摘要
背景:CD34+细胞和许旺细胞在单独移植情况下均有促进脊髓修复的功能,且各自分泌和表达成纤维细胞生长因子受体及配体。目的:探讨CD34+细胞与许旺细胞联合移植对脊髓全横断损伤大鼠的后肢运动及感觉功能恢复的影响,并观察体内移植后细胞的存活、迁移、分化及表型变化。设计、时间及地点:随机对照动物实验,于2007-07/2008-09在昆明医学院神经科学研究所完成。材料:SPF级健康雌性SD大鼠60只,随机分为模型对照组、免疫对照组、CD34+细胞组、许旺细胞组、细胞联合组,12只/组。SPF级6～8周龄ICR小鼠2只,用于分离培养骨髓CD34+细胞。SPF级一两天龄ICR新生鼠30只,用于分离培养许旺细胞。方法:各组大鼠均建立脊髓全横断损伤模型。造模后2周,CD34+细胞组、许旺细胞组将体外培养并荧光标记好的对应细胞悬液经多点注射入横断瘢痕及瘢痕上下各一个脊髓节段,共6个注射位点,每点注射5μL,每只大鼠共移植60万个细胞量;细胞联合组移植方法相同,但CD34+细胞、许旺细胞注射量各60万个。移植后除模型对照组外,余4组均使用免疫抑制剂环孢素腹腔给药。主要观察指标:通过BBB评分判定大鼠后肢运动功能的恢复情况,测定热痛阈值和机械痛阈值的变化,形态学及组织学鉴定移植细胞的存活、迁移、分化、表型变化。结果:移植后12周内CD34+细胞组BBB评分最高(P<0.05),至移植后24周时细胞联合组BBB评分最高,恢复效果最佳,许旺细胞组次之,CD34+细胞组最差(P<0.05)。与CD34+细胞组比较,移植后12周细胞联合组热痛阈值明显降低(P<0.05),随移植时间延长,各组机械痛阈值均明显降低(P<0.05),但热痛阈值没有发现类似规律。CD34+细胞在有许旺细胞参加的情况下存活数量较多,在移植后12周尤为明显。移植后4周荧光细胞集中在针道附近,第12周荧光细胞迁移到瘢痕上下一两个节段,且较集中分布于脊髓灰质,白质中少见。与CD34+细胞组比较,移植后4,8,12周细胞联合组成熟神经元特异性标记物Neu-N阳性率、胶质纤维酸性蛋白阳性率均明显升高(P<0.05),成熟少突胶质细胞标记物APC阳性率明显下降(P<0.05);CD34+细胞组、细胞联合组早期少突胶质细胞标记物O4阳性率均明显下降(P<0.05)。移植后4,8,12周,许旺细胞胶质纤维酸性蛋白阳性表达率逐渐增高。结论:CD34+细胞联合许旺细胞移植对脊髓全横断大鼠后肢功能及感觉功能恢复效果优于单纯CD34+细胞移植,并得到了体内实验的证实。
BACKGROUND： CD34^＋ cell or Schwann cell transplantation can promote spinal cord repair, and secrete and expression fibroblast growth factor receptor and ligand. OBJECTIVE： To explore the engrafted effect of CD34^＋ cells and Schwann cells on the recovery of function in hind limbs of rats subjected to spinal cord transected injury and observe the migration, differentiation and survival of the transplanted cells. DESIGN, TIME AND SETTING： Randomized, controlled, animal experiment was performed at the Institute of Neuroscience, Kunming Medical College from July 2007 to September 2008. MATERIALS： Sixty SPF healthy female SD rats were randomly divided into model control, immunity control, CD34^＋ cells, Schwann cells and combination group （n=12）. In addition, 2 SPF ICR mouse aged 6-8 weeks were used to isolate and culture bone marrow-derived CD34^＋ cells and 30 ICR mouse aged 1-2 days were selected for isolation and culture of Schwann cells. METHODS： Complete spinal cord transected injury model was established in all group. Two weeks after spinal cord transected injury, the cells cultured in vitro were separately injected in the scar and one segment of the scar up and down in CD34^＋ cells and Schwann cells group through multi-point injection for a total of 6 injection points, with 5μL at each point and a total of 600000 cells for each rat. And using the same method, 600000 CD34^＋ cells and 600000 Schwann cells were injected into the combination group. Except model control group, all groups were intraperitoneally injected with ciclosporin. MAIN OUTCOME MEASURES： The locomotor functions of rats were detected by BBB score. The mechanical withdrawal threshold and the thermal withdrawal threshold were measured, followed by morphological and histological observation to know the survival, migration and differentiation of grafted cells. RESULTS： At 12 weeks, CD34^＋ cells group had the highest BBB scores （P 〈 0.05）; at 24 weeks, the combination group had the highest scores, followed by Schwann cells group and CD34^＋ cells group （P〈0.05）. Compared with CD34~ cells group, the thermal withdrawal threshold of combination group at 12 weeks was significantly decreased （P 〈 0.05）; with increasing time, the withdrawal threshold was gradually reduced in each group （P 〈 0.05）, but no similar changes were found in the thermal withdrawal threshold. CD34^＋ cells had a higher fraction survival in the co-transplantation condition, especially at the 12^th week. Four weeks after transplantation, fluorescence-marked cells were aggregated near the injection hole, migrated to 1-2 segments up or lower the scar, extensively distributing at gray substance of spinal cord, but rare in white matter. Compared with CD34^＋ cells group, Neu-N and glial fibrillary acidic protein （GFAP）-positive rate were significantly increased in combination group at postoperatively weeks 4, 8 and 12 （P 〈 0.05）, while APC was significantly decreased （P 〈0.05）; O4 was also reduced in CD34＋ cells and combination groups （P 〈 0.05） The expression of GFAP of Schwann cells was gradually increased at postoperatively weeks 4, 8 and 12. CONCLUSION： Co-transplantation of CD34^＋ cells and Schwann cells is more effective than CD34^＋ cells transplantation alone in improving the recovery of function in hind limbs of rats subjected to spinal cord transected injury.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第32期6211-6218,共8页
Journal of Clinical Rehabilitative Tissue Engineering Research
