腺病毒介导人心肌营养素-1基因转移对大鼠脊髓损伤的修复作用
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摘要
脊髓损伤(spinal cord injury,SCI)的治疗目标有三个：挽救(rescue)受损神经元的迟发性损伤和死亡；促进神经元轴突的再生(regeneration)；组织移植替代(replacement)神经缺损，以求重建损伤脊髓地解剖连接和功能康复。实验性治疗中，组织或细胞移植(胚胎脊髓、神经干细胞、雪旺氏细胞移植等)、神经营养因子运用(BDNF、NGF、NT3等)、再生相关因子基因治疗(GAP43、Bc12等)，均直接或间接为受损脊髓提供了抑制脊髓的继发性损伤及促进神经元存活和轴突再生的神经营养因子。神经营养因子通过与靶细胞上受体结合而发挥其生物效应，由于神经元及其受体的差异性，向脊髓损伤区有效地提供特异性神经营养因子非常重要。
心肌营养素-1(cardiotrophin-1 CT-1)是1995年由Pennica等发现的一种细胞因子，与LIF、CNTF、OSM、IL-6、L-11同属于IL-6家族。CT-1能长时间的支持运动神经元、多巴胺神经元和交感神经元的存活，诱导培养交感神经元递质表型的连接。CT-1基因治疗大鼠进行性运动神经元病取得较好的效果。本研究的目的是原核表达huCT-1重组蛋白和构建huCT-1的腺病毒载体，观察SCI后huCT-1能否支持红核神经元的存活、促进红核脊髓束的再生和功能恢复。
主要方法及技术路线：
1．设计与pGEX-2T读框适配的引物，采用PCR及T-A克隆将huCT-1开放阅读框基因克隆到pMD-18T载体上，测序正确后，酶切并回收DNA片段，构建pGEX2T-huCT1。在不同温度不同时间，通过IPTG诱导pGEX2T-huCT1在DH5a大肠杆菌中表达，SDS-PAGE分析表达量和可溶性蛋白比例。GST亲和柱纯化huCT-1融合蛋白，凝血酶酶切融合蛋白并
纯化。坐骨神经切断模型观察重组huCT－l对脊髓前角神经元的存活作用。
2．构建穿梭质粒pDC316－huCTI和 pDC316－eGFP，CsCI梯度离心制
备高纯度的病毒基因组质粒pBHloxdeltal，3Cre、PHG140。培养293细胞，
CaCI。法质粒共转染293 细胞构建并筛选腺病毒AdCMV－huCTI 和
AdCMV－eGFP。病毒原液转染293细胞及NIH 3T3细胞，PCR、RT－PCR
及免疫组化鉴定。CSCI梯度离心纯化病毒，空斑试验检测病毒滴度。
3．制备大鼠C3外侧索横切模型，损伤区植入明胶海棉饱和的不同成
分治疗及对照溶液。分为CTl组、AdCMV上 组、空白对照组（单纯
注射缓冲液X AdCMV七GFP对照组及正常对照组。治疗 Iw后观察
AdCMV－huCTI在脊髓中的表达。4W后HE、GFAP、P75染色组织学观察
脊髓损伤区的神经缺损及胶质形成。FG于 CZ注射，Iw、4W、SW时行
脑切片，荧光显微镜观察红核神经元存活。FG于CS注射，4W脑切片，
荧光显微镜下红核神经元记数观察RST再生。BDA于红核注射，脊髓切
片观察红核脊髓束再生。前肢的不对称试验观察行为学的改变。
主要结果及结论：
1．成功构建了hU＊l1 的融合表达载体…EX 2＊hU＊TI，克服了
hllCT－l基因高GC含量对hllCT－l表达量和对载体启动子的影响。通过IPTG
诱导，发现29oC诱导4hr，目的蛋白表达量占全菌的1／5，可溶性蛋白与包
涵体比为2乃。对其进行亲和柱纯化，GST凡此T．1融合蛋白纯度达到“％
以上。融合蛋白酶切后，重组hU＊T，1蛋白纯度达到80％以上。
2．发现原核表达重组huCT－1蛋白能挽救55％成年大鼠脊髓运动神经
元的存活，表明原核表达重组hU＊l1 蛋白具有生物学活性。同时，说明
hU＊T．1对大鼠神经元的作用无种属特异性。
3．采用双质粒共转染293细胞Cre／loxP位点同源重组方法构建了EI
和 E3缺失的含 MCMV启动子才源基因和 SV40 PIOyA的 AdCMV上
和 AdCMV－eGFP载体。经过PCR、RT－PCR和免疫组化证实腺病毒载体构
建成功。病毒经过CSCI梯度离心纯化后，AdCMV－hllCTI 滴度达到
10x10’‘～30x10’‘，适合进行体内直接基因治疗（in viv。）。
·VI·
4．C3HX损伤后1＊周，各组FG标记红核神经元下降不明显。损伤后
8周，AdV－CTI组神经元存活率为89％，损伤组仅为69％。与损伤组相比，
明显支持红核神经元的存活…＜0刀5＼表明脊髓损伤后，红核神经元存在迟
发性损伤。AdV－CTI在脊髓中长期表达，能支持红核神经元的存活。
5．大鼠C3HX损伤下区注射FG逆行示踪，AdV－CTI组标记的红核神
经元为1．2％，明显多于对照组①．4仰伊＜0刀5卜＊盯的＊＊AI’厕于示踪，损
伤上、下区BDA标记的神经元也多于对照组，表明CT－1治疗促进RST再
生。CT4治疗组和对照损伤区内胶质癫痕的形成无明显差别，均未见明显
再生的轴突，说明这种再生不是通过损伤区，而是可能通过向未损伤区白
质或灰质再生。CT－1治疗促进RST再生原因可能为CT－1挽救的存活红核
神经元存在或重建了旁支通路和CT－l有较弱促进RST再生的能力。
6．大鼠 C3HX 4周后对照组约 4％的时间使用伤肢，AdV－CTI组约 9％
的时间使用伤肢，明显高于对照组o＜0刀5卜 说明AdV－CTI促进SCI功能
的恢复。这种功能恢复可能与CT－1挽救红核神经元存活和RST再生及其
他传导束代偿有关。
