人脐血干细胞移植修复大鼠脊髓损伤的功能评价

Functional evaluation of human umbilical cord blood mesenchymal stem cell transplantation for spinal cord injury in rats

背景：在脊髓损伤模型的研究中，BBB评分系统和斜板试验均与脊髓损伤程度高度相关。目的：建立改进大鼠脊髓全横断模型，对比观察BBB评分和斜板试验在人脐血干细胞移植术后功能评价中的作用。设计、时间及地点：观察对比实验，于2007-04／2008—07在广东省组织构建与检测重点实验室完成。材料：脐血来自健康足月产妇。实验动物为SPF级健康成年雌性SD大鼠50只，随机分为假手术对照组（n=10）、磷酸盐注射组（n=20）、脐血干细胞移植组（n=20）。方法：分离和体外培养人脐血干细胞。在显微镜下纵行剖开SD大鼠硬脊膜囊后，于硬脊膜内、蛛网膜外将超薄刀片刀尖直抵椎体骨质，将蛛网膜、脊髓、两侧壁和腹侧的硬脊膜作为一个整体完全划断，制作大鼠脊髓全横断模型，假手术对照组仅打开椎板，脐血干细胞移植组大鼠于脊髓两断端分别显微注射人脐血干细胞悬液6×10^9～7×10^9L^-1，磷酸盐注射组注射等量磷酸盐缓冲液。主要观察指标：术后12周内每2周分别应用BBB评分和斜板试验进行1次后肢运动功能评价。结果：假手术对照组后肢运动功能于手术前后无显著变化。从术后第2周开始，磷酸盐注射组和脐血干细胞移植组逐渐恢复部分后肢运动功能，两组的BBB评分和斜板试验检测结果均表现出一致的增长趋势，呈线性正相关关系。从术后4周开始，BBB评分〈13分的磷酸盐注射组和BBB评分≥13分的脐血干细胞移植组大鼠，在斜板试验中差异非常显著（P〈0．01）。到术后第12周时，磷酸盐注射组和脐血干细胞移植组斜板试验角度和恢复程度分别为34．25。和52．94％、53．85。和83．23％，并且脐血干细胞移植组高于磷酸盐注射组（P〈0．01），而同期磷酸盐注射组和脐血干细胞移植组对应的BBB评分和恢复程度仅分别为8．15和38.81％、13．90和66．19％，可见两组斜板试验角度恢复程度亦明显高于其自身对应的BBB评分结果。结论：与BBB评分系统相比，斜板试验能更敏感地反映大鼠爪的功能恢复，但不能特异性地反映爪的精细运动。而作为主观评分体系的BBB评分系统，虽然特异性很高，但容易受主观因素的影响，敏感性较低。因此，联合应用BBB评分和斜板试验能更好地反映脊髓神经功能恢复情况。

BACKGROUND： Basso, beattie and bresnahan （BBB） score and tilt table test are associated with the height of spinal cord injury degree in study of spinal cord models. OBJECTIVE： To establish modified complete spinal cord transected rat models, and to observe effects of BBB score and tilt table test on function following human umbilical cord blood mesenchymal stem cell transplantation. DESIGN, TIME AND SETTING： The observation control experiments were performed at the Key Laboratory of Tissue Construction and Detection of Guangdong Province from April 2007 to July 2008. MATERIALS： Cord blood was collected from healthy puerperants after full-term delivery. Fifty SPF healthy adult female Sprague Dawley rats were randomly divided into group A （sham operation） （n=10）, group B （spinal cord transection and phosphate buffered saline, PBS injection） （n=20）, group C （spinal cord transection and hMSCs transplantation） （n=20）. METHODS： Human umbilical cord blood stem cells were harvested and cultured in vitro. After incising rat dural sac, arachnoid membrane, spinal cord, bilateral wall and ventral dura mater of spinal cord were completely incised in the dural mate of spinal cord to establish complete spinal cord transected rat models. Vertebral plates were only opened in the group A. Rats in the group C were injected with human umbilical cord blood mesenchymal stem cells （6× 10^9 -7×10^9 L^-1） at two broken ends of fractured spinal cord. Phosphate buffer saline was injected into rats in the group B. MAIN OUTCOME MEASURES： At 12 weeks after surgery, a behavioral testing was performed every 2 weeks upon each hindlimb for all animals by using the BBB scoring system and tilt table test. RESULTS： No significant difference in hindlimb motor function was detected in the group A. From the second weeks after surgery, motor function of hindlimbs was gradually recovered in the groups B and C. BBB scoring system and tilt table test showed a consistent increasing tendency in a positive correlation. From the fourth week after surgery, significant difference in tilt table test was detected between rats with BBB score 〈13 points in the group B and rats with BBB score ≥13 points in the group C （P 〈 0.01）. At the twelfth week, tilt table angle and recovery degree were respectively 34.25° and 52.94%, 53.85° and 83.23% in the groups B and D It was higher in the group C than in the group B （P 〈 0.01）. Simultaneously, BBB score and recovery degree were respectively 8.15 and 38.81%, 13.90 and 66.19% in the groups B and C. It is indicated that the recovery of tilt table test was significantly higher compared to the recovery of BBB score in the groups B and C. CONCLUSION： Compared to the BBB score system, tilt table test can reflect the function of rat claw, but cannot reflect the precise movement of claw. The positive BBB score system has a high specificity, but it is easily affected by subjective factors, with a low sensitivity. Thus, the combining application of the BBB score system and the tilt test is superior to reflect the functional recovery of the injured spinal cord.