RNA干扰沉默神经干细胞NgR基因、阻断Nogo-66抑制神经突生长作用的研究
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摘要
RNA干扰沉默神经干细胞NgR基因、阻断Nogo-66抑制神经突生长作用的研究
目的
中枢神经系统损伤后的再生是世界性的难题。中枢神经系统髓鞘相关抑制因子包括Nogo-A，髓磷脂相关糖蛋白myelin- associated glycoprotein(MAG)和少突胶质细胞髓磷脂糖蛋白oligodendrocyte myelin glycoprotein(OMgp)，它们都具有轴突生长抑制作用，在中枢神经系统神经再生的研究中倍受关注。研究表明Nogo蛋白分子羧基端两个跨膜区之间存在一个66个氨基酸的结构域Nogo-66，Nogo-66通过与Nogo-66受体(Nogo-66 Receptor，NgR)相结合具有重要的轴突生长抑制活性。MAG和OMgp也先后被发现是NgR的配体。在寻找NgR协同受体的研究中和直接结合试验都证实NgR是MAG的受体。Wang的研究表明NgR也是OMgp的受体。体外试验表明Nogo-66，MAG和OMgp的神经突生长抑制作用都通过NgR介导。NgR成为治疗脊髓损伤研究的焦点。
神经干细胞移植是治疗脊髓损伤最有前途的方案之一，神经干细胞是否表达NgR及移植后的神经干细胞在其分化形成神经元的过程中是否受到脊髓损伤局部Nogo-66的影响目前尚未见报道。本研究检测了大鼠脊髓来源神经干细胞NgR的表达情况，在体外培养的神经干细胞分化过程中加入Nogo-66的活性片段Nogo-P4，显微测量神经元神经突长度，观察Nogo-66对神经干细胞分化形成的神经元神经突生长是否同样存在抑制作用，并进一步观察能否用RNAi的方法沉默神经干细胞NgR基因表达，阻断Nogo-66的抑制作用。
实验方法
一、实验动物生后24h内的Wistar大鼠由中国医科大学动物实验中心提供。
二、主要试剂
表皮生长因子(epidermal growth factor，EGF)、NgR和nestin兔抗鼠单克隆抗体购自Chemicon公司，碱性成纤维细胞生长因子(basal fibroblast growth factor，bFGF)、DMEM／F12培养基、B27、PI、Alexa Fluor~(?) 488羊抗兔荧光二抗、Block-It Fluorescent Oligo、Lipofectamine 2000和Opti MEM I均购自Invitrogen公司。兔抗鼠神经元特异性烯醇化酶(neuron specific enolase，NSE)、胶质纤维酸性蛋白质(glial fibrillary acidic protein，GFAP)和髓鞘碱性蛋白(myelin basic protein，MBP)抗体均购自武汉博士德生物公司。Nogo-P4购自ADI公司。
三、神经干细胞的培养和鉴定
(一)细胞培养取4只出生24h内的Wistar大鼠，处死后在超净台内取出脊髓。将脊髓剪碎，胰酶消化后加入添加有B27、20ng／ml bFGF和20ng／ml EGF的DMEM-F12培养基，重悬浮，200目细胞筛过滤后接种于培养瓶中，置于37℃5％CO_2培养箱中悬浮培养。
(二)Nestin免疫荧光染色培养2周后，取神经球固定后，以nestin兔抗鼠单克隆抗体标记，常规方法免疫荧光染色，荧光显微镜下观察照相。
(三)诱导分化取神经球尽量吹打分散成单细胞，将神经干细胞悬液加入预先已置入玻璃盖片的6孔培养板中，加入10％胎牛血清。1周后取出盖片，分别以NSE，GFAP和MBP抗体标记，常规方法免疫荧光染色。
四、NgR表达的检测
培养2周后，取神经球固定后，以NgR兔抗鼠单克隆抗体标记，常规方法免疫荧光染色，荧光显微镜下观察照相。
五、小分子干扰RNA(small interfering RNA，siRNA)的设计和合成
利用Invitrogen公司网上siRNA设计软件设计3条大鼠NgR mRNA的siRNA，由Invitrogen公司进行合成相应的双链RNA(double stranded RNA，dsRNA)。经预试验筛选出阻断NgR基因最有效的一条siRNA，其序列为：
Sequence(5′to 3′)：-UGC AGU ACC UCU ACC UAC AAG ACA A
Sequence(5′to 3′)：-UUG UCU UGU AGG UAG AGG UAC UGC A
六、实验分组
将神经干细胞分为siRNA组，Nogo-P4组，siRNA+Nogo-P4组和对照组。对照组加入10％胎牛血清使神经干细胞开始分化；Nogo-P4组加入10％胎牛血清和4μmol／l的Nogo-P4；siRNA组的神经干细胞先经过siRNA转染，24h后加入10％胎牛血清开始分化；siRNA+Nogo-P4组的神经干细胞先经过siRNA转染，24h后加入10％胎牛血清和4μmol／l的Nogo-P4开始分化。
七、siRNA的转染
取1ml含神经干细胞的培养液加入离心管，800r／min离心5min，弃上清，加入900ul不含抗生素的培养基，机械吹打分散细胞，加入siRNA和转染试剂的混合物进行转染。24h和72h后检测NgR基因表达。另取部分神经干细胞以荧光标记的dsRNA(Block-It Fluorescent Oligo)进行相同条件下的转染，荧光显微镜下观察并计算转染效率。
八、NgR基因沉默效率的检测
(一)免疫荧光检测转染24h和72h后的神经干细胞固定后，加入NgR兔抗鼠单克隆抗体标记，常规方法免疫荧光染色，荧光显微镜下观察照相。每张盖片随机取10个视野，计数NgR阳性细胞数N和总细胞数T，计算阻断效率(T-N)／T。实验重复3次。
(二)Western blot检测将培养液倒至15ml离心管中，1000rpm离心5min。弃上清，加入4ml PBS并轻轻吹打洗涤，然后1000rpm离心5min。弃上清后用PBS重复洗涤一次。弃去上清，加400μl裂解液，冰上裂解30min。将裂解液4℃、12000rpm离心5min，取上清分装于0.5ml离心管中并置于-20℃保存。用Bradford法进行蛋白质定量。蛋白上样量30μg，上样前将样品于沸水中煮5min。电泳后转印到硝酸纤维素膜。5％的脱脂牛奶封闭非特异性背景，室温1h。将兔抗鼠NgR抗体用TBST稀释至1∶400，将膜放入一抗稀释液中，4℃过夜。用TBS在室温下洗两次，每次10min；再用TBS洗一次，10min。准备二抗稀释液并与膜接触，室温下孵育2h。用TBS在室温洗两次，每次10min；再用TBS洗一次，10min。用KPL公司的蛋白检测试剂盒显色。
九、神经元神经突长度的测量
神经干细胞分化第3d，以NSE抗体标记，常规方法免疫荧光染色，荧光显微镜下观察照相。使用Image-Pro Plus 5.0软件进行神经元细胞神经突长度的测量。
十、统计分析
实验数据以SPSS11.5软件进行处理，差异显著性采用单因素方差分析。
实验结果
悬浮培养的细胞形成典型的神经球，经免疫荧光检测nestin和NgR表达阳性。加入10％胎牛血清分化1周后，可观察到NSE，GFAP和MBP标记阳性的细胞。这表明培养出的神经球具备分化成神经元，星形胶质细胞和少突胶质细胞的能力，是神经干细胞。
NgR抗体标记染色后，分化前的神经球和散在的单细胞均呈现强表达。
分化第3d，NgR表达阳性的细胞比率明显下降，只有(35.3±4.31)％的细胞NgR表达阳性。分化细胞的形态趋于成熟，可以识别出部分具有典型形态的神经元和胶质细胞。经双重免疫荧光染色证实，NgR表达阳性的细胞均为NSE表达阳性的细胞，多数NgR~+／NSE~+的细胞具有典型的神经元形态，双极或多级，有较长的突起。
分化第3d Nogo-P4组神经元神经突长度为80.54±6.75μm；对照组神经元神经突长度为97.80±6.97μm。Nogo-P4组和对照组神经元神经突长度有显著差异，P＜0.01。Nogo-P4组神经元神经突长度较对照组缩短约17％。
siRNA组神经干细胞经siRNA转染后大部分神经干细胞存活，少量细胞死亡，存活的细胞活力良好，超过24h后不再出现细胞死亡。部分神经干细胞以荧光标记的dsRNA(Block-It Fluorescent Oligo)进行相同条件下的转染，在荧光显微镜下观察证实dsRNA已进入神经干细胞内，转染获得成功，转染效率为94.3％。
转染后24h神经干细胞经NgR抗体标记染色，荧光显微镜下观察发现大量神经干细胞的NgR表达阴性，这表明RNA干扰可以引起NgR基因表达沉默。用随机打乱顺序的siRNA转染作为阴性对照，神经干细胞的NgR表达没有减弱，镜下显示为明亮的绿色荧光。Western blot检测的结果证实对照组和经随机打乱顺序的siRNA(scrambled siRNA)转染后24小时的神经干细胞NgR均显著表达，经siRNAC转染后24 h的神经干细胞NgR表达显著降低。转染后24h的NgR基因阻断效率为(90.35±3.1)％。转染后72h的NgR基因阻断效率为(89.96±3.9)％。
分化第3d，siRNA组神经元神经突长度为92.14±7.27μm，siRNA+Nogo-P4组神经元神经突长度为94.01±8.37μm。siRNA组及siRNA+Nogo-P4组和对照组神经元神经突长度无显著差异；siRNA+Nogo-P4组和Nogo-P4组神经元神经突长度有显著差异，P＜0.01。
结论
1、大鼠脊髓来源神经干细胞显著表达NgR。
2、在神经干细胞分化成神经元的过程中始终表达NgR。
3、神经干细胞分化过程中神经元的神经突生长会受到Nogo-P4的抑制。
4、Nogo-P4的神经突生长抑制作用随浓度升高而升高，在4μm／L时达到最大抑制作用。
5、RNA干扰的方法能够高效率地使神经干细胞的NgR基因表达沉默。
6、NgR基因表达沉默能够阻断Nogo-P4的神经突生长抑制作用。
Disinhibition of neuronal neur ite outgrowth in the presence of Nogo-66 by siRNA mediated knockdown of NgR of neural stem cells
Objectives
Several proteins of CNS myelin possess axon growth- inhibiting properties, notably Nogo-A, myelin- associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp). The 66 aa surface loop of Nogo is inhibitory for axonal growth in culture and acts via a Nogo-66 Receptor (NgR) expressed by neurons and localized to their axons in vitro. Remarkably, the NgR protein mediates the inhibitory action of MAG and OMgp, as well as Nogo-66. NgR is a target for treatment to promote axon recovery because Nogo, MAG, and OMgp all bind to NgR to mediate their inhibitory effects on neurite outgrowth.
