骨髓间充质干细胞经蛛网膜下腔移植治疗大鼠脊髓损伤及其对T细胞亚群影响的研究

目的将异体骨髓间充质干细胞（marrow mesenchymal stem cells,MSCs）移植至脊髓损伤（spinalcord injury,SCI）大鼠蛛网膜下腔,探讨其治疗效果以及对T细胞亚群的影响。方法取6周龄SD大鼠10只,采用密度梯度离心法进行细胞分离及培养,传至第6代后收集细胞。雌性8周龄SD大鼠60只,采用重物打击法制备SCI模型,从BBB后肢功能评分为0的存活大鼠随机取40只分为两组,每组20只。实验组（A组）：经蛛网膜下腔注入经BrdU标记的细胞浓度为2×106/ml的MSCs细胞悬液1ml;对照组（B组）：注入等量L-DMEM。另取20只正常雌性8周龄SD大鼠作为空白组（C组）。于第1、2和3周行BBB后肢功能评分,并采外周血经流式细胞仪检测T细胞亚群;第3周时,A、B组大鼠取损伤处脊髓,行组织切片观察。结果A组BBB后肢功能评分第3周优于B组,但仍低于C组,差异均有统计学意义（P〈0.05）;CD4＋T细胞在第1、2和3周均较B、C组少,CD8＋T细胞在第2、3周较B、C组少,CD4＋/CD8＋T细胞比值在第1周较B、C组少,上述差异均有统计学意义（P〈0.05）;但CD4＋/CD8＋T细胞比值在第2、3周与B、C组比较差异无统计学意义（P〉0.05）。组织学观察,A组第3周脊髓损伤处空泡缩小,有组织长入,较B组有轻微改善,但未明确见到BrdU阳性标记的MSCs。结论MSCs经蛛网膜下腔移植对SCI有一定治疗作用,且在一定时间内不引起机体排斥反应,在第1周对T细胞亚群的抑制更为明显,为临床利用MSCs治疗SCI及其他疾病提供了免疫学实验依据。

EFFECT OF TRANSPLANTING MARROW MESENCHYMAL STEM CELLS VIA SUBARACHNOID SPACE ON SPINAL CORD INJURY AND T CELL SUBPOPULATION IN RATS

OBJECTIVE: To investigate the therapeutic effects of transplanting allogeneic marrow mesenchymal stem cells (MSCs) via subarachnoid space on spinal cord injury(SCI) and the T cell subpopulation. METHODS: Density gradient centrifugation was used to isolate and expand MSCs from bone marrow of 10 six-week-old SD rats. The SCI model was produced by weight-beating from 60 eight-week-old female SD rats. Forty survival SCI rats, which BBB scores were zero, were divided randomly into 2 groups:experimental group (group A) and control group (group B). In addition, 20 normal eight-week-old SI) rats were used as blank group (group C). In group A, 1 ml cells suspention containing MSCs (the 6th generation, 2 x 10(6)/ml) was injected via subarachnoid space. In group B, equal volume of L-DMEM was injected in the same way. The BBB score was obtained after 1st, 2nd and 3rd weeks of injection. At the same time,T cell subpopulation was detected by flow cytometry. RESULTS: The BBB score in group A was better than that in group B, but fewer than that in group C in the 3rd week. CD4+ T cells in group A were less than those in groups B and C in the 1st, 2nd, and 3rd weeks. CD8+ T cells in group A were less than those in groups B and C in the 2nd and 3rd weeks. The ratio of CD4+/CD8+ T cells in group A was less than those in groups B and C in the 1st week. Above differences showed statistically significant difference (P < 0.05). However, there were no statistically significant differences in the ratio of CD4+/CD8+T cells between group A and groups B, C in the 2nd and 3rd weeks (P>0.05). CONCLUSION: The above results suggest that allogeneic MSCs transplantation via subarachnoid space is beneficial to SCI to some extend, do not result in rejection in vivo. Furthermore, it can lead to immunosuppression in short time. So, it provides clues to apply MSCs to treat SCI and other diseases.