C3转移酶联合雪旺细胞修复脊髓损伤的实验研究
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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。
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.
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。
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.
引文
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[2].Lakatos A, Franklin RJ, Barnett SC. Olfactory ensheathing cells and Schwann cells differ in their in vitro interactions with astrocytes. Glia.2000;32(3):214-225.
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[1].Schwab ME. Repairing the injured spinal cord. Science. 2002;295(5557):1029-1031.
[2].Lakatos A, Franklin RJ, Barnett SC. Olfactory ensheathing cells and Schwann cells differ in their in vitro interactions with astrocytes. Glia.2000;32(3):214-225.
[3].Ohta M, Suzuki Y, Noda T, Ejiri Y, Dezawa M, Kataoka K, et al. Bone marrow stromal cells infused into the cerebrospinal fluid promote functional recovery of the injured rat spinal cord with reduced cavity formation. Experimental neurology. 2004;187:266-78.
[4].Bakshi A, Barshinger AL, Swanger SA, Madhavani V, Shumsky JS, Neuhuber B, et al. Lumbar puncture delivery of bone marrow stromal cells in spinal cord contusion:a novel method for minimally invasive cell transplantation. J Neurotrauma.2006;23:55-65.
[5].Khalatbary AR, Tiraihi T. Localization of bone marrow stromal cells in injured spinal cord treated by intravenous route depends on the hemorrhagic lesions in traumatized spinal tissues. Neurol Res.2007;29:21-6.
[6].Vaquero J, Zurita M, Oya S, Santos M. Cell therapy using bone marrow stromal cells in chronic paraplegic rats:systemic or local administration? Neuroscience letters.2006;398:129-34.
[7].Martin D,Schoene J,Delree P,etal.Grafts of syngenic cultured adult dorsal root ganglion derived cells to the injured spinal cord of adult rat:Preliminary morphological studies[J].NeurosciLett,1991,124(1):44-8.
[8].Xu XM,XChenA,KleitmanN,etal.Bridging schwann cell Transplants promote axonal regeneration from both the rostral and caudal stumps of transected adult rats spinal cord[J].JNeurocytol,1997,26(1):1-16.
[9].TakamiT,OugegaM,BatesML,etal. Schwann cell but not olfactoryen sheathing glia transplants improve hind limb locmotor performance in the moderately confused adult rat thoracic spinal cord[J].Neurosci,2002,22(34):6670-6681.
[10].Lj Y,Field PM,Raisman G. Repair of adult rat corticospinal tract by transplan ts of olfactory ensheathing cells.Science,1997,277(5334):2000-2002.
[11].黄红云.嗅鞘细胞移植治疗脊髓损伤分子基础与临床研究.中国临床康复,2002,6(14)：2027～2028.
[12].冯世庆.雪旺细胞移植治疗脊髓损伤研究进展[J].中国现代神经疾病杂志,2004.24：276—279.
[13].Satake K,Lou J, Lenke LG, et al. Migration of mesenchymal stem cells through cerebro spinal fluid into injuried spinal caod tissue. Spine,2004,29(18):1971-1979.
[14].Bakshi A, Hunter C, Swanger S, et al. mininally invasive delievery of stem cells for spinal cord injury:advantages of the lumbar puncture technique. J Neurosurg. Spine,2004,1(3):330-337.
[15].Callera F, do Nascimento RX.delivery of autologous bone marrow precursor cells into the spinal cord via lumbar puncture technique in patients with spinal cord injury:a preliminary safety study. Exp Hematol.2006,34(2):130-131.
[16].Ohta M,Suzuki Y,Noda T,el al. Bone marrow stroma| cells infused into the cerebrospinal fluid promote functional recovery of the injured rat spinal cord with reduced cavity formation. Exp Neurol,2004,187(2):266—278.
[17].Firouzi M, Moshayedi P, Saberi H, Mobasheri H, Abolhassani F, Jahanzad I, Raza M. Transplantation of Schwann cells to subarachnoid space induces repair in contused rat spinal cord. Neurosci Lett.2006 Jul 10;402(1-2):66-70.
