NgR特异性RNAi对EAE大鼠脑内NgR表达、小胶质细胞及髓鞘修复的影响
摘要
目的多发性硬化是中枢神经系统(Central nervous system, CNS)常见的自身免疫性疾病,以慢性炎性脱髓鞘为主要病理改变。在青壮年发病率相对较高,其具体的发病机制不明,缺乏有效的特异性治疗,具有较高的致残率。实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis, EAE)是研究多发性硬化常用的动物模型,该模型在临床、病理、生化等方面均能很好地模拟多发性硬化的发病过程。Nogo受体(nogo receptor,NgR)是轴突再生抑制因子Nogo-A,髓鞘相关糖蛋白(Myelin-associated glycoprotein, MAG),少突胶质细胞髓鞘糖蛋白(Oligodendrocyte Myelin glycoprotein, OMgp)的共受体,因而在CNS损伤后轴突再生障碍中起重要作用。近来有研究表明在周围神经损伤的动物模型中,NgR可能可引导损伤部位巨噬细胞的清除,从而起到调节炎症的作用。此外,有研究表明在多发性硬化病人的脑组织中NgR的表达是增高的。这些都提示NgR在CNS免疫性疾病中可能具有免疫调节的作用。近年来,基因沉默技术中的短链RNA干扰(short interfering RNAs, siRNAs)技术可高度特异性降解目的基因,且不引起非特异性干扰反应,因而对揭示某疾病中表达异常增高的基因或蛋白的功能是非常有用。小胶质细胞是中枢神经系统中常驻的单核吞噬细胞之一,其在功能及形态上与巨噬细胞具有许多相似之处,参与脑内的炎症反应,并可能参与髓鞘的损伤与修复过程。因此,通过重组腺病毒为载体的NgR特异性shRNA转染EAE大鼠脑组织,并观察转染前后小胶质细胞及髓鞘的情况,可有助于阐述NgR在EAE中是否具有调节小胶质细胞及髓鞘再生的作用。
本研究拟探讨：1在EAE动物模型中,NgR的的表达是否增高,以及小胶质细胞的数量变化及激活等情况；2以重组腺病毒为载体的NgR特异性shRNA转染EAE大鼠的侧脑室,转染后EAE大鼠脑组织NgR基因及蛋白表达的变化,以及转染后EAE脑组织小胶质细胞的数量及激活情况的变化；3该转染对EAE大鼠临床发病及脑组织炎性细胞聚集、髓鞘缺失情况的影响。从而揭示NgR在多发性硬化中可能具有的调节小胶质细胞及髓鞘再生的作用。为阐明多发性硬化等中枢神经免疫性疾病的发病机制提供理论依据,并为其基因治疗的临床应用奠定基础。
方法1.建立Wistar大鼠EAE模型,运用MRI扫描、HE染色、Luxol Fast Blue染色等方法鉴定EAE模型制备是否成功；通过免疫荧光染色观察EAE脑组织中NgR表达是否上调,以及巨噬/小胶质细胞的数量是否增加及其活化情况。
2.应用人胚肾293细胞扩增重组腺病毒,采用赛多利斯腺病毒纯化试剂盒纯化、收集重组腺病毒,通过TCID50测定病毒滴度；用等体积的高、中、低滴度的重组腺病毒转染EAE大鼠侧脑室,荧光显微镜观察绿色荧光蛋白(green fluorescence protein,GFP)的表达,采用HE染色观察局部炎症反应,从而筛选出最佳的转染滴度。
3.用中滴度的Ad-shRNA-NgR、Ad-shRNA-Hk分别转染重组腺病毒载体Ad-shRNA-NgR干预组(特异性RNAi组)及重组腺病毒载体Ad-shRNA-HK干预组(阴性对照组),空白对照组以等量的PBS代替。在转染后第7及14天取材,通过RT-PCR观察转染后脑组织NgRmRNA的表达,免疫荧光染色观察转染后脑组织NgR蛋白的表达情况以及小胶质细胞的数量变化及激活情况,行HE、Luxol Fast Blue染色以观察炎性反应、髓鞘缺失等组织病理学变化,并对各组大鼠的临床转归进行记录。
结果1.所制备的EAE模型在MRI及病理学方面的改变符合经典EAE表现。EAE脑组织中NgR的表达上调,小胶质细胞数量增加,并以激活的小胶质细胞为主。
2.以GFP标记了的重组腺病毒转染293细胞后,转染后第24-72小时,荧光显微镜下观察发现：GFP表达逐渐增多。经扩增、纯化后得重组腺病毒Ad-shRNA-NgR及Ad-shRNA-Hk的滴度分别为：2.93×1010pfu/ml,2.84×1010pfu/ml。高、中、低滴度组EAE大鼠脑组织均出现GFP阳性表达,且高、中滴度组转染效率明显高于低滴度组,但高滴度组与中滴度组的GFP表达无明显差异。HE染色显示,高滴度组大鼠脑组织转染局部有明显急性炎性反应,其余组转染局部未发现明显炎性反应。
3.转染后第7天(d14)：特异性RNAi组脑组织中NgR mRNA及蛋白的表达明显下降(p<0.01),小胶质细胞的表达及髓鞘缺失情况与阴性及空白对照组未见明显差异。转染后第14天(d21)：特异性RNAi组脑组织中NgR mRNA及蛋白表达仍低于空白及阴性对照组(p<0.01),但较该组转染后第7天时有所增加(p<0.05)；小胶质细胞的数量较空白及阴性对照组多(p<0.05),且以活化状态为主；髓鞘缺失的恢复较空白及阴性对照组好(p<0.05)。
4.与空白及阴性对照组比较,特异性RNAi组大鼠的发病潜伏期延长(p<0.05)、临床症状评分减轻(p<0.05)；d21时的髓鞘缺失缓解略明显；d21时,病灶部位内的炎性细胞消退较慢。
结论EAE模型的制备是成功的。中滴度的腺病毒可高效、安全地转染EAE脑组织。NgR特异性RNAi可明显降低EAE脑组织中NgR基因及蛋白的表达,并有利于髓鞘缺失的修复,但对于激活的小胶质细胞的早期清除不利。该现象可能是通过病灶内激活的小胶质细胞启动了髓鞘再生的修复机制,从而促进髓鞘再生。
Objective Multiple sclerosis (MS) is a inflammatory demyelinating disease in central nervous system (CNS). With the development of magnetic resonance (MRI), The incidence of MS is increasing. The pathogenesis of MS is unclear. Furthermore there is still no good way to treat this disease. So to search for a new method for nerve protection in this disease is very important. EAE is a classical model of MS. It can simulate MS no matter in clinic or in pathology. Nogo receptor is the same receptor for MAG, OMgp and NgR. The three proteins above are the inhibitors for axons regeneration in CNS. Right now some projects indicate that NgR may participate in inflammatory. And in the CNS tissue of MS, the expression of NgR is increased. It seems that NgR may play some role in MS. RNAi can reduce the expression of objective gene. Ad-shRNA-NgR can down regulation the expression of NgR. Microglias is the principal member of macrophage family in CNS. These cells might sustain and propagate inflammation within the CNS during autoimmune inflammation. So by transfecting the Ad-shRNA-NgR into the experimental autoimmune encephalomyelitis (EAE) model'lateral cerebral ventricle and observation the microglias and remyelination could help us to find the effects of NgR on microglias and remyelination.