Exclude approaches aimed at disinhibiting axon outgrowth to treat spinal injury, regenerative medicine using stem cell biology is attracting a lot of attention because this is considered a particularly important therapeutic strategy for regeneration of the CNS. But if NSCs express NgR and could be affected by myelin associated inhibitory proteins in the progress of differentiation has not been proved.
Nogo-P4 is an active segment of Nogo-66 and has the core inhibitory activity of Nogo-66. Here, we use immunocytochemistry and Western blot methods to detect NgR in NSCs derived from spinal cord of rat, compare the average neuronal neurite length、differentiated neuron percentage and percentage of neurite possessing neuron during the differentiation of NSCs with and without Nogo-P4, before and after siRNA-mediated knockdown of NgR on NSCs.
Experimental methods
1. Animals
Postnatal 1 day Wistar rats were supplied by animal center of China Medical University.
2. Main reagents
Monoclonal NgR antibody (Chemicon) was used to label NSCs at 10μg/ml for immunocytochemistry(ICC) and Western blots. Polyclonal neuron specific enolase (NSE) antibody, glial fibrillary acidic protein (GFAP) antibody and myelin basic protein (MBP) antibody were purchased from Boster Company and were used to label neurons and neurites, astrocytes and oligodendrocytes respectively at 1:300. Monoclonal nestin antibody (Chemicon) was used to label NSCs at 1:200. Second antibody Alexa Fluor(?) 488 was purchased from Invitrogen. PI(Sigma) was used for counterstaining to identify nuclei at 1:3000. DMEM/F12 medium、B27、PI、Block-It Fluorescent Oligo、LIPOFECTAMINE 2000 REAGENT and OPTI MEM were purchased from Invitrogen company.
3. Culture and identification of NSCs
3.1 Culture of NSCs
The culture methods were based on those described previously. Spinal cords of P1(postnatal 1 day) Wistar rats(animal center of China Medical University) were dissected and dissociated into single cells using a solution of 0.125% trypsin for 45min at 37℃. The suspension was centrifuged at 1000 rpm for 10 min at 4℃. Decant as much of the supernatant as possible. Resuspend the cells in a total volume of 10 mL of DMEM/F12 (Invitrogen) serum-free stem cell medium containing freshly added epidermal growth factor (EGF) (Chemicon) 20ng/ml, basal fibroblast growth factor(bFGF) (Invitrogen) 20ng/ml and B27((Invitrogen, 1:50) (prewarmed to 37℃). Cell suspension was cultured in a atmosphere containing 5% CO_2 at 37℃.
3.2 Expression of nestin
Neurospheres in primary cultures or subcultures were collected by centrifugation at 1000 rpm for 10 min at 4℃. These spheres were fixed in acetone for 15 min after brief washes in 0.01M phosphatebuffered saline (PBS). Some were dropped on poly-d-lysine coated coverslips and immunostained intact to detect nestin immunoreactivity of the spheres.
3.3 Differentiation of NSCs
NSCs suspension was added to 6 well culture plate with glass cover slips pre-coated with poly-D-lysine in it and cultured in the presence of 10% FBS to initiate differentiation. After 1 week of differentiation cover slips were taken out for immunocytochemisty assay. The differentiated cells were incubated with primary antibodies against GFAP, MBP and NSE, respectively, and then exposed to secondary antibodies.
Microscopic images of the immunostained neurospheres and differentiated cells were captured using a SpotRT digital camera (Diagnostic Instruments Inc.) mounted on an Olympus BX60 microscope (Olympus Optical Co.).
4. Detection of NgR expression
Neurospheres in primary cultures or subcultures were collected by centrifugation at 1000 rpm for 10 min at 4℃. These spheres were fixed in acetone for 15 min after brief washes in 0.01M phosphatebuffered saline (PBS). Some were dropped on poly-d-lysine coated coverslips and immunostained intact to detect NgR immunoreactivity of the spheres.
5. Design and synthesis of siRNA
SiRNA directed against NgR was designed and synthesized by Invitrogen Company. The siRNA sequences used were:
siRNAA
Sequence (5′to 3′): - UUU GAG UGC AGC CAC AGG AUG GUG A
Sequence (5′to 3′): - UCA CCA UCC UGU GGC UGC ACU CAAA
siRNA B
Sequence (5′to 3′): - UCA GUG AGC UGA UUG GUC UGG AAG G
Sequence (5′to 3′): - CCU UCC AGA CCAAUC AGC UCA CUG A
siRNA C
Sequence (5′to 3′): - UGC AGU ACC UCU ACC UAC AAG ACAA
Sequence(5′to 3′): - UUG UCU UGU AGG UAG AGG UAC UGC A
6. Group
NSCs were divided into control group, Nogo-P4 group, siRNA group and siRNA+Nogo-P4 group. In control group, NSCs differentiated in the presence of 10% FBS; in Nogo-P4 group NSCs differentiated in the presence of 10% FBS and 4μmol/l Nogo-P4; in siRNA group, NSCs were transfected with siRNA before they differentiated in the presence of 10% FBS; in siRNA+Nogo-P4 group, NSCs were transfected with siRNA before they differentiated in the presence of 10% FBS and 4μmol/l Nogo-P4.
7. SiRNA preparation and transfection
Before transfection the neurospheres were triturated into single cells as much as possible. In one tube, 50pmol of siRNA was mixed in 50μl Opti-MEM, while a second tube contained 1μl Lipofectamine 2000 reagent and 50μl Opti-MEM and each was incubated at room temperature for 15min before the two solutions were combined and incubate for complex formation. Then the complex was added to NSCs suspension without antibiotics. After 1d, 3d and 5d the NSCs were collected and immunostained with NgR antibody to detect the knockdown efficiency.
Block-It Fluorescent Oligo was fluorescence labeled duplex siRNA used for observation of transfection efficiency. The transfection procedures are as described above.
8. Detection of knockdown efficiency
8.1 Immunohistochemistry
After 1d, 3d and 5d of transfection, the NSCs were collected and immunostained with NgR antibody to detect the knockdown efficiency. The cells attached to coverslips in differential cultures were briefly riNSEd with 0.01M PBS and fixed in 4% paraformaldehyde in 0.1M phosphate buffer for 30 min at 4℃.
Before staining, cover slips were incubated sequentially in 0.3% Triton X-100 in PBS (PBS-T; 30 min), 10% goat serum in PBS (30 min), and then with primary antibodies for 24hr at 4℃. After incubating in the primary antibodies for 24hr, sections were washed in PBS and then incubated for 2 hr at room temperature with second antibody (Alexa Fluor(?) 488, Invitrogen).
8.2 Western blots
The NgR protein expression was detected by Western blot method. Cells were collected by centrifugation(1000rpm, 5min) and washed three times with ice-cold PBS, then lysed in buffer(50mM Tris-HCl, pH8.0, 150mM NaCl, 100μg/ml PMSF, 1%TritonX-100) for 30 rain at ice. After removal of cell debris by centrifugation (12, 000rpm, 4℃), the protein concentration of lysates was meatured by Bradford method, 40μg of each lysate sample was boiled for 5 min in sample buffer and were separated by 10% SDS-PAGE and transferred onto nitrocellulose membrane (Pall Corporation). Nonspecific reactivity was blocked in 5% nonfat dry milk in TBST (10mM Tris-HCl, pH7.5, 150mM NaCl, 0.05%Tween-20) for 1h at room temperature. The membrane was then incubated overnight at 4℃with monoclonal anti-NgR antibody (Chemicon) followed by reaction with alkaline phosphatase conjugated second antibody. Reactive protein was detected by Protein Detector BCIP/NBT Western Blotting kit (KPL).
9. Measurement of neurite outgrowth
NSE was used to label the neuron by immunocytoflurescence method. Neurite length was measured by Image-Pro Plus 5.0 software.
