Upregulation of Nogo receptor expression induces apoptosis of retinal ganglion cells in diabetic rats

The Nogo receptor is an essential factor for neuronal apoptosis, but the changes in Nogo receptor expression in the retina and the effects of the Nogo receptor on retinal ganglion cell apoptosis in diabetes mellitus remain unclear. We found that Nogo receptor expression was mainly visible in retinal ganglion cells of a rat model of diabetes mellitus induced by streptozotocin. At 12 weeks after onset of diabetes mellitus, Nogo receptor and Rho kinase expression signiifcantly increased in the retina, and retinal ganglion cell apoptosis was apparent. When RNA interference was used to suppress Nogo receptor expression in rat retina, Rho kinase expression was obviously inhibit-ed, and retinal ganglion cell apoptosis was evidently reduced in rats with diabetes mellitus. These results indicate that upregulation of Nogo receptor expression is an important mechanism of retinal ganglion cell apoptosis in rats with diabetes mellitus.

Nogo receptor expression in microglia/macrophages during experimental autoimmune encephalomyelitis progression

Myelin-associated inhibitory factors within the central nervous system（CNS） are considered to be one of the main obstacles for axonal regeneration following disease or injury. The nogo receptor 1（NgR1） has been well documented to play a key role in limiting axonal regrowth in the injured and diseased mammalian CNS. However, the role of nogo receptor in immune cell activation during CNS inflammation is yet to be mechanistically elucidated. Microglia/macrophages are immune cells that are regarded as pathogenic contributors to inflammatory demyelinating lesions in multiple sclerosis（MS）. In this study, the animal model of MS, experimental autoimmune encephalomyelitis（EAE） was induced in ngr1^＋/＋ and ngr1^–/– female mice following injection with the myelin oligodendrocyte glycoprotein（MOG_（35–55）） peptide. A fatemap analysis of microglia/macrophages was performed throughout spinal cord sections of EAE-induced mice at clinical scores of 0, 1, 2 and 3, respectively（increasing locomotor disability） from both genotypes, using the CD11 b and Iba1 cell markers. Western immunoblotting using lysates from isolated spinal cord microglia/macrophages, along with immunohistochemistry and flow cytometric analysis, was performed to demonstrate the expression of nogo receptor and its two homologs during EAE progression. Myelin protein engulfment during EAE progression in ngr1^＋/＋ and ngr1^–/– mice was demonstrated by western immunblotting of lysates from isolated spinal cord microglia/macrophages, detecting levels of Nogo-A and MOG. The numbers of M1 and M2 microglia/macrophage phenotypes present in the spinal cords of EAE-induced ngr1^＋/＋ and ngr1^–/– mice, were assessed by flow cytometric analysis using CD38 and Erg-2 markers. A significant difference in microglia/macrophage numbers between ngr1^＋/＋ and ngr1^–/– mice was identified during the progression of the clinical symptoms of EAE, in the white versus gray matter regions of the spinal cord. This difference was unrelated to the expression of Ng R on these macrophage/microglial cells. We have identified that as EAE progresses, the phagocytic activity of microglia/macrophages with myelin debris, in ngr1^–/– mice, was enhanced. Moreover, we show a modulation from a predominant M1-pathogenic to the M2-neurotrophic cell phenotype in the ngr1^–/– mice during EAE progression. These findings suggest that CNS-specific macrophages and microglia of ngr1^–/– mice may exhibit an enhanced capacity to clear inhibitory molecules that are sequestered in inflammatory lesions.

Experience-dependent expression of Nogo-A and Nogo receptor in the developing rat visual cortex

Nogo-A and Nogo receptor （NgR） expression in the visual cortex following a critical developmental period （postnatal days 20-60） has been previously shown. However, little is known regarding Nogo-A and NgR expression between postnatal day 0 and initiation of the critical period. The present study analyzed Nogo-A and NgR expression at four different time points： postnatal day 0 （P0）, before critical period （P14）, during critical period （P28）, and after critical period （P60）. Results showed significantly increased Nogo-A mRNA and protein expression levels in the visual cortex following birth, and expression levels remained steady between P28 and P60. NgR mRNA or protein expression was dramatically upregulated with age and peaked at P14 or P28, respectively, and maintained high expression to P60. In addition, Nogo-A and NgR expression was analyzed in each visual cortex layer in normal developing rats and rats with monocular deprivation. Monocular deprivation decreased Nogo-A and NgR mRNA and protein expression in the rat visual cortex, in particular in layers Ⅱ-Ⅲ and Ⅳ in the visual cortex contralateral to the deprived eye. These findings suggested that Nogo-A and NgR regulated termination of the critical period in experience- dependent visual cortical plasticity.

Lateral olfactory tract usher substance(LOTUS) protein,an endogenous Nogo receptor antagonist,converts a non-permissive to permissive brain environment for axonal regrowth

It is well known that primates,including humans,hardly recover motor function after spinal cord injury（SCI）when compared with non-primate mammals such as rodents.This limited functional recovery is in part due to a non-permissive environment of the central nervous system（CNS）inhibiting axonal regrowth.

