甲钴胺联合BMSCs移植治疗大鼠脊髓损伤的基础研究

现代医药卫生2019 年#月第35 卷第2 期JModMedHealthJanuaiy 2019，V〇1.35，N〇.2*183 *•论著•甲钴胺联合BM SCs移植治疗大鼠脊髓损伤的基础研究！彭立军，刘骞，羊明智，谢中(南华大学附属第一医院脊柱外科，湖南衡阳421001)[摘要]目的在前期成功利用曱钴胺体外定向诱导大鼠骨髓间充质干细胞（B M SC s)向神经元样细胞分化的基础上，进一步探讨大鼠脊髓损伤模型中曱钴胺局部注射诱导移植后的大鼠B M SC s向神经元样细胞分化的可行性；并观察对脊髓损伤的修复作用％方法将％0只S D大鼠制备T10节段脊髓横断模型后随机抽样分为A组（模型对照组）、B组（曱钴胺治疗组）、C组(B M SC s移植组）和V组（曱钴胺联合BM SCs移植组），每组10只％于术后1、7、14、21d采用肢体功能评判标准（BBB评分）、免疫荧光显微镜观测绿色荧光蛋白标记的B M SC s的存活及分布情况，苏木精-伊红染色观察脊髓白质、灰质及细胞间质损伤情况，尼氏（N issl)染色观察残存神经元状态、分布及再生情况％结果造模后％组大鼠均出现了严重的双下肢瘫痪，随着时间的推移，双下肢功能无明显恢复，4组大鼠BBB评分比较，差异均无统计学意义（P>0.05)%脊髓组织病理切片显示，A组大鼠脊髓受损严重，细胞结构紊乱，大量炎症细胞浸润，修复程度低且缓慢；B、C组大鼠脊髓受损程度均优于A组；D组大鼠可见组织受损较轻，且修复较快；免疫荧光显微镜下C、D组大鼠均可见带示踪标记的神经元样细胞表达，呈双染色表现，且D组大鼠荧光强度及密度较C组明显；在N issl染色切片中，D组大鼠尼氏小体再生数量最多％结论BM SCs移植和曱钴胺治疗对改善脊髓损伤大鼠双下肢运动功能不明显％BM SCs在脊髓组织微环境中能分化为神经元样细胞，曱钴胺能协同B M SC s向神经元样细胞分化％曱钴胺联合BM SCs移植可减轻大鼠脊髓损伤后的病理改变％[关键词]骨髓间充质干细胞+神经元样细胞+脊髓损伤+曱钴胺D O I：10. 3969". issn. 1009-5519. 2019. 02. 008 中图法分类号：C744 ; Q254文章编号：1009-5519(2019)02-0183-05 文献标识码：ABasic study on mecobalamin combined with BM S Cs transplantation for treating rat spinal cord injury*PEN G L iju n，L IU Q ian，YAN G M in g zh i，X IE Zhong{Department o f Spinal S u rg ery，First A f f ilia ted H osp ita l，University o f South China，H engyang，Hunan421001 , China) [Abstract] Objective To further research the feasibility of mecobalamin local injection for inducing BMSCs to differentiate into neuron-like cells in rat spinal cord injury model on the basis of successfully using mecobalamin for in vitro directionally inducing bone marrow mesenchymal stem cells (BM SCs) to differentiate into neuron cells in the previous stage , and to observe its repair effect on spinal cord injury. Methods Forty SD rats were made into the spinal cord transection model at T10 segment and then di­vided into the group A(model control group) , B (mecobalamin treatment group) , C (BMSCs transplantation group) and D (m eco­balamin ,BMSCs combined transplantation group) , 10 cases in each group. The lower-extremity f score) and immunofluorescence microscope were used to observe the survival and distribution situations of BMSCs labeled with the green fluorescence protein(GFP) on postoperative 1,7,14,21d；the injury situations of white matter , gray matter a lular interstitial were observed by the HE staining , t h e status；the status，distribution and regeneratio neuron were observed by theN issl staining. Results The paralysis of both lower extremities in all the rats of the four groups ap­peared after constructing the model , t h eir function had no obvious recovery with the time elapse , t h e BBB scores had no statistical difference among 4 g rou p s(P>0. 05). The pathological sections of the spinal tissue indicated that t group A was serious with the cell structure distortion , a large infiltration of inflammatory cells , t h e repair degree was low and slow；the injury degree of spinal cord in the group B and C were better than that in the group A；t h e group D showed that the ti s sue was damaged lighter and repaired faster；the expression of neuron-like cells with t race markers and d the immunofluorescence microscope in the group C and D , moreover the fluorescence intensity and density in the group D were more significant than those in the group C；the regenerated quantity of Nissl bodies in the group D was maximal in Nissl staining sec­tions. Conclusion The BMSCs transplantation and mecobalamin treatment are unobvious for improving the movement function of lower extremities in spinal cord injury rats. BMSCs can differentiate into neuron-like cells in m tissue , and mecobalamin can collaborate BMSCs to differentiate into neuron-like cells. The mecobalamin combined with BMSCs transplantation can alleviate the pathological changes of rat spinal cord injury.[Key words] Bone marrow mesenchymal stem cells；Neuron-like cells；Spinal cord injury；Mecobalamin脊髓损伤是中枢神经系统严重的损伤，常导致损 伤以下节段感觉、运动功能障碍。

早期通常采用手术基金项目：湖南省衡阳市科学技术发展项目资助课题（2016K J2)。

作者筒介：彭立军（1982 — ％硕士研究生，副主任医师，主要从事脊柱外科基础与临床研究。

治疗，对相应脊髓损伤节段进行减压，为神经功能的 恢复创造条件。

然而，手术只能阻止病变的进展和促 进功能的恢复，对损伤坏死的神经却无能为力，许多 截瘫患者神经功能不能得到有效的恢复，严重影响其 生活质量。

随着干细胞研究的不断深人，干细胞的高效自我 更新能力和向神经细胞的分化潜能为脊髓损伤的治 疗带来了新的希望。

由于骨髓间充质干细胞（BM-SCs)可以很方便地从成人体内获得，易于分离、培养，体外扩增迅速，能在体内外存活、迁移、分化[13]。

因此，在脊髓损伤的细胞移植方面具有良好前景。

既往 研究方法多数是将BMSCs直接移植到损伤脊髓上，尽管在组织及细胞学方面具有促进神经修复的作用，但在症状改善方面仍远远不够。

本研究对BMSCs进 行干预、移植，以期提高对脊髓损伤的疗效。

1材料与方法1.1材料与试剂健康清洁S D大鼠40只，雌雄不 限，体重（240±10)g，由南华大学动物实验中心提供，许可证号：SCXK(湘）2015，实验过程均在该中心操 作，并严格遵守《关于善待实验动物的指导性意见》的规定。