课题背景：课题主要是与原上海第二医科大学发育生物学中心实验室合作的课题，同时也得到黑龙江省自然基金、省教育基金资助。前期的实验工作主要是胚胎干细胞移植修复脊髓损伤的实验研究，论文已发表，主要研究不同种类的干细胞修复脊髓损伤疗效分析及机制探讨。

相关链接：脊髓损伤治疗领域研究热点和重点主要是证实干细胞移植治疗脊髓损伤的有效性，以及探讨这些干细胞是否分化为神经细胞，是否提高脊髓的功能。目前在脊髓损伤亟待解决的问题主要明确脊髓损伤的机制，即是有哪些基因和神经生长因子及各种蛋白在起作用及对干细胞对这些基因是否有调控作用。

应用要点：现已有很多有关干细胞治疗脊髓损伤的相关文章报道，但对骨髓单个核细胞治疗脊髓损伤报道还很少，作者选择此切入点进行研究，实验证实其移植的单个核细胞移植后是有效的，同时也发现骨髓单个核细胞能在受损的脊髓内存活且能分化为血管的标志，这个结果提示在脊髓急性损伤的过程中，骨髓单个核细胞能分化为新生的血管，这对急性脊髓损伤的治疗是非常重要的。

摘要
目的：研究已证实，各种干细胞移植治疗损伤脊髓，都可以一定程度的恢复脊髓中枢神经的功能。但对骨髓单个核细胞移植治疗损伤脊髓以及长期效果的研究少见。利用大鼠抽取新鲜分离的骨髓单个核细胞,将其移植入脊髓损伤大鼠模型,评价脊髓功能恢复情况、神经再生和新生血管形成及长期预后效果。
方法：实验于2005-10/2006-04在上海第二医科大学发育生物学研究中心实验室完成。实验室级别：生物安全一级。①实验材料：8周龄SD雌性大鼠，体质量200~220 g，清洁级，由中国科学院实验动物中心提供，实验过程中对动物处置符合动物伦理学标准。②实验方法：从大鼠胫骨及股骨采集骨髓细胞,密度梯度离心法分离骨髓单个核细胞备用。制作大鼠脊髓损伤动物模型，将造模成功22只大鼠随机分成2组:模型+细胞组（n =11）：脊髓完全横断T9~10后，椎管内注射骨髓单个核细胞；模型+DMEM组（n =11）：脊髓完全横断T9~10后在损伤邻近区注射DMEM。假手术组（n =9）：仅剪除T9-10棘突和椎板后，不损伤脊髓，逐层缝合。③实验评估：采用原位杂交和免疫组织化学技术检测移植细胞在宿主脊髓内的存活情况，BBB评分评估大鼠脊髓神经功能恢复情况。
结果：①假手术组在术后各时间点观察中评分无明显差异，属评分正常。模型+DMEM组评分为0分，其脊髓功能无明显恢复。模型+细胞组在2，4，6，8周脊髓功能处于逐渐恢复的过程。与模型+DMEM组及假手术组比较，差异有显著性意义 (P < 0.01)。②原位杂交和免疫组织化学检测显示，移植的细胞能在宿主体内存活，并嵌合到宿主脊髓组织表达血管标志。
结论：骨髓单个核细胞移植后8周，不仅能够在损伤脊髓内存活，而且还能分化新生血管，促进脊髓功能的恢复。
关键词：急性脊髓损伤；骨髓单个核细胞；细胞移植

李长德，杨建华，刘铮，李东平．骨髓单个核细胞移植治疗急性脊髓损伤[J].中国组织工程研究与临床康复，2008，8(8):1431-1434 [www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1431(ps).pdf]

Bone marrow mononuclear cell transplantation in the treatment of acute spinal cord injury

Abstract

AIM：Some researches show that stem cell transplantation for damaged spinal cord can improve the function of damaged spinal cord. But the studies about bone marrow mononuclear cell transplantation for injuried spinal cord are seldom. We transplanted fresh bone marrow mononuclear cells isolated from rats into rat models of injured spinal cord to explore the effect of bone marrow mononuclear cells for injured spinal cord functions, nerve regeneration, neovascuarization and long-term effect.

METHODS: Experiments were performed in the Experiment Center of Developmental Biology of Shanghai Second Medical University from October 2005 to April 2006. The laboratory is Specific-pathogen free grade. ①Female clean SD rats aged 8 weeks weighting 200-220 g were offered by Animal Experimental Centre of Chinese Academy of Sciences. Animal intervention met the animal ethical standard. ②Rat bone marrow mononuclear cells were isolated from the tibia and the femur by Ficoll-Paque density gradient centrifugation. Rat models of spinal injury were established. The 22 successfully established rat models were divided into 2 groups. Rat models in a model plus cell group (n =11) received the complete T9-10 transection of spinal cord, and then bone marrow mononuclear cells were transplanted into the vertebral canal. Rat models in a model plus DMEM group (n =11) received the complete T9-10 transection of spinal cord, and then DMEM was injected into adjacent region. Rat models in a sham operation group (n =9) received T9-10 spinous process and lamina of vertebra incision and then the incision was sutured. ③Hybridization in situ and immunohistochemistry technique were used to determine the survival of implanted cells in host spinal cord. BBB scale system was applied to assess the functional recovery of spinal cord nerves.

RESULTS: ①There was no significant difference in postoperative score at each time point in the sham operation group. The score was 0 point in the model plus DMEM group. The function of spinal cord did not recover. The function of spinal cord became better at weeks 2, 4, 6 and 8 in the model plus cell group. There were significant differences as compared with the model plus DMEM group and the sham operation group (P < 0.01). ②Hybridization in situ and immunohistochemistry technique showed that transplanted cells survived after the transplantation and expressed specific markers for neovascularization.

CONCLUSION: After 8-week transplantation, bone marrow mononuclear cells can survive, differentiate into neovascularization and improve function recovery of spinal cord.

Li CD, Yang JH, Liu Z, Li DP.Bone marrow mononuclear cell transplantation in the treatment of acute spinal cord injury.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(8):1431-1434(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1431(ps).pdf]