In this research, after transfection the Ad-shRNA-NgR into the experimental autoimmune encephalomyelitis(EAE) model'lateral cerebral ventricle, we investigated:the expression of nogo receptor(NgR)mRNA and protein, the aggregation of inflammation cells, the depletion of myelin sheath and the clinical invasion of the EAE model. By doing this we can find the contribution of NgR myelin regeneration and microglias. This project can provide a therapeutic approach for MS and other inflammation diseases of neurology.
Method 1.Established the EAE model and MRI scanning, the dyeing of HE, Luxol fast blue, microglias were carried out for evaluation.
2. The recombinated adenovious was amplified in 293 cells(human embroyonic kidney cells)and purified by Sartorius Vivapure AdenoPACK 20 kit.The virus titer was determined by TCID50. Three different titers of recombinated adenovious(high titer, middle titer, low titer) were used to transfect the brain tissue of EAE. Histopathological examination was performed to evaluate the local inflammatory reaction. And green fluorescent protein (GFP) expression was observed under fluorescence microscope. The optimal transfective virus titer was selected;
3.48 female Wistar rats were randomed into normal control group、blank control group, Ad-shRNA-HK intervention group(negative control group), Ad-shRNA-NgR intervention group (specific RNAi group). Reverse transcription polymerases chain reaction (RT-PCR) was used to determine the changes of NgR mRNA expression at the 7th and 14th day after transfection.The stain of HE、Luxol Fast Blue、NgR and CDllb/C was also carried out at the 7th and 14th day after transfection.
Result 1.The MRI scanning and pathological observations of the rats were consistent with those of classical EAE model.
2. After 24 hours of transfection the fluorescence was observed in 293 cells. The titer of amplified recombined adenovirus Ad-shRNA-NgR and Ad-shRNA-HK were 2.93×1010pfu/ml and 2.84×1010pfu/ml respectively. Green fluorescence protein(GFP) could be found in the brain of the high, middle and low titer group at the 7th day of transfection. GFP expression in the high and middle titer group was obviously higher than that in the low titer group. While there was no obvious difference between the high and middle titer group. HE staining showed that acute inflammatory infiltration was only observed in the high titer group;
3. NgR protein in the brain of EAE was significantly increased at the 14th and 21th day after induction (p<0.01). NgR mRNA and protein was reduced in the brain of EAE at the 7th and 14th day after RNAi intervention (p<0.01);
4.Compared with the blank control group, the Ad-shRNA-NgR specific intervention group had longer latency (p<0.05)、lower clinical score;at the d21,the more significant regeneration of myelin sheath, while the slower extinction of inflamation.