There are three main types of therapeutic strategies for spinal cord injury (SCI): rescue of injured neurons from secondary injury with prevention of neuronal death; promotion of target-directed axonal regeneration; and neural replacement by transplantation. In experimental study, examples are various cells or tissue transplantation (e.g. fetal CNS tissue, neural stem cells, and Schwann cells), application of neurotrophic factors, and overexpression of growth-associated gene (e.g. GAP-43, c-Jun, and Bcb). All of these strategies are directly or indirectly provided neurotrophic factors, which promote axonal regeneration and rescue neurons for SCI. The survival of central neurons and axonal regeneration depend on multiple neurotropic factors produced by different neuronal targets. Therefore, besides NTs, it is important to local effective and specific factors into the injured spinal cord.
Cardiotrophin-1 (CT-1), an IL-6-related cytokine, cause hypertrophy of cardiac myocytes and has pleiotropic effects on various other cell types. CT-1 has potent survival promoting effects on motor neurons in vitro and in vivo. CT-1-treated progressive motor neuronopathy (pmn) mice showed a significantly reduce degeneration of facial motoneuron cytons and phrenic nerve myelinated axons. This study examined whether adenovirus huCT-1 gene transfer had neuroprotective effect, promotion rubrospinal axons regeneration, and recovery of forelimb function after spinal cord injury in adult rats.
Main methods and techniques:
1. To express human CT-1, the coding region from plasmid pBSSK-huCTl was cloned into plasmid pMD 18-T by PCR and T-A cloning, then cloned into
prokaryotic GST-fusion expression vector pGEX-2T to give pGEX2T-huCTl. After IPTG induced pGEX2T-huCTl expression in E.coli at different temperature and different time, the soluble GST/huCTl was purified by immobilized glutathione columns. The GST-fusion protein was cleaved by thrombin and purified again.
2. The huCT-1 gene was cloned into shuttle plasmid pDC316 to give pDC316-huCTl. Recombinant replication-defective adeno virus vector AdCMV-huCTl was rescued in 293 packaging cells by co-transfection and Cre-mediated recombination of both plasmids pDC316-huCTl and pBHGloxdeltal,3Cre containing Ad5 genome with deletions of packaging signal El and E3 regions. The insert gene and its expression were identified by PCR, RT-PCR, and immunohistochemistry. Another control recombinant adeno virus vector AdCMV-eGFP was constructed by same protocol. Recombinant adenovirus vectors were purified by CsCl banding and titrated by plaque forming test.
