大鼠骨髓基质细胞移植治疗急性脊髓损伤的实验研究

Experimentation of Acute Injured Spinal Cord by transplanting the bone marrow stromal cells

目的:本实验在大鼠脊髓损伤后经原位移植骨髓基质细胞(bone marrow stroma cell-s,BMSCs),证明植入的BMSCs在损伤的脊髓内能分化为神经元样细胞,并通过对远期运动能力测试,探讨移植后神经功能障碍恢复的情况。方法:①采用Alien重物坠落法制作改良型大鼠Alien’s脊髓损伤模型(重量10g,高度30mm),苏木精-伊红(Haematoxyli-n/eosin,HE)染色鉴定脊髓损伤情况。②采用体外骨髓贴壁法培养大鼠BMSCs,进行细胞染色观察细胞形态,检测CD44从而对骨髓基质细胞进行鉴定。细胞进行三次培养传代后用携带绿色荧光蛋白(Green Fluorescent Protein,GFP)基因的慢病毒转染。③传代细胞移植及移植后细胞存活:将GFP标记的BMSCs原位(损伤区)植入损伤模型,分别对2w、4w、6w后处死大鼠行脊髓组织切片,免疫荧光显微镜下观察表达GFP的细胞。④随机入组的各组大鼠行改良Rivlin斜板实验和脊髓运动功能BBB评分法,比较单纯损伤组、移植后2w组、移植后4w组及移植后6w组大鼠的运动能力的改变。结果:①采用Alien’s重物坠落法造模,制造改良型大鼠Alien’s脊髓损伤模型,效果可靠、稳定。②BMSCs经过3次传代后增殖能力已趋于稳定,细胞在体外培养、扩增后,细胞形态改变为胞体呈锥形,突起交织成网,故可作为脊髓损伤后细胞移植的来源;染色观察细胞形态,检测CD44呈阳性。③由携带GFP基因的慢病毒转染BMSCs移植入脊髓损伤模型后,在6w内可见到细胞表达GFP。④通过改良Rivlin斜板实验和脊髓运动功能BBB评分法对单纯损伤组大鼠测试结果均明显差于移植对照组,差异具有统计学意义(P<0.05);移植后2w、4w及6w组均能改善远期运动功能,以6w后神经功能障碍恢复最好,差异具有统计学意义(P<0.05)。结论:BMSCs移植能促进SCI后神经功能障碍的恢复,其机制可能与BMSCs分化为神经元和神经胶质细胞及移植诱导后的BMSCs可与周围的神经细胞相互作用,产生某些细胞因子有关。

Objective ：The experimental spinal cord injury in rats after orthotopic transplantation of bone marrow stromal cells BMSCs,That bone marrow stromal cells implanted in the spinal cord injury can be differentiated into neuron-like cells,And through the test of long-term exercise capacity.Study on the recovery of neural function after transplantation.Methods： ①Alien＇s free-falling （weight lOg, heigbt 30mm ）method was used to establish Spinal cord injury model of rats was improved, and then to determine the degree of injury by hematoxylin-feosin staining. ② We used adherent whole bone marrow method to separate and culture bone marrow stroma cells in vitro. Observe cell form with swiss dyeing, the special mark antigen of CD44 was detected by immunocytochemistry. The passage cells were tested and prepare to label the GFP by lentiviral veetors. GFP was examined under fluorescent microscope and fluorescence-activated cell sorter （FACS）. ③Cells transplantation and survival test; Transplanted the GFP-BMSCs for SCI rats in situ injury, and after transplantations 2,4,6 weeks respectively the animals were sacrificed and the pathological sections underwent fluoroscopy process to detecte the transplanted labeled cells at the site of injured area. ④The rats ran- domly of each group underwent Oblique Rivlin＇s board test and BBB spinal motor function score, Compared the differenees of simple injury group, 2w transplantation group, 4w transplantation group and 6w transplantation group.Results：①Alien＇s free-falling （weight 10g, height 30mm）method was used to establish SCI moderately models in stabilize and reliability.②Pass three generations of cell proliferation of bone marrow stromal cells has stabilized, bone marrow stromal cells in vitro, amplified, cell morphology, cell takes the shape of cone, protruding in- terwoven into a network, can be treated as an ideal cell source of cell transplantation ; Switzerland staining of eell morphology, CD44 was de- tected by immunocytochemistry. ③By the lentivirus carrying GFP gene-transfected bone marrow stromal cells transplanted into mice can still be seen behind the 6w cells expressing GFP. ④Oblique Rivlin＇s board test and BBB spinal motor function score, were found the simple injury group are significantly worse than the transplantation group （P〈O.05）; Two weeks after transplantation, but can improve the long-term results of behavior ,and afier 6W Nervous system function is the best recovery. （P〈0.05）Conclusion：BMSCs transplantation could pronmte the recovery of the neurological dysfunction after SCI, the mechanism maybe related with BMSCs can differentiation into NSE and GFAP,and transplantation of bone marrow stromal cells after peripheral nerve cells and interaction related with some cytokines.