低温保存的嗅鞘细胞移植治疗脊髓损伤的实验研究

Cryopreserved olfactory ensheathing cells transplantation for the treatment of spinal cord injury,an experimental study

目的:观察低温保存复苏后嗅鞘细胞(OECs)局部移植治疗脊髓损伤(SCI)的疗效,探讨低温保存对嗅鞘细胞活性的影响。方法:建立大鼠T10节段脊髓半切损伤模型56只,随机分为四组,每组12只:新鲜OECs移植组(A组),冻存OECs移植组(B组),D/F12培养液移植组(C组)和空白对照组(D组)。A组损失伤局部行新鲜OECs移植,B组将处于对数生长期的OECs冻存3个月,复苏后(用双苯亚甲胺标记)移植到脊髓半切模型大鼠脊髓损伤区。术后第1天、1、2、3、4、6、8及10周时进行联合行为学(CBS)综合评分,术后5周和10周时取材观察移植细胞存活及迁移情况;HE染色及嗜银染色观察脊髓组织病理及轴突情况;NGFRp75免疫组织化学染色情况。结果:术后第1天,2、10周时CBS评分,A组分别为85.78±1.07、58.80±5.00、6.52±2.37;B组分别为86.12±1.29、60.06±6.51、8.15±2.26;C组分别为86.4±1.03、66.28±7.00、30.65±5.60;D组分别为86.75±1.37、69.85±6.61、34.13±5.38。A、B两组间无明显差别(P>0.05);C组与D组间比较无差异(P>0.05);A组及B组较C组和D组在2周后各时段差别有显著意义(P<0.05)。组织学方面,HE染色和嗜银染色A、B两组在术后5周可见多量突起经近侧断端向损伤区域生长,10周时可见神经纤维跨越损伤区域,而C、D组未见有神经纤维跨越损伤区;NGFRp75免疫组化染色,无论5周还是10周,A、B两组在损伤部位均可见阳性染色区域,C、D组未见有阳性着色。术后5周时A、B两组可检测到荧光标记细胞,且可见细胞发生迁移。结论:低温保存的OECs脊髓局部移植治疗SCI与新鲜OECs移植治疗效果无差别。

Objectives： To observe the effect of cryopreserved olfactory ensheathing cells（OECs） transplantation in situ on spinal cord injury （SCI） and the effect of hypothermia on OECs activity. Methods： 56 Wistar rats undergoing T10 spinal cord hemisection were divided into 4 groups： fresh OECs group（A）, cryopreserved OECs group（B）, D/F12 medium group（C） and control group（D）. In group B, the OECs in logarithmic phase were cryopreserved for 3 months, after resuscitate, they （labeled with hoechst） were transplanted into the injured region, while fresh OECs were transplanted in group A. At dayl and 1, 2, 3, 4, 6, 8, 10 weeks after transplantation, combined behavioral score（CBS） was used to evaluate the locomotion. The tissues were sectioned to observe the survival and migration of OECs, the nerve fiber regeneration and NGFRp75 immunohistochemistry staining at 5 and 10 weeks after transplantion. Results： At day/, 2weeks, 10 weeks after trans- plantation, the combined behavioral score （CBS） for group A was 85.78±1.07, 58.80±5.00, 6.52＋2.37 respectively; 86.12±1.29, 60.06±6.51, 8.15±2.26 for group B; 86.4±1.03, 66.28±7.00, 30.65±5.60 for group C; 86.75±1.37, 69.85±6.61, 34.13±5.38 for group D. Group A and B had more improvement than group C and D at each timepoint after transplantation for 2 weeks （P〈0.05）, while no significant difference was noted between group A and B as well as group C and D （P〉0.05）.At 5 weeks after transplantation, axons were noted to grow into the injury site in group A and B, and some nerve fiber reached the distal end of injury site at 10 weeks after transplantation, no nerve fiber was noted in the injury site in group C and D. Both at 5 and 10 weeks, under NGFRp75 irnmunohistochemicl staining, the OECs could be observed around the injury area in group A and B, while no similar findings was noted in group C and D. OECs were detected in the spinalcord after 5 weeks of transplantation in group A and B, and their migration was evidenced. Conclusions： As for the treatment outcome for SCI, no difference exists between the fresh OECs and the cryopreserved OECs transplantation.