Conclusion The EAE model was induced successfully. The middle titers of adenovirus can transfect the brain tissue of EAE model efficiently and safely. The expression of NgR was increased in the brain of EAE. Ad-shRNA-NgR interfering can degrade the expressing of NgR remarkably and enhance the regeneration of myelin sheath, but it's not good for extinction of activated microglias.
本研究拟探讨：1在EAE动物模型中,NgR的的表达是否增高,以及小胶质细胞的数量变化及激活等情况；2以重组腺病毒为载体的NgR特异性shRNA转染EAE大鼠的侧脑室,转染后EAE大鼠脑组织NgR基因及蛋白表达的变化,以及转染后EAE脑组织小胶质细胞的数量及激活情况的变化；3该转染对EAE大鼠临床发病及脑组织炎性细胞聚集、髓鞘缺失情况的影响。从而揭示NgR在多发性硬化中可能具有的调节小胶质细胞及髓鞘再生的作用。为阐明多发性硬化等中枢神经免疫性疾病的发病机制提供理论依据,并为其基因治疗的临床应用奠定基础。
方法1.建立Wistar大鼠EAE模型,运用MRI扫描、HE染色、Luxol Fast Blue染色等方法鉴定EAE模型制备是否成功；通过免疫荧光染色观察EAE脑组织中NgR表达是否上调,以及巨噬/小胶质细胞的数量是否增加及其活化情况。
2.应用人胚肾293细胞扩增重组腺病毒,采用赛多利斯腺病毒纯化试剂盒纯化、收集重组腺病毒,通过TCID50测定病毒滴度；用等体积的高、中、低滴度的重组腺病毒转染EAE大鼠侧脑室,荧光显微镜观察绿色荧光蛋白(green fluorescence protein,GFP)的表达,采用HE染色观察局部炎症反应,从而筛选出最佳的转染滴度。
3.用中滴度的Ad-shRNA-NgR、Ad-shRNA-Hk分别转染重组腺病毒载体Ad-shRNA-NgR干预组(特异性RNAi组)及重组腺病毒载体Ad-shRNA-HK干预组(阴性对照组),空白对照组以等量的PBS代替。在转染后第7及14天取材,通过RT-PCR观察转染后脑组织NgRmRNA的表达,免疫荧光染色观察转染后脑组织NgR蛋白的表达情况以及小胶质细胞的数量变化及激活情况,行HE、Luxol Fast Blue染色以观察炎性反应、髓鞘缺失等组织病理学变化,并对各组大鼠的临床转归进行记录。
结果1.所制备的EAE模型在MRI及病理学方面的改变符合经典EAE表现。EAE脑组织中NgR的表达上调,小胶质细胞数量增加,并以激活的小胶质细胞为主。
2.以GFP标记了的重组腺病毒转染293细胞后,转染后第24-72小时,荧光显微镜下观察发现：GFP表达逐渐增多。经扩增、纯化后得重组腺病毒Ad-shRNA-NgR及Ad-shRNA-Hk的滴度分别为：2.93×1010pfu/ml,2.84×1010pfu/ml。高、中、低滴度组EAE大鼠脑组织均出现GFP阳性表达,且高、中滴度组转染效率明显高于低滴度组,但高滴度组与中滴度组的GFP表达无明显差异。HE染色显示,高滴度组大鼠脑组织转染局部有明显急性炎性反应,其余组转染局部未发现明显炎性反应。
3.转染后第7天(d14)：特异性RNAi组脑组织中NgR mRNA及蛋白的表达明显下降(p<0.01),小胶质细胞的表达及髓鞘缺失情况与阴性及空白对照组未见明显差异。转染后第14天(d21)：特异性RNAi组脑组织中NgR mRNA及蛋白表达仍低于空白及阴性对照组(p<0.01),但较该组转染后第7天时有所增加(p<0.05)；小胶质细胞的数量较空白及阴性对照组多(p<0.05),且以活化状态为主；髓鞘缺失的恢复较空白及阴性对照组好(p<0.05)。
4.与空白及阴性对照组比较,特异性RNAi组大鼠的发病潜伏期延长(p<0.05)、临床症状评分减轻(p<0.05)；d21时的髓鞘缺失缓解略明显；d21时,病灶部位内的炎性细胞消退较慢。
结论EAE模型的制备是成功的。中滴度的腺病毒可高效、安全地转染EAE脑组织。NgR特异性RNAi可明显降低EAE脑组织中NgR基因及蛋白的表达,并有利于髓鞘缺失的修复,但对于激活的小胶质细胞的早期清除不利。该现象可能是通过病灶内激活的小胶质细胞启动了髓鞘再生的修复机制,从而促进髓鞘再生。
Objective Multiple sclerosis (MS) is a inflammatory demyelinating disease in central nervous system (CNS). With the development of magnetic resonance (MRI), The incidence of MS is increasing. The pathogenesis of MS is unclear. Furthermore there is still no good way to treat this disease. So to search for a new method for nerve protection in this disease is very important. EAE is a classical model of MS. It can simulate MS no matter in clinic or in pathology. Nogo receptor is the same receptor for MAG, OMgp and NgR. The three proteins above are the inhibitors for axons regeneration in CNS. Right now some projects indicate that NgR may participate in inflammatory. And in the CNS tissue of MS, the expression of NgR is increased. It seems that NgR may play some role in MS. RNAi can reduce the expression of objective gene. Ad-shRNA-NgR can down regulation the expression of NgR. Microglias is the principal member of macrophage family in CNS. These cells might sustain and propagate inflammation within the CNS during autoimmune inflammation. So by transfecting the Ad-shRNA-NgR into the experimental autoimmune encephalomyelitis (EAE) model'lateral cerebral ventricle and observation the microglias and remyelination could help us to find the effects of NgR on microglias and remyelination.
