【作者】 江波；

【导师】 沈忆新；

【作者基本信息】 苏州大学 ， 骨外科学， 2007， 硕士

【摘要】 脊髓损伤（spinal cord injury, SCI）是临床上常见病多发病,尤其是严重的脊髓损伤,致残率很高,严重危及人类的健康,对个人和社会都造成了极大的损失。近年来,大量实验证明嗅鞘细胞（OEC_S）移植能促进脊髓损伤神经纤维的再生。OEC_S促进脊髓再生的作用是通过在损伤段为再生神经纤维形成髓鞘和分泌一些促进脊髓损伤再生的因子而实现的。嗅鞘细胞移植将成为解决脊髓损伤这一临床世纪难题的重要方法之一。目的:在差速贴壁+Thy1.1抗体及补体方法基础上,用无血清培养液换液纯化培养条件下OEC_S的生长,并观察其移植后在体内存活情况和对脊髓横断伤的修复作用。方法:1.细胞培养:用Sprague-Dawley大鼠嗅球（OB）做OEC_S原代培养,然后利用差速贴壁+Thy1.1抗体及补体和无血清培养液换液方法做纯化培养。倒置相差显微镜下观察OECs的形态变化特点及生长情况,利用中枢神经系统中仅有OEC_S细胞表现为神经胶质元纤维酸性蛋白（GFAP）和低亲和力神经生长因子受体(P⁷⁵)免疫染色双阳性的特性,鉴定OEC_S。2.动物实验:四月龄Sprague-Dawley大鼠,体重250g左右。显露T₉段脊髓,在中间横行完全切断脊髓。A组（20只）在损伤远近端注射的OEC_S,B组（20只）在相同位置注射DMEM培养液,C组（10只）单纯做T₉节段的椎板减压。术后观察动物脊髓功能恢复情况,每两周测定一次后肢运动功能（BBB评分法）,共12周。术后三个月取损伤段及其临近脊髓标本,做HE染色、嗜银染色、抗NF和P⁷⁵免疫组化染色。利用P⁷⁵免疫组化染色来观察OEC_S在体内存活情况。结果:1.差速贴壁+Thy1.1抗体及补体和无血清纯化培养方法,OEC_S增殖迅速,是一种效率较高的培养方法。2.手术后4周开始有运动功能恢复,在各个时间段BBB运动功能得分A组均高于B组。比较12周时两组BBB得分,差异明显（P<0.001）。3.A、B组都有神经纤维进入损伤区,排列紊乱,数量不等。在损伤近端,神经纤维数量A组多于B组（P<0.001）,但都少于相应节段的C组（P<0.001）。4.通过显微镜能观察到经过P⁷⁵免疫组化的OEC_S存在于脊髓损伤段周围,最远可到距离损伤区边缘1.0cm处。5.统计学分析表明,脊髓损伤再生神经纤维的数量与运动功能恢复之间存在正相关（r=0.981）。结论:1.差速贴壁+Thy1.1抗体及补体和无血清纯化的培养方法是一种能使细胞增殖迅速,可获得较高纯度OEC_S的培养方法2. OEC_S植入体内可以长时间存活,并有自我迁移能力。3.OEC_S移植具有明显促进脊髓横断伤神经纤维再生和功能恢复的作用。更多还原

【Abstract】 Spinal cord injury （SCI） is a frequently encountered clinical problem. It can easily lead to crippledom and seriously damages to people’s health,especially serious spinal cord injury.It’s treatment is still a century problem.Recent studies have proved that olfactory ensheating cells （OEC_S） can promote the regeneration of spinal cord axons by excreting several factors and forming myelin sheathes for regenerating axons.It is believed that the transplantation of olfactory ensheathing cells would be effective option for the treatment of SCI.Objcctive:To explore a effective method for OEC_S culture and purification and observe OEC_S survival and repair in vivo for spinal cord transection after the OEC_S transplantation.Methods:1. Cell culture: The primary OEC_S was cultivated with the olfactory bulb of Sprague-Dawley rats, and then the purification was done by the differing rates of attachment of the various harvested cell types and anti-Thy1.1-mediated complement lysis and free serum medium. The morphological changes of the cultured OB-OECs were observed under a phase contrast microscope at different culture time.As a result, the majority of neuron and other mixed cells were eliminated. Because only OEC_S express both GFAP and P⁷⁵ in the CNS, we can identify it by this characteristic. The purity of the OB-OECs was evaluated according to the percentage of NGFRp75 immunostaining cells.2. Animal experiment: The T₉ spinal cords of 4 months Sprague-Dawley rats was exposed, then complete transection was performed in the middle of the area in Group A and Group B. In group A （20 rats） ,OEC_S was transplanted. In group B （20 rats）, DMEM were injected. In Group C （10 rats）, T₉ laminectomies were done without cord transection. The functional recovery of the spinal cord injury was observed with BBB locomotion score in each double weeks. The tissue sections were done 3 months postoperatively. HE staining, silver staining ,NF and P⁷⁵ immunohistochemical staining were performed respectively to observe the pathological changes and axon regeneration. The survival of OEC_S was observed by P⁷⁵ staining under the microscope.Results:1. The differing rates of attachment of the various harvested cell types with anti-Thy1.1-mediated complement lysis and free serum medium was a efficient method of OB-OECs puritification and culture.2. The experiment showed that locomotion behaviour had improved in 4 weeks postoperatively. The BBB locomotion score of group A was higher than that of group B in all periods, statistically significant difference in 12 week after operation.3. Nerve fiber innervated injury area in the group A and B.In the cephal lesion, the number of nerve fibers of group A was more than that of group B（P<0.001）, but less than that of group C（P<0.001）.4. The OEC_S by P⁷⁵ immunohistochemical staining could be observed around spinal injury area through the microscope. OEC_S could migrate to 1.0cm far from the injury area.5. There was positive correlation between the number of regeneration axons and the recovery of locomotion function（r=0.981）.Conclusion:1. The cell culture of the differing rates of attachment of the various harvested cell types with anti-Thy1.1-mediated complement lysis and free serum medium can obtain high purity OEC_S. Furthermore, the method is simple and economical.2. After transplantation, OEC_S can survive for at least 3 months in vivo, and can migrate from the injection sites.3. For spinal cord transection, OEC_S can promote the axons regeneration and the recovery of locomotion function in our study.更多还原