10. Statistical analysis
The numbers of NgR~+ cells were determined in at least three samples in each culture. The total cell number was determined by PI counterstaining. About 400 cells of each culture were counted. One-Way Analysis of Variance was used for the comparison of different cultures. All the data were treated by SPSS 11.5 software.
Results
1. Expression of NgR on NSOs
Growth of neurospheres derived from spinal cord of rat is relatively slow. Neurospheres could be seen after suspension culture for 1 week and obtained in large amount after 2 weeks. The neurospheres express nestin positively and they could differentiate into NSE, GFAP and MBP positive cells after mitogens were removed and they were cultured in the presence of 10% fetal bovine serum. So the neurospheres are capable to generate neurons, astrocytes as well as oligodendrocytes and they have pluripotency.
Both neurospheres and single neural stem cells express NgR positively after they were treated by immunocytochemistry.
In the test of Western blot, NSCs were confirmed to express NgR apparently.
2. Inhibition effect of Nogo-P4
In Nogo-P4 group, Nogo-P4 was added to the culture medium during the differentiation progress of NSCs at the concentration of 4μmol/L. In control group, NSCs differentiated without Nogo-P4. In both Nogo-P4 group and control group, 10% FBS was added to the culture to initiate and promote differentiation. NSE was used to mark the neuron by immunocytofluorecence method. Neurite length of neurons differentiated from NSCs was measured under fluorescence microscope.
In control group, the average neuron neurite length was 97.80±6.97(μm/cell) three days after the neural stem cell began to differentiate. The differentiated neuron percentage (NP) was 34.73±5.21%; the percentage of neurite possessing neuron (NPNP) was 58.67+4.31%. In Nogo-P4 group, the average neurite length was 80.54±6.75(μm/cell). NP was 38.97±5.79%, NPNP was 88.52±3.96%. The neurite length has significant difference between the Nogo-P4 group and control group, P＜0.01. The neurite length was shortened about 17% in Nogo-P4 group. So Nogo-P4 inhibits neurites outgrowth during the differentiation progress of neural stem cell. NP has no differences between control group and Nogo-P4 group, while NPNP of Nogo-P4 group is significantly higher than that of control group (p＜0.05).
3. siRNA-mediated knockdown of NgR on NSCs
After transfection, most NSCs survived. Some NSCs were transfected with fluorescence labeled dsRNA (Block-It Fluorescent Oligo) at same condition to observe the transfection efficiency. The total transfection efficiency was 94.3%.
We tested the NgR immunoreactivity at 1, 3 and 5 days after transfection. NgR immunoreactivity was markedly reduced in NSCs transfected with siRNA sequence C. The scrambled NgR siRNA sequence did not alter NgR immunoreactivity.
In Western blot test, we detect the expression of NgR 1d after transfection of sequence A～C and scrambled siRNA. NSCs without transfection were detected too as control group. NgR gene was knocked down by the three siRNA in different degree. NgR protein was expressed significantly in control group and NSCs transfected by scrambled siRNA.
The knockdown efficiency decreased as time elongated after transfection. The most effective siRNA (Sequence C) almost completely knocked down NgR (90.35±3.1% knockdown, P＜0.01vs scrambled siRNA) 1d after transfection and kept high knock-down efficiency (85.22±3.1%) even at 5 days after transfection.
The NgR~- NSCs were defined as siRNA group and differentiated in the presence of 10% FBS. After 3 days, the neurite length was 92.14±7.27 (μm/cell); the differentiated neuron percentage (NP) was 32.01±4.82%; the percentage of neurite possessing neuron (NPNP) was 53.62±6.53 %.
4. Disinhibition of neurite outgrowth of neurons
differentiated from neural stem cells by siRNA-mediated knockdown of NgR
In siRNA+Nogo-P4 group NSCs were transfected with siRNA sequence C to knockdown NgR before they differentiate in the presence of Nogo-P4 and 10% FBS. The neurite length was 94.01±8.37 (μm/cell) three days after the neural stem cell began to differentiate. It has no difference with control group and has significant difference with Nogo-P4 group, P＜0.01. NP was 22.74±4.56%; NPNP was 86.61+2.94 %. Significantly, NPNP of siRNA group was higher than NPNP of control group (p＜0.05) and has no difference with that of Nogo-P4 group.
Conelusion
1. Neural stem cells express NgR apparently.
2. Neural stem cells express NgR apparently during the differentiation progress to neurons.
3. Nogo-P4 could inhibit neuron neurite outgrowth differentiated from neural stem cell by interacting with NgR.
4. The inhibition effect of Nogo-P4 reached apex in 4μmol/L.
5. NgR expression of neural stem cells could be knocked down effectively by RNAi.
6. Inhibition effect of Nogo-P4 could be removed by siRNA mediated knockdown of NgR.
目的
中枢神经系统损伤后的再生是世界性的难题。中枢神经系统髓鞘相关抑制因子包括Nogo-A，髓磷脂相关糖蛋白myelin- associated glycoprotein(MAG)和少突胶质细胞髓磷脂糖蛋白oligodendrocyte myelin glycoprotein(OMgp)，它们都具有轴突生长抑制作用，在中枢神经系统神经再生的研究中倍受关注。研究表明Nogo蛋白分子羧基端两个跨膜区之间存在一个66个氨基酸的结构域Nogo-66，Nogo-66通过与Nogo-66受体(Nogo-66 Receptor，NgR)相结合具有重要的轴突生长抑制活性。MAG和OMgp也先后被发现是NgR的配体。在寻找NgR协同受体的研究中和直接结合试验都证实NgR是MAG的受体。Wang的研究表明NgR也是OMgp的受体。体外试验表明Nogo-66，MAG和OMgp的神经突生长抑制作用都通过NgR介导。NgR成为治疗脊髓损伤研究的焦点。
神经干细胞移植是治疗脊髓损伤最有前途的方案之一，神经干细胞是否表达NgR及移植后的神经干细胞在其分化形成神经元的过程中是否受到脊髓损伤局部Nogo-66的影响目前尚未见报道。本研究检测了大鼠脊髓来源神经干细胞NgR的表达情况，在体外培养的神经干细胞分化过程中加入Nogo-66的活性片段Nogo-P4，显微测量神经元神经突长度，观察Nogo-66对神经干细胞分化形成的神经元神经突生长是否同样存在抑制作用，并进一步观察能否用RNAi的方法沉默神经干细胞NgR基因表达，阻断Nogo-66的抑制作用。
实验方法
一、实验动物生后24h内的Wistar大鼠由中国医科大学动物实验中心提供。
二、主要试剂
表皮生长因子(epidermal growth factor，EGF)、NgR和nestin兔抗鼠单克隆抗体购自Chemicon公司，碱性成纤维细胞生长因子(basal fibroblast growth factor，bFGF)、DMEM／F12培养基、B27、PI、Alexa Fluor~(?) 488羊抗兔荧光二抗、Block-It Fluorescent Oligo、Lipofectamine 2000和Opti MEM I均购自Invitrogen公司。兔抗鼠神经元特异性烯醇化酶(neuron specific enolase，NSE)、胶质纤维酸性蛋白质(glial fibrillary acidic protein，GFAP)和髓鞘碱性蛋白(myelin basic protein，MBP)抗体均购自武汉博士德生物公司。Nogo-P4购自ADI公司。
三、神经干细胞的培养和鉴定
(一)细胞培养取4只出生24h内的Wistar大鼠，处死后在超净台内取出脊髓。将脊髓剪碎，胰酶消化后加入添加有B27、20ng／ml bFGF和20ng／ml EGF的DMEM-F12培养基，重悬浮，200目细胞筛过滤后接种于培养瓶中，置于37℃5％CO_2培养箱中悬浮培养。
(二)Nestin免疫荧光染色培养2周后，取神经球固定后，以nestin兔抗鼠单克隆抗体标记，常规方法免疫荧光染色，荧光显微镜下观察照相。
(三)诱导分化取神经球尽量吹打分散成单细胞，将神经干细胞悬液加入预先已置入玻璃盖片的6孔培养板中，加入10％胎牛血清。1周后取出盖片，分别以NSE，GFAP和MBP抗体标记，常规方法免疫荧光染色。
四、NgR表达的检测
培养2周后，取神经球固定后，以NgR兔抗鼠单克隆抗体标记，常规方法免疫荧光染色，荧光显微镜下观察照相。
五、小分子干扰RNA(small interfering RNA，siRNA)的设计和合成
