BMSCs联合人发角蛋白移植治疗陈旧性脊髓损伤效果

TRANSPLANTATION OF BONE MESENCHYMAL STEM CELLS AND HUMAN HAIR KERATINS IN THE TREATMENT OF OLD SPINAL CORD INJURY

目的研究骨髓间充质干细胞（BMSCs）联合人发角蛋白支架复合体移植治疗陈旧性脊髓损伤可行性。方法体外采用全骨髓贴壁筛选法原代培养大鼠BMSCs,移植前应用5溴-2脱氧尿嘧啶（BrdU）标记。采用改良Allen打击法制备大鼠陈旧性脊髓损伤模型,随机分为对照组、单纯BMSCs移植组、BMSCs联合人发角蛋白移植组,每组10只,采用立体定位移植方法分别于各组脊髓损伤部位植入细胞培养基、BMSCs悬液、人发角蛋白＋BMSCs细胞悬液。移植后第1、2、4、8周应用动物后肢运动功能评分法（BBB）对大鼠后肢运动功能进行评分;移植后第8周处死大鼠,取T9~T11节段脊髓组织制备切片,采用苏木精-伊红（HE）染色法观察脊髓组织大体形态,免疫组化方法检测BrdU阳性细胞的存活及移植细胞的分化情况。结果单纯BMSCs移植组、BMSCs联合人发角蛋白移植组各时段大鼠BBB评分均优于对照组,BMSCs联合人发角蛋白移植组优于单纯BMSCs移植组,差异均有显著性（F=25.64,q=11.86~20.79,P〈0.05）。HE染色观察见3组脊髓均严重受损,灰白质界限模糊,均有空洞和坏死瘢痕组织,BMSCs联合人发角蛋白移植组空洞较其余两组少。BMSCs联合人发角蛋白移植组移植部位以及头尾端距离移植中心约1 cm处均可见BrdU染色阳性细胞及BrdU＋神经元特异性烯醇化酶（NSE）阳性细胞。结论 BMSCs联合人发角蛋白移植后移植细胞可以存活并分化为神经元样和神经胶质样细胞,有效促进陈旧性脊髓损伤大鼠模型后肢功能的恢复,其效果较单纯干细胞移植显著。

ObjectiveTo study the feasibility of transplantation of bone marrow mesenchymal stem cells（BMMSCs） and human hair keratin（HHK） scaffold complex in the treatment of old spinal cord injury.MethodsPrimary culture of BMMSCs in a rat was done in vitro.Before transplantation,the BMMSCs were labeled by 5 bromo-2 deoxyuridine（BrdU）.Models of old spinal cord injury in rats were created by modified Allen method,which were divided into group 1（control group）,group 2（pure BMMSC-transplantation group）,and group 3（BMMSC＋HHK-transplantation group）,with 10 rats in each group.By employing stereotactic transplantation technique,cell culture medium,BMMSC suspensions,and HHK＋BMSC suspension were separately implanted into the injured site of spinal cord.At weeks 1,2,4 and 8 after transplantation,the motor function of hindlimbs of the rats was score by BBB method;the rats were sacrificed at week 8 after the transplantation,T9-T11 segments of spinal cord were taken for tissue sections,macropathology of the cord was observed with HE stain,the survival of positive BrdU cells,and differentiation of the implanted cells were detected immunohistochemically.ResultsBBB scores of groups 2 and 3 were higher than that of the group 1（P0.05）;the score of group 3 was higher than that of the group 2,the differences among them were significant（F=25.64,q=11.86-20.79,P0.05）.With HE stain,severe damages of the spinal cord were observed,the line of gray matter was blurred,cavities and necrotic scar tissues could be seen,but the cavities in group 3 were smaller than that of the other two groups.In group 3,positive BrdU-stain cells and BrdU-positive neuron-specific enolase cells could be seen.ConclusionAfter transplantation of BMMSC＋HHK,the transplanted cells can survive and differentiate into neurons-like and neuroglia-like cells,which effectively promote recovery of hindlimb function in rats with old spinal cord injury,the efficacy is better than pure stem cell transplantation.