In this research, after transfection the Ad-shRNA-NgR into the experimental autoimmune encephalomyelitis(EAE) model'lateral cerebral ventricle, we investigated:the expression of nogo receptor(NgR)mRNA and protein, the aggregation of inflammation cells, the depletion of myelin sheath and the clinical invasion of the EAE model. By doing this we can find the contribution of NgR myelin regeneration and microglias. This project can provide a therapeutic approach for MS and other inflammation diseases of neurology.
Method 1.Established the EAE model and MRI scanning, the dyeing of HE, Luxol fast blue, microglias were carried out for evaluation.
2. The recombinated adenovious was amplified in 293 cells(human embroyonic kidney cells)and purified by Sartorius Vivapure AdenoPACK 20 kit.The virus titer was determined by TCID50. Three different titers of recombinated adenovious(high titer, middle titer, low titer) were used to transfect the brain tissue of EAE. Histopathological examination was performed to evaluate the local inflammatory reaction. And green fluorescent protein (GFP) expression was observed under fluorescence microscope. The optimal transfective virus titer was selected;
3.48 female Wistar rats were randomed into normal control group、blank control group, Ad-shRNA-HK intervention group(negative control group), Ad-shRNA-NgR intervention group (specific RNAi group). Reverse transcription polymerases chain reaction (RT-PCR) was used to determine the changes of NgR mRNA expression at the 7th and 14th day after transfection.The stain of HE、Luxol Fast Blue、NgR and CDllb/C was also carried out at the 7th and 14th day after transfection.
Result 1.The MRI scanning and pathological observations of the rats were consistent with those of classical EAE model.
2. After 24 hours of transfection the fluorescence was observed in 293 cells. The titer of amplified recombined adenovirus Ad-shRNA-NgR and Ad-shRNA-HK were 2.93×1010pfu/ml and 2.84×1010pfu/ml respectively. Green fluorescence protein(GFP) could be found in the brain of the high, middle and low titer group at the 7th day of transfection. GFP expression in the high and middle titer group was obviously higher than that in the low titer group. While there was no obvious difference between the high and middle titer group. HE staining showed that acute inflammatory infiltration was only observed in the high titer group;
3. NgR protein in the brain of EAE was significantly increased at the 14th and 21th day after induction (p<0.01). NgR mRNA and protein was reduced in the brain of EAE at the 7th and 14th day after RNAi intervention (p<0.01);
4.Compared with the blank control group, the Ad-shRNA-NgR specific intervention group had longer latency (p<0.05)、lower clinical score;at the d21,the more significant regeneration of myelin sheath, while the slower extinction of inflamation.
Conclusion The EAE model was induced successfully. The middle titers of adenovirus can transfect the brain tissue of EAE model efficiently and safely. The expression of NgR was increased in the brain of EAE. Ad-shRNA-NgR interfering can degrade the expressing of NgR remarkably and enhance the regeneration of myelin sheath, but it's not good for extinction of activated microglias.
引文
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[19]Carsoa, Sutdife JG, Canal IL. Micmglia stimulate naive T-cell Differenciation without stimulating T-cell prolifexation[J]. J Neurosci Res,1999.55(1):127-134.
[20]Etty N. Benveniste.Role of macrophages/microglia in multiple sclerosis and experimental allergic encephalomyelitis. [J]. Journal of Molecular Medicine 1997, 3(2):165-173
[21]Koh CS, Paterson PY. Suppression of clinical signs of celltransferred experimental allergic encephalomyelitis and altered cerebrovascular permeability in Lewis rats treated with a p lasminogen activator inhibitor[J]. Cell Immunol,1987,107 (1): 52-63.
[22]M. Rausch, et al.MRI-Based Monitoring of Inflammation and Tissue Damage in Acute and Chronic Relapsing EAE[J]Magnetic Resonance in Medicine,2003, 50:309-314.
[23]B. Brochet, M.S.A. Deloire,IT. Touil, al ect. Early macrophage MRI of inflammatory lesions predicts lesion severity and disease development in relapsing EAE[J].NeuroImage,2006,32:266-274.
[24]Chih-Liang Chinet al ect.Distinct spatiotemporal pattern of CNS lesions revealed by USPIO-enhanced MRI in MOG-induced EAE rats implicates the involvement of spino-olivocerebellar pathways [J]. Journal of Neuroimmunology. 2009,211:49-55.