3. Gel foam saturated with AdCMV-huCTl was left into a C3-4 lateral funiculus hemisection cavity that completely interrupted one RST. 1,4, 8 weeks after lesion, RN neurons survival and RST regeneration were demonstrated with retrograde and anterograde tracing techniques, and function recovery was examined forelimb use asymmetries.
Main results and conclusions:
1. After the pGEX2T-huCTl-DH5 a cells induced by IPTG at 29癈 for 4hr, the highest expression of level of the recombinant protein is about 1/5 of total cell proteins, and the soluble portion is about 2/5 of fusion protein. Purification of soluble portion and thrombin cleaved fusion protein resulted in 85% and 80% purified recombinant GST-fusion protein and huCT-1 protein respectively.
2. Recombinant huCT-1 protected 55% motoneurons in spinal cord against sciatic axotomy in vivo in adult rats, indicating recombinant huCT-1 has biological activity in rat neurons.
3. We have constructed two recombinant adeno viral vectors: AdCMV-huCTl and AdCMV-eGFP, which expression cassette with MCMV promoter, foreign DNA, and SV40 PolyA was inserted into El of Ad5 genomic DNA with deletions of E3 regions. The positive huCT-1 mRNA and protein were identified in AdCMV-huCTl transfected NIH 3T3 cells. The titer of virus stocks was generally up to 1010 phaque forming units (pfu) per milliliter.
4. Following injury t
心肌营养素-1(cardiotrophin-1 CT-1)是1995年由Pennica等发现的一种细胞因子，与LIF、CNTF、OSM、IL-6、L-11同属于IL-6家族。CT-1能长时间的支持运动神经元、多巴胺神经元和交感神经元的存活，诱导培养交感神经元递质表型的连接。CT-1基因治疗大鼠进行性运动神经元病取得较好的效果。本研究的目的是原核表达huCT-1重组蛋白和构建huCT-1的腺病毒载体，观察SCI后huCT-1能否支持红核神经元的存活、促进红核脊髓束的再生和功能恢复。
主要方法及技术路线：
1．设计与pGEX-2T读框适配的引物，采用PCR及T-A克隆将huCT-1开放阅读框基因克隆到pMD-18T载体上，测序正确后，酶切并回收DNA片段，构建pGEX2T-huCT1。在不同温度不同时间，通过IPTG诱导pGEX2T-huCT1在DH5a大肠杆菌中表达，SDS-PAGE分析表达量和可溶性蛋白比例。GST亲和柱纯化huCT-1融合蛋白，凝血酶酶切融合蛋白并
纯化。坐骨神经切断模型观察重组huCT－l对脊髓前角神经元的存活作用。
2．构建穿梭质粒pDC316－huCTI和 pDC316－eGFP，CsCI梯度离心制
备高纯度的病毒基因组质粒pBHloxdeltal，3Cre、PHG140。培养293细胞，
CaCI。法质粒共转染293 细胞构建并筛选腺病毒AdCMV－huCTI 和
AdCMV－eGFP。病毒原液转染293细胞及NIH 3T3细胞，PCR、RT－PCR
及免疫组化鉴定。CSCI梯度离心纯化病毒，空斑试验检测病毒滴度。
3．制备大鼠C3外侧索横切模型，损伤区植入明胶海棉饱和的不同成
分治疗及对照溶液。分为CTl组、AdCMV上 组、空白对照组（单纯
注射缓冲液X AdCMV七GFP对照组及正常对照组。治疗 Iw后观察
AdCMV－huCTI在脊髓中的表达。4W后HE、GFAP、P75染色组织学观察
脊髓损伤区的神经缺损及胶质形成。FG于 CZ注射，Iw、4W、SW时行
脑切片，荧光显微镜观察红核神经元存活。FG于CS注射，4W脑切片，
荧光显微镜下红核神经元记数观察RST再生。BDA于红核注射，脊髓切
片观察红核脊髓束再生。前肢的不对称试验观察行为学的改变。
主要结果及结论：
1．成功构建了hU＊l1 的融合表达载体…EX 2＊hU＊TI，克服了
hllCT－l基因高GC含量对hllCT－l表达量和对载体启动子的影响。通过IPTG