利用Invitrogen公司网上siRNA设计软件设计3条大鼠NgR mRNA的siRNA，由Invitrogen公司进行合成相应的双链RNA(double stranded RNA，dsRNA)。经预试验筛选出阻断NgR基因最有效的一条siRNA，其序列为：
Sequence(5′to 3′)：-UGC AGU ACC UCU ACC UAC AAG ACA A
Sequence(5′to 3′)：-UUG UCU UGU AGG UAG AGG UAC UGC A
六、实验分组
将神经干细胞分为siRNA组，Nogo-P4组，siRNA+Nogo-P4组和对照组。对照组加入10％胎牛血清使神经干细胞开始分化；Nogo-P4组加入10％胎牛血清和4μmol／l的Nogo-P4；siRNA组的神经干细胞先经过siRNA转染，24h后加入10％胎牛血清开始分化；siRNA+Nogo-P4组的神经干细胞先经过siRNA转染，24h后加入10％胎牛血清和4μmol／l的Nogo-P4开始分化。
七、siRNA的转染
取1ml含神经干细胞的培养液加入离心管，800r／min离心5min，弃上清，加入900ul不含抗生素的培养基，机械吹打分散细胞，加入siRNA和转染试剂的混合物进行转染。24h和72h后检测NgR基因表达。另取部分神经干细胞以荧光标记的dsRNA(Block-It Fluorescent Oligo)进行相同条件下的转染，荧光显微镜下观察并计算转染效率。
八、NgR基因沉默效率的检测
(一)免疫荧光检测转染24h和72h后的神经干细胞固定后，加入NgR兔抗鼠单克隆抗体标记，常规方法免疫荧光染色，荧光显微镜下观察照相。每张盖片随机取10个视野，计数NgR阳性细胞数N和总细胞数T，计算阻断效率(T-N)／T。实验重复3次。
(二)Western blot检测将培养液倒至15ml离心管中，1000rpm离心5min。弃上清，加入4ml PBS并轻轻吹打洗涤，然后1000rpm离心5min。弃上清后用PBS重复洗涤一次。弃去上清，加400μl裂解液，冰上裂解30min。将裂解液4℃、12000rpm离心5min，取上清分装于0.5ml离心管中并置于-20℃保存。用Bradford法进行蛋白质定量。蛋白上样量30μg，上样前将样品于沸水中煮5min。电泳后转印到硝酸纤维素膜。5％的脱脂牛奶封闭非特异性背景，室温1h。将兔抗鼠NgR抗体用TBST稀释至1∶400，将膜放入一抗稀释液中，4℃过夜。用TBS在室温下洗两次，每次10min；再用TBS洗一次，10min。准备二抗稀释液并与膜接触，室温下孵育2h。用TBS在室温洗两次，每次10min；再用TBS洗一次，10min。用KPL公司的蛋白检测试剂盒显色。
九、神经元神经突长度的测量
神经干细胞分化第3d，以NSE抗体标记，常规方法免疫荧光染色，荧光显微镜下观察照相。使用Image-Pro Plus 5.0软件进行神经元细胞神经突长度的测量。
十、统计分析
实验数据以SPSS11.5软件进行处理，差异显著性采用单因素方差分析。
实验结果
悬浮培养的细胞形成典型的神经球，经免疫荧光检测nestin和NgR表达阳性。加入10％胎牛血清分化1周后，可观察到NSE，GFAP和MBP标记阳性的细胞。这表明培养出的神经球具备分化成神经元，星形胶质细胞和少突胶质细胞的能力，是神经干细胞。
NgR抗体标记染色后，分化前的神经球和散在的单细胞均呈现强表达。
分化第3d，NgR表达阳性的细胞比率明显下降，只有(35.3±4.31)％的细胞NgR表达阳性。分化细胞的形态趋于成熟，可以识别出部分具有典型形态的神经元和胶质细胞。经双重免疫荧光染色证实，NgR表达阳性的细胞均为NSE表达阳性的细胞，多数NgR~+／NSE~+的细胞具有典型的神经元形态，双极或多级，有较长的突起。
分化第3d Nogo-P4组神经元神经突长度为80.54±6.75μm；对照组神经元神经突长度为97.80±6.97μm。Nogo-P4组和对照组神经元神经突长度有显著差异，P＜0.01。Nogo-P4组神经元神经突长度较对照组缩短约17％。
siRNA组神经干细胞经siRNA转染后大部分神经干细胞存活，少量细胞死亡，存活的细胞活力良好，超过24h后不再出现细胞死亡。部分神经干细胞以荧光标记的dsRNA(Block-It Fluorescent Oligo)进行相同条件下的转染，在荧光显微镜下观察证实dsRNA已进入神经干细胞内，转染获得成功，转染效率为94.3％。
转染后24h神经干细胞经NgR抗体标记染色，荧光显微镜下观察发现大量神经干细胞的NgR表达阴性，这表明RNA干扰可以引起NgR基因表达沉默。用随机打乱顺序的siRNA转染作为阴性对照，神经干细胞的NgR表达没有减弱，镜下显示为明亮的绿色荧光。Western blot检测的结果证实对照组和经随机打乱顺序的siRNA(scrambled siRNA)转染后24小时的神经干细胞NgR均显著表达，经siRNAC转染后24 h的神经干细胞NgR表达显著降低。转染后24h的NgR基因阻断效率为(90.35±3.1)％。转染后72h的NgR基因阻断效率为(89.96±3.9)％。
分化第3d，siRNA组神经元神经突长度为92.14±7.27μm，siRNA+Nogo-P4组神经元神经突长度为94.01±8.37μm。siRNA组及siRNA+Nogo-P4组和对照组神经元神经突长度无显著差异；siRNA+Nogo-P4组和Nogo-P4组神经元神经突长度有显著差异，P＜0.01。
结论
1、大鼠脊髓来源神经干细胞显著表达NgR。
2、在神经干细胞分化成神经元的过程中始终表达NgR。
3、神经干细胞分化过程中神经元的神经突生长会受到Nogo-P4的抑制。
4、Nogo-P4的神经突生长抑制作用随浓度升高而升高，在4μm／L时达到最大抑制作用。
5、RNA干扰的方法能够高效率地使神经干细胞的NgR基因表达沉默。
6、NgR基因表达沉默能够阻断Nogo-P4的神经突生长抑制作用。
Disinhibition of neuronal neur ite outgrowth in the presence of Nogo-66 by siRNA mediated knockdown of NgR of neural stem cells
Objectives
Several proteins of CNS myelin possess axon growth- inhibiting properties, notably Nogo-A, myelin- associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp). The 66 aa surface loop of Nogo is inhibitory for axonal growth in culture and acts via a Nogo-66 Receptor (NgR) expressed by neurons and localized to their axons in vitro. Remarkably, the NgR protein mediates the inhibitory action of MAG and OMgp, as well as Nogo-66. NgR is a target for treatment to promote axon recovery because Nogo, MAG, and OMgp all bind to NgR to mediate their inhibitory effects on neurite outgrowth.
Exclude approaches aimed at disinhibiting axon outgrowth to treat spinal injury, regenerative medicine using stem cell biology is attracting a lot of attention because this is considered a particularly important therapeutic strategy for regeneration of the CNS. But if NSCs express NgR and could be affected by myelin associated inhibitory proteins in the progress of differentiation has not been proved.
Nogo-P4 is an active segment of Nogo-66 and has the core inhibitory activity of Nogo-66. Here, we use immunocytochemistry and Western blot methods to detect NgR in NSCs derived from spinal cord of rat, compare the average neuronal neurite length、differentiated neuron percentage and percentage of neurite possessing neuron during the differentiation of NSCs with and without Nogo-P4, before and after siRNA-mediated knockdown of NgR on NSCs.
Experimental methods
1. Animals
Postnatal 1 day Wistar rats were supplied by animal center of China Medical University.
2. Main reagents
Monoclonal NgR antibody (Chemicon) was used to label NSCs at 10μg/ml for immunocytochemistry(ICC) and Western blots. Polyclonal neuron specific enolase (NSE) antibody, glial fibrillary acidic protein (GFAP) antibody and myelin basic protein (MBP) antibody were purchased from Boster Company and were used to label neurons and neurites, astrocytes and oligodendrocytes respectively at 1:300. Monoclonal nestin antibody (Chemicon) was used to label NSCs at 1:200. Second antibody Alexa Fluor(?) 488 was purchased from Invitrogen. PI(Sigma) was used for counterstaining to identify nuclei at 1:3000. DMEM/F12 medium、B27、PI、Block-It Fluorescent Oligo、LIPOFECTAMINE 2000 REAGENT and OPTI MEM were purchased from Invitrogen company.
3. Culture and identification of NSCs
3.1 Culture of NSCs
The culture methods were based on those described previously. Spinal cords of P1(postnatal 1 day) Wistar rats(animal center of China Medical University) were dissected and dissociated into single cells using a solution of 0.125% trypsin for 45min at 37℃. The suspension was centrifuged at 1000 rpm for 10 min at 4℃. Decant as much of the supernatant as possible. Resuspend the cells in a total volume of 10 mL of DMEM/F12 (Invitrogen) serum-free stem cell medium containing freshly added epidermal growth factor (EGF) (Chemicon) 20ng/ml, basal fibroblast growth factor(bFGF) (Invitrogen) 20ng/ml and B27((Invitrogen, 1:50) (prewarmed to 37℃). Cell suspension was cultured in a atmosphere containing 5% CO_2 at 37℃.
3.2 Expression of nestin
Neurospheres in primary cultures or subcultures were collected by centrifugation at 1000 rpm for 10 min at 4℃. These spheres were fixed in acetone for 15 min after brief washes in 0.01M phosphatebuffered saline (PBS). Some were dropped on poly-d-lysine coated coverslips and immunostained intact to detect nestin immunoreactivity of the spheres.
3.3 Differentiation of NSCs
NSCs suspension was added to 6 well culture plate with glass cover slips pre-coated with poly-D-lysine in it and cultured in the presence of 10% FBS to initiate differentiation. After 1 week of differentiation cover slips were taken out for immunocytochemisty assay. The differentiated cells were incubated with primary antibodies against GFAP, MBP and NSE, respectively, and then exposed to secondary antibodies.
