenchymal stem cells for repairing spinal cord injury

作　　者：付海涛 戚超[1] 陈进利 高甲科[1] 李海峰[1] 赵夏[1] 张益 申友亮 张英泽[2] 于腾波[1] Fu Haitao;Qi Chao;Chen Jinli;Gao Jiake;Li Haifeng;Zhao Xia;Zhang Yi;Shen Youliang;Zhang Yingze;Yu Tengbo(Department of Sports Medicine,Affiliated Hospital of Qingdao University,Qingdao 266000,China;Department of Trauma Emergency Center,the Third Hospital of Hebei Medical University,Orthopaedics Research Institution of Hebei Province,Key Laboratory of Biomechanics of Hebei Province,Shijiazhuang 050051,China)

机构地区：[1]青岛大学附属医院运动医学科,266000 [2]河北医科大学第三医院创伤急救中心,河北省骨科研究所,河北省骨科生物力学重点实验室,石家庄050051

出　　处：《中华骨科杂志》2021年第24期1803-1812,共10页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金(82102558,31872310);山东省自然科学基金青年基金(ZR2020QH117);中国博士后科学基金(2021M691690)。

摘　　要：目的探讨小神经胶质细胞去除联合骨髓间质干细胞(bone marrow mesenchymal stem cells,BMSCs)移植修复脊髓损伤(spinal cord injury,SCI)的效果。方法培养表达绿色荧光蛋白(green fluorescent protein,GFP)的BMSCs并进行干细胞鉴定,体外检测GFP-BMSCs分泌营养因子情况,与背根神经节(dorsal root ganglions,DRGs)共培养,观察其促进轴突生长效果;应用集落刺激因子1受体(colony stimulating factor 1 receptor,CSF1R)抑制剂PLX3397去除小鼠脊髓中小神经胶质细胞,并对脊髓进行钳夹损伤(Crush损伤),成功建立去除小神经胶质细胞小鼠SCI模型,将GFP-BMSCs移植到去除小神经胶质细胞小鼠SCI区域。将小鼠随机分为单纯饲料组和PLX3397组。于术后7、30、60 d分别取材,进行HE染色、免疫荧光染色对比观察移植GFP-BMSCs存活情况及神经组织修复情况;应用Basso mouse scale(BMS)评分来评价小鼠运动功能恢复情况。结果体外GFP-BMSCs能够分泌大量的神经营养因子,其与DRGs共培养,促进了DRGs神经轴突的生长。去除小神经胶质细胞明显提高SCI区域移植GFP-BMSCs存活,与单纯GFP-BMSCs移植相比,两者联合作用能够在一定程度上减少损伤区域面积,但差异无统计学意义(t=2.141,P=0.065)。免疫荧光染色显示单纯移植GFP-BMSCs或去除小神经胶质细胞后移植GFP-BMSCs均未能使皮质脊髓束(corticospinal tract,CST)轴突跨过损伤中心到达脊髓远端,在损伤后1、7、14、21、28 d行BMS评分,单纯饲料组分别为(1.20±0.45)、(3.20±0.45)、(3.80±0.45)、(4.20±0.45)、(4.60±0.55)分,PLX3397组分别为(0.60±0.55)、(3.00±0.71)、(3.80±0.84)、(4.20±0.84)、(4.40±0.89)分,两组各个时间点BMS评分差异无统计学意义(均P>0.05)。结论去除小神经胶质细胞提高了SCI区域移植GFP-BMSCs存活,但是未能促进CST轴突再生及脊髓损伤小鼠运动功能恢复,可能需要联合促轴突再生策略以提高SCI后神经功能恢复。Objective:To study the effect of microglia depletion combined with bone marrow mesenchymal stem cells(BMSC)transplantation for spinal cord injury(SCI)repair.Methods:GFP-BMSCs were cultured,identified and detected for expression levels of growth factors.The effects of BMSCs ondorsal root ganglion(DRG)axon outgrowth were observed by the co-culture of BMSCs with DRGs.Mice were depleted of microglia by administrating the colony stimulating factor 1 receptor(CSF1R)inhibitor PLX3397.The spinal cords of these microglia-depleted mice were subjected to crush injury.BMSCs were transplanted into SCI area after microglia depletion.Mice were randomly divided into control group(SCI+BMSCs)and experimental group(PLX3397+SCI+BMSCs).Mice were sacrificed at corresponding time points after transplantation for observing the survival of transplanted BMSCs and the repair of spinal cord.BMS score was used for evaluation of motor function recovery.Results:BMSCs secreted a large number of neurotrophic factors and promoted the growth of DRG axons when co-cultured with DRGs.Depletion of microglia significantly improved the survival of transplanted BMSCs.Compared with BMSCs transplantation alone,the combined treatments slightly but non-significantly reduced the area of the lesion(t=2.141,P=0.065).Immunofluorescence staining showed that both BMSC transplantation alone and the combined treatments did not cause the corticospinalaxons across the lesion and into distal spinal cord.BMS scores were(1.20±0.45),(3.20±0.45),(3.80±0.45),(4.20±0.45),and(4.60±0.55)points in control group at 1,7,14,21 and 28 d after injury.The experimental groups were(0.60±0.55),(3.00±0.71),(3.80±0.84),(4.20±0.84),and(4.40±0.89)points,respectively.Conclusion:Depletion of microglia improves the survival of transplanted cells,depletion of microglia combined with BMSC transplantation did not result in a significant reduction in lesion area.At the same time,the damaged CST axons were notregenerated.Thus,combining cell transplantation with axon-promoting strategy may

关 键 词：小神经胶质细胞 间质干细胞 脊髓损伤 动物实验 

分 类 号：R651.2[医药卫生—外科学]