[25]Fire A, Xu S, Montomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature.1998,391:806 811
[26]Zamore PD, Tuschl T, Sharp PA, et al. RNAi:double. stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals[J]. Cell. 2000,101:25-33
[27]Brummel Kamp TR, Bernards R, Agami R, et al. A system for stable expression of short interfering RNAs inmammalian cells [J]. Science 2002:296(5567):5555
[28]Roy I, Holle L, Song W, et al. Em cient translocation an d apoptosis induction by adenovirus encoded VP22. p53 fusion protein in human tumor cells in vitro [J]. Antican cer Res 2002,22(6):3185-3189
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[30]王敬.腺病毒介导的RNAi对缺血再灌注大鼠脑内NgR表达、轴突再生和神经行为响.[D].重庆：重庆医科大学,2009
[31]Langer SJ, Schaack J.293 cell lines that inducibly express high levels of adenovirus type 5 precursor terminal protein [J].Virology.1996,222(1):172-179
[32]Brough DE, Lizonova A, Hsu C, et al. A gene transfer vector-cell line system for complete functional fomplementation of adenovirus early regions El and E4[J]. J Virol.1996,70(9):6497-6501
[33]Oualikene W, Lamoureux L, Weber J M, et al. Protease-deleted aden-ovirus vectors and complementing cell lines:potential applications of single-round replication mutants for vaccination and gene therapy[J].Human Gene Therapy. June.2000, 11(9):1341-1353
[34]Abea K, Setoguchib Y, Hayashia T, et al.In vivo adenovirus-mediated gene transfer and the expression in ischemic and reperfused rat brain[J].Brain Research. 1997,763(2):191-201
[35]Fournier AE,GrandPre T,Strittmatter SM. Identification of a receptor mediating Nogo-66 inhibition of axonal regeremtion[J]. Nature.2001,409:341 -6
[36]M. Domeniconi et al., Myelin-associated glycoprotein interacts with the Nogo66 receptor to inhibit neurite outgrowth [J].Neuron 35 (2002),35:283-290.
[37]Wang KC, Koprivica V, Kim JA, et al. Oligodendrocyte myelin glycoprotein is a Nogo receptor ligand that inhibits neurite out growth [J]. Nature.2002.417: 941-944.
[38]Lee JK, Kim JE, Sivula M, et al. Nogo receptor antagonism promotes stroke recovery by enhancing axonal plasticity [J]. J Neurosci.2004,24:6209-17
[39]Li S. Strittmatter SM. Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury [J].J Neurosci.2003,23:4219-4227.
[40]Etty N. Benveniste.Role of macrophages/microglia in multiple sclerosis and experimental allergic encephalomyelitis [J]. J Mol Med.1997,75:165-173
[41]Selmaj KW, Raine CS. Tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro [J].Ann Neurol 1988,23:339-346
[42]Luca Muzio, Gianvito Martino, Roberto Furlan. Multifaceted aspects of inflammation in multiple sclerosis:The role of microglia.[J] Journal of Neuroimmunology 2007,191:39-44
[43]Town T, Nikolic V, Tan J. The microglial "activation" continuum:from innate to adaptive responses [J]. Neuroinflammation 2005,24(2):221-223.
[44]Chari, D.M., Zhao, C., Kotter, M.R., Blakemore, W.F., Franklin, R.J.,2006. Corticosteroids delay remyelination of experimental demyelination in the rodent central nervous system. J. Neurosci. Res.83,594-605.
[45]Setzu, A., Lathia, J.D., Zhao, C., Wells, K., Rao, M.S., Ffrench-Constant, C., Franklin, R.J.,2006. Inflammation stimulates myelination by transplanted oligodendrocyte precursor cells. Glia 54,297-303.
[1]Fournier AE,GrandPre T,Strittmatter SM. Identification of a receptor mediating Nogo-66 inhibition of axonal regeremtion[J]. Nature.2001,409:341-346
[2]Oertle T,van der Haar ME,Banddow CE,et al. Nogo-A inhibits neurite outgrowth and cell spreading with three discrete regions[J].Neurosci.2003,23(13):5393-5406
[3]Ng CE,Tang BL. Nogos and the Nogo-66 receptor:factors inhibiting CNS neuron regeneration[J]. J Neurosci Res.2002,67(5):559-565
[4]Pignot V, Hein AE, Barske C, et al. Characterization of two novel proteins, NgRH1 an d NgRH2, structurally an d biochemieally homolous to the Nogo - 66 receptor. J Neurochem,2003,85:717-728
[5]Hasegawa T,Ohno K,Sano M,et al. The differential expression patterns of messenger RNAs encoding Nogo-A and Nogo-receptor in the rat central nervous system[J]. Mol Brain Res.2005,133(1):119-130
[6]胡泽岚刘莹莹金卫林等.Nogo-66受体在成年大鼠脊髓白质内胶质细胞的分布[J].军医大学学报.2004,25(16)：1444-1447
[7]Elizabeth J. Fry, Carole Ho, Samuel David. A Role for Nogo Receptor in Macrophage Clearance from Injured Peripheral Nerve.[J]Neuron.2007,53(5):649-642
[8]王华,姚裕家,陈大鹏.新生鼠缺氧缺血性脑损伤脑组织NgR mRNA和NgR蛋白表达及意义[J].四川大学学报医学版.2007,38(1)：68-72
[9]Satoh, Jun-Ichi, Onoue Hiroyuki,Arima Kunimasa ect. Nogo-A and Nogo Receptor Expression in Demyelinating Lesions of Multiple Sclerosis.[J] Journal of Neuropathology & Experimental Neurology.2005,64(5):129-138
[10]M. Domeniconi et al., Myelin-associated glycoprotein interacts with the Nogo66 receptor to inhibit neurite outgrowth, Neuron 35 (2002),35:283-290.
