静脉移植骨髓间充质干细胞治疗大鼠脊髓损伤

目的 研究静脉移植骨髓间充质干细胞(bone martow stromal cells,BMSCs)对大鼠脊髓损伤后功能恢复的影响.方法 对10只同种异体SD大鼠的BMSCs进行体外分离、培养、扩增、纯化后用5-溴脱氧尿嘧啶核苷(5-bromodeoxyuridine,BrdU)进行标记;重物打击致瘫痪造模法制成完全性截瘫的同种大鼠脊髓损伤动物模型,制模后7 d完全随机分为A、B、C三组:A组22只,作为BMSCs移植组,经鼠尾静脉注射2×106/ml的BMSCs细胞悬液1 ml;B组22只,作为对照组,注射1 ml培养液;C组22只,作为空白手术对照组.注射后2,3,6周,免疫组织化学检测BMSCs在损伤段脊髓的存活、分布、分化并计数,观察损伤段脊髓内生长相关蛋白-43(growth associated protein-43,GAP-43)、神经丝蛋白200(neurofilament 200,NF200)和巢蛋白的表达,注射后1,2,3,4,5,6周Basso-Beattie-Bresnaban(BBB)评分评估各组后肢运动功能.结果 移植后2,3,6周BrdU阳性的BMSCs主要分布于损伤段及邻近节段脊髓内.计数发现,移植后2周BrdU阳性的BMSCs约占移植细胞数的4.9%,3周占4.4%,6周占2.9%;移植后2周,BMSCs形态大多变为圆形或椭圆形,部分阳性标记的细胞长出神经元样突起,约12.6%表达胶质纤维酸性蛋白(glial fribrillary acidic protein,GAFP),约5.4%表达神经元特异性核蛋白(neuronal nuclear antigen,NeuN);移植后2,3,6周,A组GAP-43、NF200、巢蛋白的表达明显高于B、C组(P<0.05);移植后3周开始,A组的BBB评分在各观察时相点明显高于B、C组(P<0.05).结论 通过静脉注射的BMSCs能向脊髓损伤灶内迁徙、存活,表现出对损伤灶的趋化性,并向神经元和星形胶质细胞分化,上调GAP-43、NF200和巢蛋白的表达,改善神经功能,可用于脊髓损伤的细胞移植治疗.

Treatment of spinal cord injury with intravenous administration of bone marrow stromal cells in rats

Objective To observe the effect of intravenous administration of bone marrow stromal cells (BMSCs) on the functional recovery after spinal cord injury in rats.Methods BMSCs harvested from 10 donor adult Sprague-Dawley rats were isolated,cultured,purified,amplified and labeled with bromodeoxyuridine (BrdU ).A total of 66 adult Sprague-Dawley rats were subjected to weight-drop impact causing complete paraplegia and randomly divided into three groups seven days after injury.Group A (22 rats) was treated with 2 × 106 BMSCs cultured in 1 ml phosphate-buffered saline by tail vein,Group B (22 rats) was treated with 1 ml Dulbecco' s modified Eagle medium and Group C (22 rats) was set as blank control group.The distribution and differentiation of donor cells in spinal cord were observed in recipient rats by using immunohistochemical staining and expressions of growth associated protein-43 ( GAP-43),neurofilament 200 (NF200) and nestin in the contused spinal cord measured at 2,3 and 6 weeks respectively after injection.The motor function of three groups was evaluated by the Basso-Beattie-Bresnahan ( BBB) scores at 1,2,3,4,5 and 6 weeks respectively after injection.Results BrdU-reactive cells were mainly distributed through the contused and near regions of spinal cord at 2,3 and 6 weeks after injection.The survived cells in contused spinal cord accounted for 4.9% ,4.4% and 2.6% of the total injection cells at 2,3 and 6 weeks respectively after injection.Two weeks after injection,BMSCs were mainly round or ellipse in shape,12.6% BrdU-reactive cells expressed glial fibrillary acidic protein (GFAP) and 5.4% expressed the neuronal nuclear antigen (NeuN).The expressions of GAP-43,NF200 and nestin were detected at 2,3 and 6 weeks after injection,with significant higher level in Group A than that in Groups B and C (P<0.05).BBB score in Group A was significantly higher than that in Groups B and C 3-6 weeks after transplantation (P<0.05).Conclusions With intravenous administration after spinal cord injury,BMSCs migrate and survive into contused spinal cord,exhibit site-dependent differentiation,up-regulate the expressions of GAP-43,NF200 and nestin,ameliorate nerve function and can be used for treatment of spinal cord injury.