诱导，发现29oC诱导4hr，目的蛋白表达量占全菌的1／5，可溶性蛋白与包
涵体比为2乃。对其进行亲和柱纯化，GST凡此T．1融合蛋白纯度达到“％
以上。融合蛋白酶切后，重组hU＊T，1蛋白纯度达到80％以上。
2．发现原核表达重组huCT－1蛋白能挽救55％成年大鼠脊髓运动神经
元的存活，表明原核表达重组hU＊l1 蛋白具有生物学活性。同时，说明
hU＊T．1对大鼠神经元的作用无种属特异性。
3．采用双质粒共转染293细胞Cre／loxP位点同源重组方法构建了EI
和 E3缺失的含 MCMV启动子才源基因和 SV40 PIOyA的 AdCMV上
和 AdCMV－eGFP载体。经过PCR、RT－PCR和免疫组化证实腺病毒载体构
建成功。病毒经过CSCI梯度离心纯化后，AdCMV－hllCTI 滴度达到
10x10’‘～30x10’‘，适合进行体内直接基因治疗（in viv。）。
·VI·
4．C3HX损伤后1＊周，各组FG标记红核神经元下降不明显。损伤后
8周，AdV－CTI组神经元存活率为89％，损伤组仅为69％。与损伤组相比，
明显支持红核神经元的存活…＜0刀5＼表明脊髓损伤后，红核神经元存在迟
发性损伤。AdV－CTI在脊髓中长期表达，能支持红核神经元的存活。
5．大鼠C3HX损伤下区注射FG逆行示踪，AdV－CTI组标记的红核神
经元为1．2％，明显多于对照组①．4仰伊＜0刀5卜＊盯的＊＊AI’厕于示踪，损
伤上、下区BDA标记的神经元也多于对照组，表明CT－1治疗促进RST再
生。CT4治疗组和对照损伤区内胶质癫痕的形成无明显差别，均未见明显
再生的轴突，说明这种再生不是通过损伤区，而是可能通过向未损伤区白
质或灰质再生。CT－1治疗促进RST再生原因可能为CT－1挽救的存活红核
神经元存在或重建了旁支通路和CT－l有较弱促进RST再生的能力。
6．大鼠 C3HX 4周后对照组约 4％的时间使用伤肢，AdV－CTI组约 9％
的时间使用伤肢，明显高于对照组o＜0刀5卜 说明AdV－CTI促进SCI功能
的恢复。这种功能恢复可能与CT－1挽救红核神经元存活和RST再生及其
他传导束代偿有关。
There are three main types of therapeutic strategies for spinal cord injury (SCI): rescue of injured neurons from secondary injury with prevention of neuronal death; promotion of target-directed axonal regeneration; and neural replacement by transplantation. In experimental study, examples are various cells or tissue transplantation (e.g. fetal CNS tissue, neural stem cells, and Schwann cells), application of neurotrophic factors, and overexpression of growth-associated gene (e.g. GAP-43, c-Jun, and Bcb). All of these strategies are directly or indirectly provided neurotrophic factors, which promote axonal regeneration and rescue neurons for SCI. The survival of central neurons and axonal regeneration depend on multiple neurotropic factors produced by different neuronal targets. Therefore, besides NTs, it is important to local effective and specific factors into the injured spinal cord.
Cardiotrophin-1 (CT-1), an IL-6-related cytokine, cause hypertrophy of cardiac myocytes and has pleiotropic effects on various other cell types. CT-1 has potent survival promoting effects on motor neurons in vitro and in vivo. CT-1-treated progressive motor neuronopathy (pmn) mice showed a significantly reduce degeneration of facial motoneuron cytons and phrenic nerve myelinated axons. This study examined whether adenovirus huCT-1 gene transfer had neuroprotective effect, promotion rubrospinal axons regeneration, and recovery of forelimb function after spinal cord injury in adult rats.