[18].Eglitis MA,Mezey E.Hematopoietic cells diferentiate into both microglia and macrogliainthe brains of adult mi ce.Proc Natl Acad Sci USA 1997,94:4080-5.
[19].Chen JL,Li Y,Wang L,et al.Therapeutic Benefit of Intravenous Administration of Bone Marrow Stromal Cells After Cerebrall schemiain Rats.Stroke 2001,4:1005-11.
[20].Li Y,Chen J,W ang L,et al.Treatment of stroke in rat with intracarotid administration of marrow stromal ce lls Neurology 2001;56 (12):1666-1672.
[21].Lu D,Mahmood A,W ang L,et al.Adult bone marrow stromal cells administrated intravenously to rats after traumatic brain jnjury migrate into brain and improve neurelogical out come Neuroreport 2001;12(3):559-563.
[22].Hofstetter CP, Schwarz EJ, Hess D, et al. Marrow stromal cells from guiding strands in the injuried spinal cord and promote recovery[J].Proc Natl Acad Sci USA,2002,99(4):2199-2204.
[23].Sykova E,Jendelova P,Glogarova K,Urzilova I,Hetynek V,Hajek M.Bone marrow stromal cells-a promising tool for therapy ot brain and spinal rord injury[J].Exp Neurol,2004,187:
[24].McKenzieAL, Hall JJ, AiharaN, et al. Immunolocalization of endothelin in the traumatized spinal cord:relationship to blood-spinal cord barrier breakdown[J]. J Neurotrauma,1995,12:257-268.
[25].Takeuchi H, Natsume A, Wakabayashi T, et al. Intravenously transplanted human neural stem cells migrate to the injured spinal cord in adult mice in an SDF-1-and HGF-dependent manner[J]. Neurosci Lett,2007,426(2):69-74.
[26].Keirstead HS,Nistor G,Bernal G,et al.Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury.J Neurosci,2005,25(19):4694-4705.
[27].Okano H,Ogawa Y,Nakamura M,et al.Transplantation of neural stem cells into the spinal cord after injury.Semin Cell Dev Biol,2003,14(3):191-198.
[28].Maikos JT, Shreiber DI. Immediate damage to the blood-spinal cord barrier due to mechanical trauma[J]. J Neurotrauma,2007,24(3):492-507.
[29].Zhang WD,Smith C, Howlett C, et al.Inflammatory activation of human brain endothelial ce lls by hypoxic in vitro is mediated by 1 L.[J] Cereb Blood Flow Metab 2000:20(6):967-968.
[30].Fleming JC,Norenberg MD,Ramsay DA,et al.The ce lluar inflammatoryrespo nse in human spinal cords after injury.Brain 2006;129(1):3249-3269.
[31].Blanc K. Immunomodulatory efects of fetal and adult mesenchymal stem cells Cytotherapy 2003;5(6):485-489.
[32].Mackenzie TC, Flake AW, Human mesenchymal stem cells persist demonstrate site-specific multipotential diferentiation and are presenten sites of wound healing and tissue regeneration after transplantation into fetal sheep.Blood Cells Mol Dis 2001;27(3):601-604.
[33].Khalatbary AR, Tiraihi T. A comparative study of therapeutic benefits of intraspinal and intravenous bone marrow stromal cell administration to spinal cord injuries. Iran Biomed J.2009; 13(1):43-48.
[34].Jing WL,Yan FX,Zuo YZ.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu.2008; 12(51):10045-10049.
[35].Takeuchi H, Natsume A, Wakabayashi T, et al. Intravenously transplanted human neural stem cells migrate to the injured spinal cord in adult mice in an SDF-1-and HGF-dependent manner. Neurosci Lett.2007;426(2):69-74.
[36].Xu ZY,Fang H,Luo YX,et al.Zhongguo Jiaoxing Waike Zazhi. 2007;15(6):459-462.
[37].Kimura H,Yoshikawa M, M atsuda R,et a 1.Transplantation of embryonic stem cell-derived neural stem cells for spinal cord injury in adult mice.Neural Res,2005,27(8):812-819.