Microscopic images of the immunostained neurospheres and differentiated cells were captured using a SpotRT digital camera (Diagnostic Instruments Inc.) mounted on an Olympus BX60 microscope (Olympus Optical Co.).
4. Detection of NgR expression
Neurospheres in primary cultures or subcultures were collected by centrifugation at 1000 rpm for 10 min at 4℃. These spheres were fixed in acetone for 15 min after brief washes in 0.01M phosphatebuffered saline (PBS). Some were dropped on poly-d-lysine coated coverslips and immunostained intact to detect NgR immunoreactivity of the spheres.
5. Design and synthesis of siRNA
SiRNA directed against NgR was designed and synthesized by Invitrogen Company. The siRNA sequences used were:
siRNAA
Sequence (5′to 3′): - UUU GAG UGC AGC CAC AGG AUG GUG A
Sequence (5′to 3′): - UCA CCA UCC UGU GGC UGC ACU CAAA
siRNA B
Sequence (5′to 3′): - UCA GUG AGC UGA UUG GUC UGG AAG G
Sequence (5′to 3′): - CCU UCC AGA CCAAUC AGC UCA CUG A
siRNA C
Sequence (5′to 3′): - UGC AGU ACC UCU ACC UAC AAG ACAA
Sequence(5′to 3′): - UUG UCU UGU AGG UAG AGG UAC UGC A
6. Group
NSCs were divided into control group, Nogo-P4 group, siRNA group and siRNA+Nogo-P4 group. In control group, NSCs differentiated in the presence of 10% FBS; in Nogo-P4 group NSCs differentiated in the presence of 10% FBS and 4μmol/l Nogo-P4; in siRNA group, NSCs were transfected with siRNA before they differentiated in the presence of 10% FBS; in siRNA+Nogo-P4 group, NSCs were transfected with siRNA before they differentiated in the presence of 10% FBS and 4μmol/l Nogo-P4.
7. SiRNA preparation and transfection
Before transfection the neurospheres were triturated into single cells as much as possible. In one tube, 50pmol of siRNA was mixed in 50μl Opti-MEM, while a second tube contained 1μl Lipofectamine 2000 reagent and 50μl Opti-MEM and each was incubated at room temperature for 15min before the two solutions were combined and incubate for complex formation. Then the complex was added to NSCs suspension without antibiotics. After 1d, 3d and 5d the NSCs were collected and immunostained with NgR antibody to detect the knockdown efficiency.
Block-It Fluorescent Oligo was fluorescence labeled duplex siRNA used for observation of transfection efficiency. The transfection procedures are as described above.
8. Detection of knockdown efficiency
8.1 Immunohistochemistry
After 1d, 3d and 5d of transfection, the NSCs were collected and immunostained with NgR antibody to detect the knockdown efficiency. The cells attached to coverslips in differential cultures were briefly riNSEd with 0.01M PBS and fixed in 4% paraformaldehyde in 0.1M phosphate buffer for 30 min at 4℃.
Before staining, cover slips were incubated sequentially in 0.3% Triton X-100 in PBS (PBS-T; 30 min), 10% goat serum in PBS (30 min), and then with primary antibodies for 24hr at 4℃. After incubating in the primary antibodies for 24hr, sections were washed in PBS and then incubated for 2 hr at room temperature with second antibody (Alexa Fluor(?) 488, Invitrogen).
8.2 Western blots
The NgR protein expression was detected by Western blot method. Cells were collected by centrifugation(1000rpm, 5min) and washed three times with ice-cold PBS, then lysed in buffer(50mM Tris-HCl, pH8.0, 150mM NaCl, 100μg/ml PMSF, 1%TritonX-100) for 30 rain at ice. After removal of cell debris by centrifugation (12, 000rpm, 4℃), the protein concentration of lysates was meatured by Bradford method, 40μg of each lysate sample was boiled for 5 min in sample buffer and were separated by 10% SDS-PAGE and transferred onto nitrocellulose membrane (Pall Corporation). Nonspecific reactivity was blocked in 5% nonfat dry milk in TBST (10mM Tris-HCl, pH7.5, 150mM NaCl, 0.05%Tween-20) for 1h at room temperature. The membrane was then incubated overnight at 4℃with monoclonal anti-NgR antibody (Chemicon) followed by reaction with alkaline phosphatase conjugated second antibody. Reactive protein was detected by Protein Detector BCIP/NBT Western Blotting kit (KPL).
9. Measurement of neurite outgrowth
NSE was used to label the neuron by immunocytoflurescence method. Neurite length was measured by Image-Pro Plus 5.0 software.
10. Statistical analysis
The numbers of NgR~+ cells were determined in at least three samples in each culture. The total cell number was determined by PI counterstaining. About 400 cells of each culture were counted. One-Way Analysis of Variance was used for the comparison of different cultures. All the data were treated by SPSS 11.5 software.
Results
1. Expression of NgR on NSOs
Growth of neurospheres derived from spinal cord of rat is relatively slow. Neurospheres could be seen after suspension culture for 1 week and obtained in large amount after 2 weeks. The neurospheres express nestin positively and they could differentiate into NSE, GFAP and MBP positive cells after mitogens were removed and they were cultured in the presence of 10% fetal bovine serum. So the neurospheres are capable to generate neurons, astrocytes as well as oligodendrocytes and they have pluripotency.
Both neurospheres and single neural stem cells express NgR positively after they were treated by immunocytochemistry.
In the test of Western blot, NSCs were confirmed to express NgR apparently.
2. Inhibition effect of Nogo-P4
In Nogo-P4 group, Nogo-P4 was added to the culture medium during the differentiation progress of NSCs at the concentration of 4μmol/L. In control group, NSCs differentiated without Nogo-P4. In both Nogo-P4 group and control group, 10% FBS was added to the culture to initiate and promote differentiation. NSE was used to mark the neuron by immunocytofluorecence method. Neurite length of neurons differentiated from NSCs was measured under fluorescence microscope.
In control group, the average neuron neurite length was 97.80±6.97(μm/cell) three days after the neural stem cell began to differentiate. The differentiated neuron percentage (NP) was 34.73±5.21%; the percentage of neurite possessing neuron (NPNP) was 58.67+4.31%. In Nogo-P4 group, the average neurite length was 80.54±6.75(μm/cell). NP was 38.97±5.79%, NPNP was 88.52±3.96%. The neurite length has significant difference between the Nogo-P4 group and control group, P＜0.01. The neurite length was shortened about 17% in Nogo-P4 group. So Nogo-P4 inhibits neurites outgrowth during the differentiation progress of neural stem cell. NP has no differences between control group and Nogo-P4 group, while NPNP of Nogo-P4 group is significantly higher than that of control group (p＜0.05).
3. siRNA-mediated knockdown of NgR on NSCs
After transfection, most NSCs survived. Some NSCs were transfected with fluorescence labeled dsRNA (Block-It Fluorescent Oligo) at same condition to observe the transfection efficiency. The total transfection efficiency was 94.3%.
We tested the NgR immunoreactivity at 1, 3 and 5 days after transfection. NgR immunoreactivity was markedly reduced in NSCs transfected with siRNA sequence C. The scrambled NgR siRNA sequence did not alter NgR immunoreactivity.
In Western blot test, we detect the expression of NgR 1d after transfection of sequence A～C and scrambled siRNA. NSCs without transfection were detected too as control group. NgR gene was knocked down by the three siRNA in different degree. NgR protein was expressed significantly in control group and NSCs transfected by scrambled siRNA.
The knockdown efficiency decreased as time elongated after transfection. The most effective siRNA (Sequence C) almost completely knocked down NgR (90.35±3.1% knockdown, P＜0.01vs scrambled siRNA) 1d after transfection and kept high knock-down efficiency (85.22±3.1%) even at 5 days after transfection.
The NgR~- NSCs were defined as siRNA group and differentiated in the presence of 10% FBS. After 3 days, the neurite length was 92.14±7.27 (μm/cell); the differentiated neuron percentage (NP) was 32.01±4.82%; the percentage of neurite possessing neuron (NPNP) was 53.62±6.53 %.
4. Disinhibition of neurite outgrowth of neurons
differentiated from neural stem cells by siRNA-mediated knockdown of NgR
In siRNA+Nogo-P4 group NSCs were transfected with siRNA sequence C to knockdown NgR before they differentiate in the presence of Nogo-P4 and 10% FBS. The neurite length was 94.01±8.37 (μm/cell) three days after the neural stem cell began to differentiate. It has no difference with control group and has significant difference with Nogo-P4 group, P＜0.01. NP was 22.74±4.56%; NPNP was 86.61+2.94 %. Significantly, NPNP of siRNA group was higher than NPNP of control group (p＜0.05) and has no difference with that of Nogo-P4 group.
Conelusion
1. Neural stem cells express NgR apparently.
2. Neural stem cells express NgR apparently during the differentiation progress to neurons.
3. Nogo-P4 could inhibit neuron neurite outgrowth differentiated from neural stem cell by interacting with NgR.
4. The inhibition effect of Nogo-P4 reached apex in 4μmol/L.
5. NgR expression of neural stem cells could be knocked down effectively by RNAi.
6. Inhibition effect of Nogo-P4 could be removed by siRNA mediated knockdown of NgR.
引文
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4 Kim HJ, Hida H, Jung CG, et al. Treatment with deferoxamine increases neurons from neural stem/progenitor cells. Brain Res, 2006, 30;1092(1):1-15.
