【作者】 李辉；

【导师】 冯世庆； 郑永发；

【作者基本信息】 天津医科大学 ， 外科学， 2010， 硕士

【摘要】 目的探讨C3转移酶联合雪旺细胞(schwann cells, SCs)尾静脉移植治疗大鼠脊髓损伤(spinal cord injury, SCI)的疗效。方法SPF级雌性Wistar大鼠,体重50±10g,脱颈处死后取双侧坐骨神经,显微镜下抽出神经束,采用植块法培养、传代、纯化,S-100染色鉴定雪旺细胞,移植前一天用Hoechst33342标记雪旺细胞。SPF级Wistar雌性大鼠60只,体重220±10g,利用Impactor model-Ⅱ打击仪垂直打击(10g×25 min),建立胸10(T10)脊髓损伤模型,造模成功后随机分为4组：单纯DMEM空白对照组(A组)、单纯C3移植组(B组)、单纯雪旺细胞移植组(C组)C3联合雪旺细胞移植组(D组)；单纯DMEM空白对照组(A组)：损伤后1周尾静脉注射DMEM 1ml;单纯C3移植组(B组)：损伤后立即和术后3天分别局部注射C3转移酶10μl；单纯雪旺细胞移植组(C组)：损伤后1周尾静脉移植雪旺细胞1ml(1×106)；C3联合雪旺细胞移植组(D组)：损伤后立即和术后3天局部分别注射C3转移酶10μl,损伤后1周尾静脉移植雪旺细胞1ml(1×106)。术前1d、术后1d、3d、1w及以后每周进行BBB评分、脚印分析行为学评价损伤后大鼠后肢功能恢复情况。细胞移植后1、2、4周各组分别取材行冰冻切片观察移植细胞的迁移情况。损伤后12周行葡聚糖胺(biotin-dextran amine, BDA)顺行示踪皮质脊髓束,2周后取出实验动物损伤段脊髓,行快速冰冻切片观察神经纤维再生情况。移植后13周取材行常规HE染色,免疫荧光染色观察损伤段神经元特异性烯醇化酶(neuron specific enolase, NSE)、胶质原纤维酸性蛋白(glial fibrillary acidic protein, GFAP)的表达。结果组织块培养第3天见少量雪旺细胞迁出,1周后可见植块周边有大量细胞迁出,经纯化、S-100染色鉴定,获得雪旺细胞的纯度达95%。移植后1、2、4周损伤段及邻近节段脊髓内可观察到Hoechst33342阳性细胞。术前所有实验动物双后肢BBB评分均为21分,术后1、3d BBB评分0-1分,随后BBB评分逐渐上升,至损伤后13周实验结束时D组最高评分达13分左右。术后第4周C、D组与A组比较差异有统计学意义(p<0.05),第5周各组组间差异均有统计学意义(p<0.05)；术后9周B、C、D组组间脚印分析结果差异有统计学意义(p<0.05)。HE染色示损伤后2周时各组均有空洞形成,B、C、D组损伤区空洞面积小于A组。免疫荧光染色示,A组GFAP反应最强,损伤区空洞明显可见,范围大于其它三组,D组GFAP阳性面积明显小于其它实验组(p<0.05)；B、C、D组NSE阳性面积与A组比较差异均有统计学意义(p<0.05)；C组和D组脊髓损伤区及其远端可见BDA标记的神经纤维通过损伤区域。结论静脉移植的雪旺细胞能够通过血脊髓屏障向脊髓损伤区域迁移,C3转移酶可以改善损伤局部微环境,C3转移酶联合雪旺细胞移植,能够促进受损神经纤维的修复和再生,对大鼠脊髓损伤的修复有较好的疗效。更多还原

【Abstract】 Objective:To investigate the effects of C3 transferase combined with intravenous transplantation of Schwann cells on spinal cord injuries in adult wistar rats.Method:Female Wistar rats weighing 50±10g, The bilateral sciatic nerves of wistar rats were separated in viro, SCs were cultured by the tissue clot method, SCs identification were positive for S-100 immunofluorescent staining, SCs were marked by Hoechst33342 before transplantation.The rat’s SCI model was made by impactor model-Ⅱtype weight drop apparatus at T10, Then sixty female Wistar rats were randomly divided into 4 groups, DMEM control group(n=15,group A), C3 Transferase group(n=15,group B),SCs transplantation group(n=15,group C), and combined transplantation group(n=15,group D), C3 Transferase group (group B) and combined transplantation group (group D):C3 transferase 10μl were injected into the injured epicenter of spinal cord after injury immediately and three days after injury respectively,SCs group (group C) and combined transplantation group (group D):1ml SCs suspension (1×106)were transplantated by tail vein respectively 1 week after injury, control group(group A):lml DMEM was injected by tail vein as control. Preoperative Id, postoperative 1d,3d,1 week and every week after injury The Basso Beattie Bresnahan(BBB)score and footprint analysis were carried out to evaluate functional recovery of injuried rat’s hind limbs. To reveal the migration of SCs, Hoechst33342 positive cells were detected in spinal cord of the injured rats at 1,2,4 weeks after intravenous transplantation. Two rats in each group were injected with 10%biotinylated dextran amine(BDA)in the double cerebra cortex for anterograde labeling of CST 12 weeks after injury. HE staining, immunofluorescence staining (NSE and GFAP) around the injured epicenter was measured respectively at 13 weeks after transplantation.Results:A amount of Schwann cells grow out three days after cultured, the final SCs purities reached 95%. After injection, Hoechst33342 positive cells were observed throughout the injured epicenter and the nearby regions of spinal cord at 1,2, and 4 weeks. statistical difference of BBB score happened among four groups four weeks after injury(p<0.05), and the BBB scores of groups C and D were higher than that of groups A and B. statistical difference of footprint analysis appeared between group D and group B C eight weeks after injury(p<0.05). HE staining showed the formation of cavity in each group, but the area of group A were the biggest. The immunofluorescence staining indicated that the expression of GFAP were more intense in group A and B than group C and D,the expression of NSE was more intense in group D than other groups.There was statistical difference in GFAP and NSE expression between groups C, D and group A, B (p<0.05). The regenerated nerve fiber stained by BDA was more intensive in groups C and D, and group D was significiant.Conclusion:Schwann cells can reach the injured epicenter of spinal cord through blood-spinal cord barrier, improve micro-environment and promote the repair of damaged nerve fibers. Schwann cells co-transplantation of C3 transferase have better effects for spinal cord injuries in rats.更多还原