[11]Wang KC, Koprivica V, Kim JA, et al. Oligodendrocyte myelin glycoprotein is a Nogo receptor ligand that inhibits neurite out growth. Nature.2002.417:941 -944.
[12]Wang KC, Kim JA, Sivasankaran R, et al. P75 interacts with the Nogo receptor as a coreceptor for Nogo, MAG and OMgp. Nature,2002,420:74-78.
[13]Wong ST, Henley JR, Kanning KC, et al. A p75(NTR)and Nogo receptor complex mediates repulsive signaling by myelin associated glycoprotein. Nat Neurosci, 2002,5:1302-1308.
[14]Hong-Yan Zhu, Hou-Fu Guo, Hai-Long Hou ect. Increased expression of the Nogo receptor in the hippocampus and its relation to the neuropathology in Alzheimer's disease [J].Human Pathology,2007,(38,3), Pages 426-434.
[15]Daniel H. S. Lee, Stephen M. Strittmatter & Dinah W. Y. Sah Targeting the Nogo Receptor to Treat Central Nervous System Injuries. [J]Nature 2003,11(2):872-879
[16]Zhou C,Li Y,Nanda A,et al.HBO suppresses Nogo-A,Ng-R,or RhoA expression in the cerebral cortex after global ischemia[J]. Bio Bio Res Com.2003,309(2):368-376
[17]GrandPre T,Li S, Strittmatter SM.Nogo-66 receptor antagonist peptide promotes axonal regeneration[J]. Nature.2002,417(6888):547-551
[18]Li S. Strittmatter SM. Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury. J Neurosci.2003,23:4219 -4227.
[19]Neumann S, Bradke F, Tessier—Lavigue M, et al. Regeneration of sensory axons within the injured spinal cord induced by intragan glionic cAMP elevation. Neuron,2002,34:885-893
[20]Qiu J, Cai D, Dai H, et al. Spinal axon regeneration induced by elevation of cyclic AMP. Neuron,2002,34:895-903
[21]Winton MJ, Dubreuil CI, Lasko D, et al. Characterization ofnew cell permeable C3 - like proteins that inactivate Rho and stimulate neurite outgrowth on inhibitory substrates. J Biol Chem,2002,277:32820-32829
[22]Teng FY, Tang BL. Why do Nogo/Nogo - 66 receptor gene knock outs result in inferior regeneration compared to treatment with neutralizing agents[J]Neurochem,2005,94:865-874
[23]Li S, Kim JE,Budel S, et al. Transgenic inhibition of Nogo - 66 receptor function allows axonal sprouting an d improved loc omotion after spinal injury. Mol Cell Neurosci,2005,29:26-39
[24]张涛,袁文,刘百峰,等.siRNA干扰大鼠神经元Nogo受体mRNA表达的时程研究[J].中国脊柱脊髓杂志,2005,15：588-591
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[14]Satoh, Jun-Ichi, Onoue Hiroyuki,Arima Kunimasa ect. Nogo-A and Nogo Receptor Expression in Demyelinating Lesions of Multiple Sclerosis.[J] Journal of Neuropathology & Experimental Neurology.2005,64(5):129-138
[15]Hong-Yan Zhu, Hou-Fu Guo, Hai-Long Hou ect. Increased expression of the Nogo receptor in the hippocampus and its relation to the neuropathology in Alzheimer's disease.[J]. Human Pathology.2007,(38,3), Pages 426-434
[16]Zamore PD, Tuschl T, Sharp PA, et al. RNAi:double. stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals[J]. Cell, 2000,101:25-33
[17]张沁丽,秦新月,殷成等.编码大鼠Nogo受体mRNA的shRNA重组腺病毒载体的构建和鉴定[J].第四军医大学学报.2008,29：55-57
[18]Aloisi F. Immunefunction of microglia[J]. Glia,2001,36(2):165-179
[19]Carsoa, Sutdife JG, Canal IL. Micmglia stimulate naive T-cell Differenciation without stimulating T-cell prolifexation[J]. J Neurosci Res,1999.55(1):127-134.