5 Carruthers S, Mason J, Papalopulu N. Depletion of the cell-cycle inhibitor p27(xic1) impairs neuronal differentiation and increases the number of ElrC+ progenitor cells in Xenopus tropicalis. Mech. Dev., 2003, 120(5), 607-616.
6 Kleber M , Sommer L. Wnt signaling and the regulation of stem cell function. Curr. Opin. Cell Biol., 2004, 16(6), 681-687
7 Ge S, Goh EL, Sailor KA, et al. GABA regulates synaptic integration of newly generated neurons in the adult brain. Nature , 2006, 439(7076), 589-593.
8 Nakamura M, Houghtling RA, MacArthur L, et al. Difference in cytokine gene expression profile between acutely and chronically injured adult rat spinal cord. Exp Neurol., 2003 , 184 (1): 313-325.
9 GrandPre T, Nakamura F, Vartanian T, et al. Identification of the Nogo inhibitor of axon regeneration as a reticulon protein. Nature, 2000, 403(6768): 439-444.
10 GrandPre T, Li S, Strittmatter SM . Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature,2002,417(6888): 547-551.
11 Fournier AE, GrandPre T, Strittmatter SM. Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration. Nature ,2001,409(6818): 341-346.
12 Liu BP, Fournier A, GrandPre T, et al. Myelin-associated glycoprotein as a functional ligand for the Nogo-66 receptor. Science, 2002, 297(5584): 1190-1193.
13 Domeniconi M, Cao Z, Spencer T, et al . Myelin-associated glycoprotein interacts with the nogo66 receptor to inhibit neurite outgrowth. Neuron, 2002, 35(2): 283.
14 Wang KC, Koprivica V, Kim JA,et al. Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature, 2002, 417(6892): 941-944.
15 Karimi-Abdolrezaee S, Eftekharpour E, Wang J,et al. Delayed transplantation of adult neural precursor cells promotes remyelination and functional neurological recovery after spinal cord injury. J Neurosci, 2006, 26(13): 3377-3389.
16 Chen MS, Huber AB, van der Haar ME, et al. Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature, 2000 ,403(6768): 434-439.
17 Prinjha R, Moore SE, Vinson M, et al. Inhibitor of neurite outgrowth in humans. Nature, 2000 ,403(6768): 383-384.
18 Wang X, Chun SJ, Treloar H, et al. Localization of Nogo-A and Nogo-66 receptor proteins at sites of axon-myelin and synaptic contact. J Neurosci., 2002, 22(13): 5505-5515.
19 Kim JE, Liu BP, Park JH, et al. Nogo-66 receptor prevents raphespinal and rubrospinal axon regeneration and limits functional recovery from spinal cord injury. Neuron, 2004 , 44(3):439-451.
20 Cafferty WB, Strittmatter SM.The Nogo-Nogo receptor pathway limits a spectrum of adult CNS axonal growth. J Neurosci, 2006, 26(47): 12242-50.
21 Schwab JM, Tuli SK, Failli V.The Nogo receptor complex: confining molecules to molecular mechanisms. Trends Mol Med., 2006 ,12(7):293-297.
22 Li W, Walus L, Rabacchi SA, et al. A neutralizing anti-Nogo66 receptor monoclonal antibody reverses inhibition of neurite outgrowth by central nervous system myelin. J Biol Chem. 2004 , 279(42):43780-43788.
23 Li S, Liu BP, Budel S, et al. Blockade of Nogo-66, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein by soluble Nogo-66 receptor promotes axonal sprouting and recovery after spinal injury. J Neurosci., 2004 , 24(46):10511-10520.
24 Ahmed Z, Dent RG, Suggate EL, et al. Disinhibition of neurotrophin-induced dorsal root ganglion cell knockdown of NgR, p75NTR and Rho-A. Mol Cell Neurosci, 2005, 28(3):509-523.
25 Wang X, Baughman KW, Basso DM, et al.Delayed Nogo receptor therapy improves recovery from spinal cord contusion.Ann Neurol, 2006 ,60(5):540-549.
26 Hou S, Tian W, Xu Q, et al.The enhancement of cell adherence and inducement of neurite outgrowth of dorsal root ganglia co-cultured with hyaluronic acid hydrogels modified with Nogo-66 receptor antagonist in vitro. Neuroscience, 2006,137(2):519-529.
27 Founder AE, Gould GC, Liu BP, et al. Truncated soluble Nogo receptor binds Nogo-66 and blocks inhibition of axon growth by myelin. J Neurosci, 2002 ,22(20):8876-8883.
1 David S, Aguayo AJ. Axonal elongation into peripheral nervous system "bridges" after central nervous system injury in adult rats. Science, 1981, 214(4523): 931-3.
2 Caroni P, Schwab ME. Two membrane protein fractions from rat central myelin with inhibitory properties for neurite growth and fibroblast spreading. J Cell Biol., 1988, 106(4): 1281-1288.
3 Spillmann AA, Bandtlow CE, Lottspeich F, et al. Identification and characterization of a bovine neurite growth inhibitor (bNI-220). J Biol Chem.,1998, 273(30): 19283-19293.
4 Chen MS, Huber AB, van der Haar ME, et al. Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature, 2000, 403(6768): 434-439.
5 Prinjha R, Moore SE, Vinson M, et al. Inhibitor of neurite outgrowth in humans. Nature, 2000, 403(6768): 383-384.
6 GrandPré T, Nakamura F, Vartanian T, et al. Identification of the Nogo inhibitor of axon regeneration as a reticulon protein. Nature, 2000, 403(6768): 439-444.
7 GrandPre T, Li S, Strittmatter SM. Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature, 2002, 417(6888): 547-551.
8 Oertle T, Marian E. van der Haar. Nogo-A Inhibits Neurite Outgrowth and Cell Spreading with Three Discrete Regions. J Neurosci., 2003, 23(13): 5393-5406.
9 Fournier AE, GrandPre T, Strittmatter SM. Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration. Nature, 2001, 409(6818): 341-346.
10 Simonen M, Pedersen V, Weinmann O, et al. Systemic deletion of the myelin-associated outgrowth inhibitor Nogo-A improves regenerative and plastic respoNSEs after spinal cord injury. Neuron, 2003, 38(2): 201-211.
11 Kim JE, Li S, GrandPre T,et al. Axon regeneration in young adult mice lacking Nogo-A/B. Neuron, 2003, 38(2):187-199.
12 Zheng B, Ho C, Li S, et al. Lack of enhanced spinal regeneration in Nogo-deficient mice. Neuron, 2003, 38(2):213-224.
13 Mckerracher L, David S, Jackson DL,et al. Identification of myelin-associated glycoprotein as a major myelin-derived inhibitor of neurite growth. Neuron, 1994,13(4): 805-811.
14 Mukhopadhyay G, Doherty P, Walsh FS, et al. A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration. Neuron, 1994, 13(3): 757-767.
15 Martini R. Expression and functional roles of neural cell surface molecules and extracellular matrix components during development and regeneration of peripheral nerves. J Neurocytol. 1994,23(1):1-28.
16 Filbin MT. The muddle with MAGMol Cell Neurosci., 1996;8(2-3):84-92.
17 Sato S, Fujita N, Kurihara T, et al. cDNA cloning and amino acid sequence for human myelin-associated glycoprotein.Biochem. Biophys. Res. Commun., 1989, 163(3):1473-1480.
18 Li C, Tropak MB, Gerlai R, et al. Myelination in the absence of myelin-associated glycoprotein. Nature, 1994, 369(6483):747-750.
19 Montag D, Giese KP, Bartsch U, et al. Mice deficient for the myelin-associated glycoprotein show subtle abnormalities in myelin. Neuron, 1994, 13(1):229-246.
20 Schafer M, Fruttiger M, Montag D, et al. Disruption of the gene for the myelin-associated glycoprotein improves axonal regrowth along myelin in C57BL/Wlds mice. Neuron, 1996, 16(6): 1107-1113.
21 Bartsch U, Bandtlow CE, Schnell L,et al. Lack of evidence that myelin-associated glycoprotein is a major inhibitor of axonal regeneration in the CNS. Neuron, 1995, 15(6): 1375-1381.
22 Wang KC, Koprivica V, Kim JA, et al. Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature, 2002, 417(6892): 941-944.
23 Barton WA, Liu BP, Tzvetkova D, et al. Structure and outgrowth inhibition binding of the Nogo-66 receptor and related proteins. The EMBO Journal, 2003,22(13): 3291-3302.
24 Wang X, Chun SJ, Treloar H, et al. Localization of Nogo-A and Nogo-66 receptor proteins at sites of axon-myelin and synaptic contact. J Neurosci .,2002, 22(13): 5505-5515.
25 Josephson A, Trifunovski A, Widmer HR,et al. Nogo-receptor gene activity: cellular localization and developmental regulation of mRNA in mice and humans. J Comp Neurol., 2002, 453(3):292-304.
26 Liu BP, Fournier A, GrandPre T,et al. Myelin-associated glycoprotein as a functional ligand for the Nogo-66 receptor. Science, 2002, 297(5584): 1190-1193.
27 Domeniconi M, Cao Z, Spencer T, et al. Myelin-associated glycoprotein interacts with the nogo66 receptor to inhibit neurite outgrowth. Neuron, 2002, 35(2): 283.
28 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(12): 1302-1308.