Main methods and techniques:
1. To express human CT-1, the coding region from plasmid pBSSK-huCTl was cloned into plasmid pMD 18-T by PCR and T-A cloning, then cloned into
prokaryotic GST-fusion expression vector pGEX-2T to give pGEX2T-huCTl. After IPTG induced pGEX2T-huCTl expression in E.coli at different temperature and different time, the soluble GST/huCTl was purified by immobilized glutathione columns. The GST-fusion protein was cleaved by thrombin and purified again.
2. The huCT-1 gene was cloned into shuttle plasmid pDC316 to give pDC316-huCTl. Recombinant replication-defective adeno virus vector AdCMV-huCTl was rescued in 293 packaging cells by co-transfection and Cre-mediated recombination of both plasmids pDC316-huCTl and pBHGloxdeltal,3Cre containing Ad5 genome with deletions of packaging signal El and E3 regions. The insert gene and its expression were identified by PCR, RT-PCR, and immunohistochemistry. Another control recombinant adeno virus vector AdCMV-eGFP was constructed by same protocol. Recombinant adenovirus vectors were purified by CsCl banding and titrated by plaque forming test.
3. Gel foam saturated with AdCMV-huCTl was left into a C3-4 lateral funiculus hemisection cavity that completely interrupted one RST. 1,4, 8 weeks after lesion, RN neurons survival and RST regeneration were demonstrated with retrograde and anterograde tracing techniques, and function recovery was examined forelimb use asymmetries.
Main results and conclusions:
1. After the pGEX2T-huCTl-DH5 a cells induced by IPTG at 29癈 for 4hr, the highest expression of level of the recombinant protein is about 1/5 of total cell proteins, and the soluble portion is about 2/5 of fusion protein. Purification of soluble portion and thrombin cleaved fusion protein resulted in 85% and 80% purified recombinant GST-fusion protein and huCT-1 protein respectively.
2. Recombinant huCT-1 protected 55% motoneurons in spinal cord against sciatic axotomy in vivo in adult rats, indicating recombinant huCT-1 has biological activity in rat neurons.
3. We have constructed two recombinant adeno viral vectors: AdCMV-huCTl and AdCMV-eGFP, which expression cassette with MCMV promoter, foreign DNA, and SV40 PolyA was inserted into El of Ad5 genomic DNA with deletions of E3 regions. The positive huCT-1 mRNA and protein were identified in AdCMV-huCTl transfected NIH 3T3 cells. The titer of virus stocks was generally up to 1010 phaque forming units (pfu) per milliliter.
4. Following injury t
引文
1. Schwab ME, Bartholdi D. Degeneration and regeneration of axons in the lesioned spinal cord. Phsyio Rev1996; 76(2): 319-3692
2. Stichel CC, Muller HW. Experimental strategies to promote axonal regeneration after traumatic central nervous system injury. Prog Neurobio 1998; 56:119-148
3. Bregman BS. Regeneration in the spinal cord. Curr Opin Neurobio 1998; 8:
800-807
4. Markats M, Bilang-Bleuil A, Buc-Caron MH, Castel-Barthe MN, Corti O, Finiels F, Horellou P, Revah F, Sabate O, Mallet J. Adenovirus in the Brain: recent advances of gene therapy for neurodegenerative diseases. Prog Neurobio 1998; 55:333-341
5. Hermens WT, Verhaagen J. Viral vectors, tools for gene transfer in the nervous system. Prog Neurobio 1998; 55:399-432
6. Huang EJ, Reichardt LF. Neurotrophins: roles in neuronal development and function. Ann Rev Neurosci 2001; 24:677-736
7. Pennica D, King KL, Shaw KJ, Luis E, Rullamas J, Luoh SM, Darbonne WC, Kuntzon DS, Yen R, Chien KR, Baker JB, Wood WI. Expression cloning of cardiotrophin 1, a cytokine that induces cardiac myocyte hypertrophy. PANS 1995; 92:1142-1146