[20]Etty N. Benveniste.Role of macrophages/microglia in multiple sclerosis and experimental allergic encephalomyelitis. [J]. Journal of Molecular Medicine 1997, 3(2):165-173
[21]Koh CS, Paterson PY. Suppression of clinical signs of celltransferred experimental allergic encephalomyelitis and altered cerebrovascular permeability in Lewis rats treated with a p lasminogen activator inhibitor[J]. Cell Immunol,1987,107 (1): 52-63.
[22]M. Rausch, et al.MRI-Based Monitoring of Inflammation and Tissue Damage in Acute and Chronic Relapsing EAE[J]Magnetic Resonance in Medicine,2003, 50:309-314.
[23]B. Brochet, M.S.A. Deloire,IT. Touil, al ect. Early macrophage MRI of inflammatory lesions predicts lesion severity and disease development in relapsing EAE[J].NeuroImage,2006,32:266-274.
[24]Chih-Liang Chinet al ect.Distinct spatiotemporal pattern of CNS lesions revealed by USPIO-enhanced MRI in MOG-induced EAE rats implicates the involvement of spino-olivocerebellar pathways [J]. Journal of Neuroimmunology. 2009,211:49-55.
[25]Fire A, Xu S, Montomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature.1998,391:806 811
[26]Zamore PD, Tuschl T, Sharp PA, et al. RNAi:double. stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals[J]. Cell. 2000,101:25-33
[27]Brummel Kamp TR, Bernards R, Agami R, et al. A system for stable expression of short interfering RNAs inmammalian cells [J]. Science 2002:296(5567):5555
[28]Roy I, Holle L, Song W, et al. Em cient translocation an d apoptosis induction by adenovirus encoded VP22. p53 fusion protein in human tumor cells in vitro [J]. Antican cer Res 2002,22(6):3185-3189
[29]DeMatteo RP, Chu G, Ahn M. Immunologic barriers to hepatic adenoviral gene therapy for transplantation:Cellular and Humoral Responses Limit Transgene Expression in Mouse Liver 1 [J]. Transplantation.1997,63(2):315-319
[30]王敬.腺病毒介导的RNAi对缺血再灌注大鼠脑内NgR表达、轴突再生和神经行为响.[D].重庆：重庆医科大学,2009
[31]Langer SJ, Schaack J.293 cell lines that inducibly express high levels of adenovirus type 5 precursor terminal protein [J].Virology.1996,222(1):172-179
[32]Brough DE, Lizonova A, Hsu C, et al. A gene transfer vector-cell line system for complete functional fomplementation of adenovirus early regions El and E4[J]. J Virol.1996,70(9):6497-6501
[33]Oualikene W, Lamoureux L, Weber J M, et al. Protease-deleted aden-ovirus vectors and complementing cell lines:potential applications of single-round replication mutants for vaccination and gene therapy[J].Human Gene Therapy. June.2000, 11(9):1341-1353
[34]Abea K, Setoguchib Y, Hayashia T, et al.In vivo adenovirus-mediated gene transfer and the expression in ischemic and reperfused rat brain[J].Brain Research. 1997,763(2):191-201
[35]Fournier AE,GrandPre T,Strittmatter SM. Identification of a receptor mediating Nogo-66 inhibition of axonal regeremtion[J]. Nature.2001,409:341 -6
[36]M. Domeniconi et al., Myelin-associated glycoprotein interacts with the Nogo66 receptor to inhibit neurite outgrowth [J].Neuron 35 (2002),35:283-290.
[37]Wang KC, Koprivica V, Kim JA, et al. Oligodendrocyte myelin glycoprotein is a Nogo receptor ligand that inhibits neurite out growth [J]. Nature.2002.417: 941-944.
[38]Lee JK, Kim JE, Sivula M, et al. Nogo receptor antagonism promotes stroke recovery by enhancing axonal plasticity [J]. J Neurosci.2004,24:6209-17
[39]Li S. Strittmatter SM. Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury [J].J Neurosci.2003,23:4219-4227.
[40]Etty N. Benveniste.Role of macrophages/microglia in multiple sclerosis and experimental allergic encephalomyelitis [J]. J Mol Med.1997,75:165-173
[41]Selmaj KW, Raine CS. Tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro [J].Ann Neurol 1988,23:339-346
[42]Luca Muzio, Gianvito Martino, Roberto Furlan. Multifaceted aspects of inflammation in multiple sclerosis:The role of microglia.[J] Journal of Neuroimmunology 2007,191:39-44
[43]Town T, Nikolic V, Tan J. The microglial "activation" continuum:from innate to adaptive responses [J]. Neuroinflammation 2005,24(2):221-223.
[44]Chari, D.M., Zhao, C., Kotter, M.R., Blakemore, W.F., Franklin, R.J.,2006. Corticosteroids delay remyelination of experimental demyelination in the rodent central nervous system. J. Neurosci. Res.83,594-605.
[45]Setzu, A., Lathia, J.D., Zhao, C., Wells, K., Rao, M.S., Ffrench-Constant, C., Franklin, R.J.,2006. Inflammation stimulates myelination by transplanted oligodendrocyte precursor cells. Glia 54,297-303.