29 He XL, Bazan JF, McDermott G,et al. Structure of the Nogo receptor ectodomain: a recognition module implicated in myelin inhibition. Neuron, 2003, 38(2):177-185.
30 Dechant G, Barde YA. The neurotrophin receptor p75(NTR): novel functions and implications for diseases of the nervous system. Nat Neurosci., 2002, 5(11): 1131-1136.
31 Roux PP, Barker PA. Neurotrophin signaling through the p75 neurotrophin receptor. Prog Neurobiol., 2002, 67(3):203-233.
32 Yamashita T, Higuchi H, Tohyama M. The p75 receptor transduces the signal from myelin-associated glycoprotein to Rho. J Cell Biol .,2002, 157(4): 565-570.
33 Niederost B, Oertle T, Fritsche J, et al. Nogo-A and myelin-associated glycoprotein mediate neurite growth inhibition by antagonistic regulation of RhoA and Racl. J Neurosci., 2002,22(23): 10368-10376.
34 Fournier AE, Takizawa BT, Strittmatter SM. Rho kinase inhibition enhances axonal regeneration in the injured CNS. J Neurosci., 2003 ,23(4): 1416-23.
35 Dergham P, Ellezam B, Essagian C, et al. Rho signaling pathway targeted to promote spinal cord repair. J Neurosci., 2002,22(15): 6570-6577.
36 Cai D, Deng K, Mellado W, et al. Arginase I and polyamines act downstream from cyclic AMP in overcoming inhibition of axonal growth MAG and myelin in vitro. Neuron, 2002, 35(4):711-9.
37 Pignot V, Hein AE, Barske C,et al. Characterization of two novel proteins, NgRH1 and NgRH2, structurally and biochemically homologous to the Nogo-66 receptor. J Neurochem., 2003 ,85(3):717-728.
38 Li W, Walus L, Rabacchi SA, et al. A neutralizing anti-Nogo66 receptor monoclonal antibody reverses inhibition of neurite outgrowth by central nervous system myelin. J Biol Chem., 2004, 279(42):43780-43788.
39 Kim JE, Liu BP, Park JH, et al. Nogo-66 receptor prevents raphespinal and rubrospinal axon regeneration and Limits functional recovery from spinal cord injury. Neuron, 2004, 44(3):439-451.
40 Zheng B, Atwal J, Ho C,et al. Genetic deletion of the Nogo receptor does not reduce neurite inhibition in vitro or promote corticospinal tract regeneration in vivo. Proc Natl Acad Sci U S A.,2005,102(4):1205-1210.
41 Bandtlow C, Schiweck W, Tai HH,et al. The Escherichia coli-derived Fab fragment of the IgM/kappa antibody IN-1 recognizes and neutralizes myelin-associated inhibitors of neurite growth. Eur J Biochem., 1996, 241(2):468-475.
42 Brosamle C, Huber AB, Fiedler M, et al. Regeneration of lesioned corticospinal tract fibers in the adult rat induced by a recombinant, humanized IN-1 antibody fragment. J Neurosci., 2000 ,20(21):8061-8068.
43 Li S, Strittmatter SM. Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury. J Neurosci. ,2003 ,23(10):4219-4227.
44 Fournier AE, Gould GC, Liu BP,et al. Truncated soluble Nogo receptor binds Nogo-66 and blocks inhibition of axon growth by myelin. J Neurosci., 2002,22(20):8876-83.
45 Li S, Liu BP, Budel S,et al. Blockade of Nogo-66, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein by soluble Nogo-66 receptor promotes axonal sprouting and recovery after spinal injury. J Neurosci., 2004,24(46): 10511-10520.
46 Lehmann M, Fournier A, Selles-Navarro l,et al. Inactivation of Rho signaling pathway promotes CNS axon regeneration. J Neurosci., 1999 ,19(17):7537-7547.
47 Neumann S, Bradke F, Tessier-Lavigne M,et al. Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation. Neuron, 2002, 34(6):885-93.
48 Qiu J, Cai D, Dai H,et al. Spinal axon regeneration induced by elevation of cyclic AMP. Neuron, 2002, 34(6):895-903.
49 Gomez TM, Spitzer NC. In vivo regulation of axon extension and pathfinding by growth-cone calcium transients. Nature, 1999 ,397(6717):350-355.
50 Hunt D, Mason MR, Campbell G, et al. Nogo receptor mRNA expression in intact and regenerating CNS neurons. Mol Cell Neurosci., 2002,20(4):537-552.
2 Ogawa Y,Sawamoto K,Miyata T,et al.Transplantation of in vitro expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord injury in adult rats . J Neurosci Res., 2002,69(6):925 - 933.
3 Lepore AC, Neuhuber B, Connors TM, et al. Long-term fate of neural precursor cells following transplantation into developing and adult CNS. Neuroscience, 2006, 139(2):513-530.
4 Kim HJ, Hida H, Jung CG, et al. Treatment with deferoxamine increases neurons from neural stem/progenitor cells. Brain Res, 2006, 30;1092(1):1-15.
5 Carruthers S, Mason J, Papalopulu N. Depletion of the cell-cycle inhibitor p27(xic1) impairs neuronal differentiation and increases the number of ElrC+ progenitor cells in Xenopus tropicalis. Mech. Dev., 2003, 120(5), 607-616.
6 Kleber M , Sommer L. Wnt signaling and the regulation of stem cell function. Curr. Opin. Cell Biol., 2004, 16(6), 681-687
7 Ge S, Goh EL, Sailor KA, et al. GABA regulates synaptic integration of newly generated neurons in the adult brain. Nature , 2006, 439(7076), 589-593.
8 Nakamura M, Houghtling RA, MacArthur L, et al. Difference in cytokine gene expression profile between acutely and chronically injured adult rat spinal cord. Exp Neurol., 2003 , 184 (1): 313-325.
9 GrandPre T, Nakamura F, Vartanian T, et al. Identification of the Nogo inhibitor of axon regeneration as a reticulon protein. Nature, 2000, 403(6768): 439-444.
10 GrandPre T, Li S, Strittmatter SM . Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature,2002,417(6888): 547-551.
11 Fournier AE, GrandPre T, Strittmatter SM. Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration. Nature ,2001,409(6818): 341-346.
12 Liu BP, Fournier A, GrandPre T, et al. Myelin-associated glycoprotein as a functional ligand for the Nogo-66 receptor. Science, 2002, 297(5584): 1190-1193.
13 Domeniconi M, Cao Z, Spencer T, et al . Myelin-associated glycoprotein interacts with the nogo66 receptor to inhibit neurite outgrowth. Neuron, 2002, 35(2): 283.
14 Wang KC, Koprivica V, Kim JA,et al. Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature, 2002, 417(6892): 941-944.
15 Karimi-Abdolrezaee S, Eftekharpour E, Wang J,et al. Delayed transplantation of adult neural precursor cells promotes remyelination and functional neurological recovery after spinal cord injury. J Neurosci, 2006, 26(13): 3377-3389.
16 Chen MS, Huber AB, van der Haar ME, et al. Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature, 2000 ,403(6768): 434-439.
17 Prinjha R, Moore SE, Vinson M, et al. Inhibitor of neurite outgrowth in humans. Nature, 2000 ,403(6768): 383-384.
18 Wang X, Chun SJ, Treloar H, et al. Localization of Nogo-A and Nogo-66 receptor proteins at sites of axon-myelin and synaptic contact. J Neurosci., 2002, 22(13): 5505-5515.
19 Kim JE, Liu BP, Park JH, et al. Nogo-66 receptor prevents raphespinal and rubrospinal axon regeneration and limits functional recovery from spinal cord injury. Neuron, 2004 , 44(3):439-451.
20 Cafferty WB, Strittmatter SM.The Nogo-Nogo receptor pathway limits a spectrum of adult CNS axonal growth. J Neurosci, 2006, 26(47): 12242-50.
21 Schwab JM, Tuli SK, Failli V.The Nogo receptor complex: confining molecules to molecular mechanisms. Trends Mol Med., 2006 ,12(7):293-297.
22 Li W, Walus L, Rabacchi SA, et al. A neutralizing anti-Nogo66 receptor monoclonal antibody reverses inhibition of neurite outgrowth by central nervous system myelin. J Biol Chem. 2004 , 279(42):43780-43788.
23 Li S, Liu BP, Budel S, et al. Blockade of Nogo-66, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein by soluble Nogo-66 receptor promotes axonal sprouting and recovery after spinal injury. J Neurosci., 2004 , 24(46):10511-10520.
24 Ahmed Z, Dent RG, Suggate EL, et al. Disinhibition of neurotrophin-induced dorsal root ganglion cell knockdown of NgR, p75NTR and Rho-A. Mol Cell Neurosci, 2005, 28(3):509-523.
25 Wang X, Baughman KW, Basso DM, et al.Delayed Nogo receptor therapy improves recovery from spinal cord contusion.Ann Neurol, 2006 ,60(5):540-549.
26 Hou S, Tian W, Xu Q, et al.The enhancement of cell adherence and inducement of neurite outgrowth of dorsal root ganglia co-cultured with hyaluronic acid hydrogels modified with Nogo-66 receptor antagonist in vitro. Neuroscience, 2006,137(2):519-529.
27 Founder AE, Gould GC, Liu BP, et al. Truncated soluble Nogo receptor binds Nogo-66 and blocks inhibition of axon growth by myelin. J Neurosci, 2002 ,22(20):8876-8883.