8. Pennica D, Shaw KJ, Luis E, Swason TA, Moore MW, Shelton DL, Zioncheck KA, Rosenthal A, Taga T, Paoni NF, Wood WI. Cardiotrophin-1, biological activities and binding to the leukemia inhibitory factor receptor /gp130 signal complex. JBC 1995; 270:10915-10922
9. Pennica D, Swanson TA, Shaw KJ, Kuang WJ, Gray CL, Beatty BG, Wood WJ. Human cardiotrophin-1: protein and gene structure, biological and binding activities, and chromosomal localization. Cytokine 1996; 8: 183-189
10. Pennica D, Arce V, Swanson A, Vejsada R, Pollock RA, Armanini M, Dudley K, Phillips HS, Rosenthal A, Kato AC, Henderson CE. Cardiotrophin-1, a cytokine present in embryonic muscle, supports long term survival of spinal motoneurons. Neuron 1996; 17:63-74
11. Arce V, Pollock RA, Philippe JM, Pennica D, Henderson CE, deLapeyriere O. Synergistic effects of Schawann-and muscle-derived factor on motoneuron survival involve GDNF and cardiotrophin-1 (CT-1). J Neurosci
1998; 18:1440-1448
12. Mitsumoto H, Klinkosz B, Pioro EP, Tsuzaka K, Ishiyama T, O'leary RM, Pennica D. Effects of cardiotrophin-1(CT-1) in a mouse motor neuron disease. Muscle Nerve 2001;24:769-777
13. Bordet T, Schmalbruch H, Pettmannn B, Hagege A, Casteinnau-Ptakhine L, Kahn A, Hasse G. Adenviral cardiotrophin-1 gene transfer protects pmn mice from progressive motor neuronopathy. J Clin Inv 1999; 104: 1077-1085
2. Stichel CC, Muller HW. Experimental strategies to promote axonal regeneration after traumatic central nervous system injury. Prog Neurobio 1998; 56:119-148
3. Bregman BS. Regeneration in the spinal cord. Curr Opin Neurobio 1998; 8:
800-807
4. Markats M, Bilang-Bleuil A, Buc-Caron MH, Castel-Barthe MN, Corti O, Finiels F, Horellou P, Revah F, Sabate O, Mallet J. Adenovirus in the Brain: recent advances of gene therapy for neurodegenerative diseases. Prog Neurobio 1998; 55:333-341
5. Hermens WT, Verhaagen J. Viral vectors, tools for gene transfer in the nervous system. Prog Neurobio 1998; 55:399-432
6. Huang EJ, Reichardt LF. Neurotrophins: roles in neuronal development and function. Ann Rev Neurosci 2001; 24:677-736
7. Pennica D, King KL, Shaw KJ, Luis E, Rullamas J, Luoh SM, Darbonne WC, Kuntzon DS, Yen R, Chien KR, Baker JB, Wood WI. Expression cloning of cardiotrophin 1, a cytokine that induces cardiac myocyte hypertrophy. PANS 1995; 92:1142-1146
8. Pennica D, Shaw KJ, Luis E, Swason TA, Moore MW, Shelton DL, Zioncheck KA, Rosenthal A, Taga T, Paoni NF, Wood WI. Cardiotrophin-1, biological activities and binding to the leukemia inhibitory factor receptor /gp130 signal complex. JBC 1995; 270:10915-10922
9. Pennica D, Swanson TA, Shaw KJ, Kuang WJ, Gray CL, Beatty BG, Wood WJ. Human cardiotrophin-1: protein and gene structure, biological and binding activities, and chromosomal localization. Cytokine 1996; 8: 183-189
10. Pennica D, Arce V, Swanson A, Vejsada R, Pollock RA, Armanini M, Dudley K, Phillips HS, Rosenthal A, Kato AC, Henderson CE. Cardiotrophin-1, a cytokine present in embryonic muscle, supports long term survival of spinal motoneurons. Neuron 1996; 17:63-74
11. Arce V, Pollock RA, Philippe JM, Pennica D, Henderson CE, deLapeyriere O. Synergistic effects of Schawann-and muscle-derived factor on motoneuron survival involve GDNF and cardiotrophin-1 (CT-1). J Neurosci
1998; 18:1440-1448
12. Mitsumoto H, Klinkosz B, Pioro EP, Tsuzaka K, Ishiyama T, O'leary RM, Pennica D. Effects of cardiotrophin-1(CT-1) in a mouse motor neuron disease. Muscle Nerve 2001;24:769-777
13. Bordet T, Schmalbruch H, Pettmannn B, Hagege A, Casteinnau-Ptakhine L, Kahn A, Hasse G. Adenviral cardiotrophin-1 gene transfer protects pmn mice from progressive motor neuronopathy. J Clin Inv 1999; 104: 1077-1085