[1]Fournier AE,GrandPre T,Strittmatter SM. Identification of a receptor mediating Nogo-66 inhibition of axonal regeremtion[J]. Nature.2001,409:341-346
[2]Oertle T,van der Haar ME,Banddow CE,et al. Nogo-A inhibits neurite outgrowth and cell spreading with three discrete regions[J].Neurosci.2003,23(13):5393-5406
[3]Ng CE,Tang BL. Nogos and the Nogo-66 receptor:factors inhibiting CNS neuron regeneration[J]. J Neurosci Res.2002,67(5):559-565
[4]Pignot V, Hein AE, Barske C, et al. Characterization of two novel proteins, NgRH1 an d NgRH2, structurally an d biochemieally homolous to the Nogo - 66 receptor. J Neurochem,2003,85:717-728
[5]Hasegawa T,Ohno K,Sano M,et al. The differential expression patterns of messenger RNAs encoding Nogo-A and Nogo-receptor in the rat central nervous system[J]. Mol Brain Res.2005,133(1):119-130
[6]胡泽岚刘莹莹金卫林等.Nogo-66受体在成年大鼠脊髓白质内胶质细胞的分布[J].军医大学学报.2004,25(16)：1444-1447
[7]Elizabeth J. Fry, Carole Ho, Samuel David. A Role for Nogo Receptor in Macrophage Clearance from Injured Peripheral Nerve.[J]Neuron.2007,53(5):649-642
[8]王华,姚裕家,陈大鹏.新生鼠缺氧缺血性脑损伤脑组织NgR mRNA和NgR蛋白表达及意义[J].四川大学学报医学版.2007,38(1)：68-72
[9]Satoh, Jun-Ichi, Onoue Hiroyuki,Arima Kunimasa ect. Nogo-A and Nogo Receptor Expression in Demyelinating Lesions of Multiple Sclerosis.[J] Journal of Neuropathology & Experimental Neurology.2005,64(5):129-138
[10]M. Domeniconi et al., Myelin-associated glycoprotein interacts with the Nogo66 receptor to inhibit neurite outgrowth, Neuron 35 (2002),35:283-290.
[11]Wang KC, Koprivica V, Kim JA, et al. Oligodendrocyte myelin glycoprotein is a Nogo receptor ligand that inhibits neurite out growth. Nature.2002.417:941 -944.
[12]Wang KC, Kim JA, Sivasankaran R, et al. P75 interacts with the Nogo receptor as a coreceptor for Nogo, MAG and OMgp. Nature,2002,420:74-78.
[13]Wong ST, Henley JR, Kanning KC, et al. A p75(NTR)and Nogo receptor complex mediates repulsive signaling by myelin associated glycoprotein. Nat Neurosci, 2002,5:1302-1308.
[14]Hong-Yan Zhu, Hou-Fu Guo, Hai-Long Hou ect. Increased expression of the Nogo receptor in the hippocampus and its relation to the neuropathology in Alzheimer's disease [J].Human Pathology,2007,(38,3), Pages 426-434.
[15]Daniel H. S. Lee, Stephen M. Strittmatter & Dinah W. Y. Sah Targeting the Nogo Receptor to Treat Central Nervous System Injuries. [J]Nature 2003,11(2):872-879
[16]Zhou C,Li Y,Nanda A,et al.HBO suppresses Nogo-A,Ng-R,or RhoA expression in the cerebral cortex after global ischemia[J]. Bio Bio Res Com.2003,309(2):368-376
[17]GrandPre T,Li S, Strittmatter SM.Nogo-66 receptor antagonist peptide promotes axonal regeneration[J]. Nature.2002,417(6888):547-551
[18]Li S. Strittmatter SM. Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury. J Neurosci.2003,23:4219 -4227.
[19]Neumann S, Bradke F, Tessier—Lavigue M, et al. Regeneration of sensory axons within the injured spinal cord induced by intragan glionic cAMP elevation. Neuron,2002,34:885-893
[20]Qiu J, Cai D, Dai H, et al. Spinal axon regeneration induced by elevation of cyclic AMP. Neuron,2002,34:895-903
[21]Winton MJ, Dubreuil CI, Lasko D, et al. Characterization ofnew cell permeable C3 - like proteins that inactivate Rho and stimulate neurite outgrowth on inhibitory substrates. J Biol Chem,2002,277:32820-32829
[22]Teng FY, Tang BL. Why do Nogo/Nogo - 66 receptor gene knock outs result in inferior regeneration compared to treatment with neutralizing agents[J]Neurochem,2005,94:865-874
[23]Li S, Kim JE,Budel S, et al. Transgenic inhibition of Nogo - 66 receptor function allows axonal sprouting an d improved loc omotion after spinal injury. Mol Cell Neurosci,2005,29:26-39
[24]张涛,袁文,刘百峰,等.siRNA干扰大鼠神经元Nogo受体mRNA表达的时程研究[J].中国脊柱脊髓杂志,2005,15：588-591
[25]Park, J.H. et al. (2006) Alzheimer precursor protein interaction with the Nogo-66 receptor reduces amyloid-b plaque deposition.[J] Neurosci.26,1386-1395