1 David S, Aguayo AJ. Axonal elongation into peripheral nervous system "bridges" after central nervous system injury in adult rats. Science, 1981, 214(4523): 931-3.
2 Caroni P, Schwab ME. Two membrane protein fractions from rat central myelin with inhibitory properties for neurite growth and fibroblast spreading. J Cell Biol., 1988, 106(4): 1281-1288.
3 Spillmann AA, Bandtlow CE, Lottspeich F, et al. Identification and characterization of a bovine neurite growth inhibitor (bNI-220). J Biol Chem.,1998, 273(30): 19283-19293.
4 Chen MS, Huber AB, van der Haar ME, et al. Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature, 2000, 403(6768): 434-439.
5 Prinjha R, Moore SE, Vinson M, et al. Inhibitor of neurite outgrowth in humans. Nature, 2000, 403(6768): 383-384.
6 GrandPré T, Nakamura F, Vartanian T, et al. Identification of the Nogo inhibitor of axon regeneration as a reticulon protein. Nature, 2000, 403(6768): 439-444.
7 GrandPre T, Li S, Strittmatter SM. Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature, 2002, 417(6888): 547-551.
8 Oertle T, Marian E. van der Haar. Nogo-A Inhibits Neurite Outgrowth and Cell Spreading with Three Discrete Regions. J Neurosci., 2003, 23(13): 5393-5406.
9 Fournier AE, GrandPre T, Strittmatter SM. Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration. Nature, 2001, 409(6818): 341-346.
10 Simonen M, Pedersen V, Weinmann O, et al. Systemic deletion of the myelin-associated outgrowth inhibitor Nogo-A improves regenerative and plastic respoNSEs after spinal cord injury. Neuron, 2003, 38(2): 201-211.
11 Kim JE, Li S, GrandPre T,et al. Axon regeneration in young adult mice lacking Nogo-A/B. Neuron, 2003, 38(2):187-199.
12 Zheng B, Ho C, Li S, et al. Lack of enhanced spinal regeneration in Nogo-deficient mice. Neuron, 2003, 38(2):213-224.
13 Mckerracher L, David S, Jackson DL,et al. Identification of myelin-associated glycoprotein as a major myelin-derived inhibitor of neurite growth. Neuron, 1994,13(4): 805-811.
14 Mukhopadhyay G, Doherty P, Walsh FS, et al. A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration. Neuron, 1994, 13(3): 757-767.
15 Martini R. Expression and functional roles of neural cell surface molecules and extracellular matrix components during development and regeneration of peripheral nerves. J Neurocytol. 1994,23(1):1-28.
16 Filbin MT. The muddle with MAGMol Cell Neurosci., 1996;8(2-3):84-92.
17 Sato S, Fujita N, Kurihara T, et al. cDNA cloning and amino acid sequence for human myelin-associated glycoprotein.Biochem. Biophys. Res. Commun., 1989, 163(3):1473-1480.
18 Li C, Tropak MB, Gerlai R, et al. Myelination in the absence of myelin-associated glycoprotein. Nature, 1994, 369(6483):747-750.
19 Montag D, Giese KP, Bartsch U, et al. Mice deficient for the myelin-associated glycoprotein show subtle abnormalities in myelin. Neuron, 1994, 13(1):229-246.
20 Schafer M, Fruttiger M, Montag D, et al. Disruption of the gene for the myelin-associated glycoprotein improves axonal regrowth along myelin in C57BL/Wlds mice. Neuron, 1996, 16(6): 1107-1113.
21 Bartsch U, Bandtlow CE, Schnell L,et al. Lack of evidence that myelin-associated glycoprotein is a major inhibitor of axonal regeneration in the CNS. Neuron, 1995, 15(6): 1375-1381.
22 Wang KC, Koprivica V, Kim JA, et al. Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature, 2002, 417(6892): 941-944.
23 Barton WA, Liu BP, Tzvetkova D, et al. Structure and outgrowth inhibition binding of the Nogo-66 receptor and related proteins. The EMBO Journal, 2003,22(13): 3291-3302.
24 Wang X, Chun SJ, Treloar H, et al. Localization of Nogo-A and Nogo-66 receptor proteins at sites of axon-myelin and synaptic contact. J Neurosci .,2002, 22(13): 5505-5515.
25 Josephson A, Trifunovski A, Widmer HR,et al. Nogo-receptor gene activity: cellular localization and developmental regulation of mRNA in mice and humans. J Comp Neurol., 2002, 453(3):292-304.
26 Liu BP, Fournier A, GrandPre T,et al. Myelin-associated glycoprotein as a functional ligand for the Nogo-66 receptor. Science, 2002, 297(5584): 1190-1193.
27 Domeniconi M, Cao Z, Spencer T, et al. Myelin-associated glycoprotein interacts with the nogo66 receptor to inhibit neurite outgrowth. Neuron, 2002, 35(2): 283.
28 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(12): 1302-1308.
29 He XL, Bazan JF, McDermott G,et al. Structure of the Nogo receptor ectodomain: a recognition module implicated in myelin inhibition. Neuron, 2003, 38(2):177-185.
30 Dechant G, Barde YA. The neurotrophin receptor p75(NTR): novel functions and implications for diseases of the nervous system. Nat Neurosci., 2002, 5(11): 1131-1136.
31 Roux PP, Barker PA. Neurotrophin signaling through the p75 neurotrophin receptor. Prog Neurobiol., 2002, 67(3):203-233.
32 Yamashita T, Higuchi H, Tohyama M. The p75 receptor transduces the signal from myelin-associated glycoprotein to Rho. J Cell Biol .,2002, 157(4): 565-570.
33 Niederost B, Oertle T, Fritsche J, et al. Nogo-A and myelin-associated glycoprotein mediate neurite growth inhibition by antagonistic regulation of RhoA and Racl. J Neurosci., 2002,22(23): 10368-10376.
34 Fournier AE, Takizawa BT, Strittmatter SM. Rho kinase inhibition enhances axonal regeneration in the injured CNS. J Neurosci., 2003 ,23(4): 1416-23.
35 Dergham P, Ellezam B, Essagian C, et al. Rho signaling pathway targeted to promote spinal cord repair. J Neurosci., 2002,22(15): 6570-6577.
36 Cai D, Deng K, Mellado W, et al. Arginase I and polyamines act downstream from cyclic AMP in overcoming inhibition of axonal growth MAG and myelin in vitro. Neuron, 2002, 35(4):711-9.
37 Pignot V, Hein AE, Barske C,et al. Characterization of two novel proteins, NgRH1 and NgRH2, structurally and biochemically homologous to the Nogo-66 receptor. J Neurochem., 2003 ,85(3):717-728.
38 Li W, Walus L, Rabacchi SA, et al. A neutralizing anti-Nogo66 receptor monoclonal antibody reverses inhibition of neurite outgrowth by central nervous system myelin. J Biol Chem., 2004, 279(42):43780-43788.
39 Kim JE, Liu BP, Park JH, et al. Nogo-66 receptor prevents raphespinal and rubrospinal axon regeneration and Limits functional recovery from spinal cord injury. Neuron, 2004, 44(3):439-451.
40 Zheng B, Atwal J, Ho C,et al. Genetic deletion of the Nogo receptor does not reduce neurite inhibition in vitro or promote corticospinal tract regeneration in vivo. Proc Natl Acad Sci U S A.,2005,102(4):1205-1210.
41 Bandtlow C, Schiweck W, Tai HH,et al. The Escherichia coli-derived Fab fragment of the IgM/kappa antibody IN-1 recognizes and neutralizes myelin-associated inhibitors of neurite growth. Eur J Biochem., 1996, 241(2):468-475.
42 Brosamle C, Huber AB, Fiedler M, et al. Regeneration of lesioned corticospinal tract fibers in the adult rat induced by a recombinant, humanized IN-1 antibody fragment. J Neurosci., 2000 ,20(21):8061-8068.
43 Li S, Strittmatter SM. Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury. J Neurosci. ,2003 ,23(10):4219-4227.
44 Fournier AE, Gould GC, Liu BP,et al. Truncated soluble Nogo receptor binds Nogo-66 and blocks inhibition of axon growth by myelin. J Neurosci., 2002,22(20):8876-83.
45 Li S, Liu BP, Budel S,et al. Blockade of Nogo-66, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein by soluble Nogo-66 receptor promotes axonal sprouting and recovery after spinal injury. J Neurosci., 2004,24(46): 10511-10520.
46 Lehmann M, Fournier A, Selles-Navarro l,et al. Inactivation of Rho signaling pathway promotes CNS axon regeneration. J Neurosci., 1999 ,19(17):7537-7547.
47 Neumann S, Bradke F, Tessier-Lavigne M,et al. Regeneration of sensory axons within the injured spinal cord induced by intraganglionic cAMP elevation. Neuron, 2002, 34(6):885-93.
48 Qiu J, Cai D, Dai H,et al. Spinal axon regeneration induced by elevation of cyclic AMP. Neuron, 2002, 34(6):895-903.
49 Gomez TM, Spitzer NC. In vivo regulation of axon extension and pathfinding by growth-cone calcium transients. Nature, 1999 ,397(6717):350-355.
50 Hunt D, Mason MR, Campbell G, et al. Nogo receptor mRNA expression in intact and regenerating CNS neurons. Mol Cell Neurosci., 2002,20(4):537-552.