转染胶质细胞源性神经营养因子基因的骨髓间充质干细胞对大鼠脊髓损伤后神经轴突再生的影响

Effect of GDNF gene transfection of BMSC on axon regeneration after spinal cord injury

目的:探讨转染胶质细胞源性神经营养因子(GDNF)基因的大鼠骨髓间充质干细胞(BMSCs)移植对大鼠脊髓损伤后神经轴突再生的影响。方法:取SD雌性大鼠的双侧股骨,冲洗髓腔分离培养得到的BMSCs,通过荧光素标记技术检测细胞表面标志物CD29、CD90、CD34、CD45,以确认是否得到BMSCs。构建GDNF过表达慢病毒用以感染BMSCs,加入感染分数分别为10、50、80、100、150、200的重组慢病毒悬液。在荧光显微镜下观察绿色荧光蛋白(GFP)的表达情况以表达细胞数量,评价转染效果,Western blot检测转染后GDNF在BMSCs中的表达,以四唑蓝比色法(MTT)法检测转染GDNF基因的BMSCs的细胞活力。将50只SD大鼠通过Allen′s打击法制备脊髓打击伤模型,造模成功后随机分为三组:A组(20只)为单纯BMSCs组,使用微量注射器移植未转染的BMSCs;B组(20只)为BMSCs转染组,使用微量注射器移植转染GDNF基因的BMSCs;C组(10只)为模型对照组,造模后脊髓内注射DMEM培养基。于造模后7d、14d、28d对大鼠进行BBB运动功能评分。各组均于造模后7d、14d、28d各取5只麻醉处死灌注取材后行HE染色并镜下观察。A、B两组采用免疫组织化学染色观察GFAP、NSE、NF-200在脊髓内的表达,以评估神经轴突生长情况。结果:经分离培养得到的细胞呈长纺锤状,以类似成纤维细胞样集落生长。流式细胞术测定结果示所培养的细胞阳性高度表达CD29、CD90,未表达CD34、CD45,表明分离培养所得细胞为BMSCs。当MOI=100时,转染12h后,BMSCs显著表达GFP,提示转染成功。Western blot结果示GDFP表达情况良好。MTT法显示转染7d后BMSCs组细胞活力明显高于未转染BMSCs组。术后7d时,三组BBB评分无显著差异。术后14d,B组的BBB评分5.80±0.19分与A组3.60±0.18分及C组3.10±0.14分相比均有显著性差异(P<0.05)。术后28d,B组的BBB评分11.90±0.28分与A组8.30±0.29分及C组5.70±0.18分相比有显著性差异(P<0.05)。GFAP、NSE、免疫组化染色光密度统计结果均显示A组和B组有显著性差异,B组明显优于A组,具有统计学意义(P<0.05)。NF-200神经纤维长度统计结果提示B组为89.98±28.31μm,A组为23.64±13.45μm,两组之间存在显著差异(P<0.05)。结论:GDNF转染BMSCs具有较高的细胞活性,能够提升BMSCs促进神经轴突再生的能力。

Objectives:To explore the effect of bone marrow derived mesenchymal stem cells(BMSCs)transfected with glial cell line-derived neurotrophic factor(GDNF)on axonal regeneration after spinal cord injury in rats.Methods:Bilateral femurs of SD female rats were collected,and the medullary cavity was rinsed to isolate and culture BMSCs.The cell surface markers CD29,CD90,CD34 and CD45 were detected by using fluoresce in labeling technology to confirm the isolation of mesenchymal stem cells.To construct GDNF overexpressed lentiviruses to infect BMSCs,the recombinant lentiviral suspensions with infection fractions of 10,50,80,100,150 and 200 were added.Green fluorescent protein(GFP)expression was observed under fluorescent microscope to evaluate transfection cells expressing effect,GDNF expression in BMSCs after transfection was detected by Western blot.BMSCs transfected with GDNF gene were detected by MTT assay.Fifty SD rats were used to establish spinal cord blow injury model by using Allen′s blow method.After successful modeling,they were randomly divided into three groups:group A was pure BMSCs group,and BMSCs without transfection were transplanted with microsyringe;group B was the BMSCs transfection group,and BMSCs transfected with GDNF gene were transplanted with microsyringe;group C was the model control group,and DMEM medium was injected into spinal cord after modeling.BBB motor function score was performed on 7,14 and 28 days after modeling.HE staining and microscopic observation were performed after anesthesia and perfusion.The expressions of GFAP,NSE and NF-200 in spinal cord were observed by immunohistochemical staining to evaluate the growth of axons.Results:BMSCs were isolated and cultured in a long spindle shape and grew in a fibroblast-like colony.The results of flow cytometry showed that CD29 and CD90 were highly expressed in BMSCs,while CD34 and CD45 were not expressed,indicating that the cells obtained in this study were mesenchymal stem cells.When MOI=100,BMSCs significantly expressed GFP at 12 hours after transfection,indicating successful transfection.Western blot results showed that GDFP was well expressed.MTT assay showed that the cell viability of BMSCs group was significantly higher than that of untransfected BMSCs group at 7 days after transfection.At 7 days after surgery,there was no significant difference in BBB score among the three groups.At 14 days after surgery,the BBB score of 5.80±0.19 in group B was significantly different from 3.60±0.18 in group A and 3.10±0.14 in group C.At 28 days after surgery,the BBB score of 11.90±0.28 in group B was significantly different from 8.30±0.29 in group A and 5.78±0.18 in group C(P<0.05).The optical density statistics of GFAP,NSE and immunohistochemical staining showed significant differences between group A and group B,and group B was significantly better than group A,with statistical significance(P<0.05).The statistical results of NF-200 nerve fiber length suggested 89.98±28.31μm in group B and 23.64±13.45μm in group A,with significant differences between these two groups(P<0.05).Conclusions:GDNF can successfully transfect BMSCs.The BMSCs have high cell viability after transfection.Transfection of GDNF gene can enhance the ability of BMSCs to promote axonal regeneration.