Soluble Nogo receptor 1 fusion protein protects neural progenitor cells in rats with ischemic stroke

Soluble Nogo66 receptor-Fc protein(sNgR-Fc)enhances axonal regeneration following central nervous system injury.However,the underlying mechanisms remain unclear.In this study,we investigated the effects of sNgR-Fc on the proliferation and differentiation of neural progenitor cells.The photothrombotic cortical injury model of ischemic stroke was produced in the parietal cortex of Sprague-Dawley rats.The rats with photothrombotic cortical injury were randomized to receive infusion of 400μg/kg sNgR-Fc(sNgR-Fc group)or an equal volume of phosphate-buffered saline(photothrombotic cortical injury group)into the lateral ventricle for 3 days.The effects of sNgR-Fc on the proliferation and differentiation of endogenous neural progenitor cells were examined using BrdU staining.Neurological function was evaluated with the Morris water maze test.To further examine the effects of sNgR-Fc treatment on neural progenitor cells,photothrombotic cortical injury was produced in another group of rats that received transplantation of neural progenitor cells from the hippocampus of embryonic Sprague-Dawley rats.The animals were then given an infusion of phosphate-buffered saline(neural progenitor cells group)or sNgR-Fc(sNgR-Fc+neural progenitor cells group)into the lateral ventricle for 3 days.sNgR-Fc enhanced the proliferation of cultured neural progenitor cells in vitro as well as that of endogenous neural progenitor cells in vivo,compared with phosphate-buffered saline,and it also induced the differentiation of neural progenitor cells into neurons.Compared with the photothrombotic cortical injury group,escape latency in the Morris water maze and neurological severity score were greatly reduced,and distance traveled in the target quadrant was considerably increased in the sNgR-Fc group,indicating a substantial improvement in neurological function.Furthermore,compared with phosphate-buffered saline infusion,sNgR-Fc infusion strikingly improved the survival and differentiation of grafted neural progenitor cells.Our findings show that sNgR-Fc regulates neural progenitor cell proliferation,migration and differentiation.Therefore,sNgR-Fc is a potential novel therapy for stroke and neurodegenerative diseases,The protocols were approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong(approval No.4560-17)in November,2015.

Anti-apoptotic efficacy of Qingnao Yizhi formula(清脑益智方)in hypoxia/reoxygenation primary cortical neurons through the promotion of synaptic plasticity by modulating the NogoA-Nogo receptor/Rho-Rho kinase signaling pathway

OBJECTIVE:To evaluate the anti-apoptotic efficacy of Qingnao Yizhi formula(清脑益智方,QNYZ)in cultured cerebral cortical neuronal cells(CNCs)and the regulation of the NogoA-Nogo receptor(NgR)/Rho-Rho kinase(ROCK)signaling pathway.METHODS:Primary cultured CNCs were randomly divided into the following groups:normal control group(N-C),hypoxia-reoxygenation group(H/R),high-dose QNYZ group(Q-H),low-dose QNYZ group(Q-L)butylphthalide(NBP)group,and Y-27632(a selective ROCK transduction pathway inhibiter)group.Except those in the N-C group,CNCs were placed in hypoxic conditions for 24 h and then in reoxygenation conditions for 24 h.Cell media was changed every 48 h,and various assays were performed on the 7 th day.Cell viability was evaluated by measuring mitochondrial dehydrogenase activity,using a CCK-8 assay,in triplicate.Synapsin(SYN)protein concentrations were evaluated by enzyme-linked immunosorbent assay.NogoA and RhoA protein expression were evaluated through Western blotting.The gene expression of NogoA,NgR,RhoA,and ROCK was evaluated by reverse transcription-polymerase chain reaction.Cell apoptosis was measured using a terminal deoxynucleotidyl transferase biotin-d UTP nick end labeling assay.RESULTS:Compared with the N-C group,the cell viability of the H/R group decreased significantly(P<0.05).The cell viability values for the Q-H and Q-L groups increased compared with that for the H/R group,and the difference was significant for the Q-H group(P<0.05).The NogoA and RhoA protein levels and the NogoA,NgR,RhoA,and ROCK m RNA expression levels increased in the H/R group,compared with the N-C group,and decreased significantly in the Q-H and Q-L groups(P<0.05)and in the Y-27632 group(P<0.05)compared with the H/R group.The SYN levels in the Q-H,Q-L,and NBP groups significantly increased compared with that in the H/R group(P<0.05).Compared with the H/R group,the numbers of apoptotic cells in the Q-H,Q-L,and NBP groups significantly decreased(P<0.05).CONCLUSION:The presented study demonstrated that QNYZ exerted anti-apoptotic effects on H/R-induced CNCs,possibly through the modulation of the NogoA-NgR/Rho-ROCK signaling pathway and the promotion of synaptic plasticity in H/R CNCs.

Nogo receptor 3,a paralog of Nogo-66 receptor 1(NgR1),may function as a NgR1 co-receptor for Nogo-66

Nogo-A is a major myelin associated inhibitor that blocks regeneration of injured axons in the central nervous system （CNS）. Nogo-66 （a 66-residue domain of Nogo-A） expressed on the surface of oligodendrocytes has been shown to directly interact with Nogo-66 receptor 1 （NgRI）. A number of additional components of NgR1 receptor complex essential for its signaling have been uncovered. However, detailed composition of the complex and its signaling mechanisms remain to be fully elucidated. In this study, we show that Nogo receptor 3 （NgR3）, a paralog of NgRI, is a binding protein for NgR1. The interaction is highly specific because other members of the reticulin family, to which Nogo-A belongs, do not bind to NgR3. Neither does NgR3 show any binding activity with Nogo receptor 2 （NgR2）, another NgRI paralog. Majority of NgR3 domains are required for its binding to NgR1. Moreover, a truncated NgR3 with the membrane anchoring domain deleted can function as a decoy receptor to reverse neurite outgrowth inhibition caused by Nogo-66 in culture. These in vitro results, together with previously reported overlapping expression profile between NgR1 and NgR3, suggest that NgR3 may be associated with NgR1 in vivo and that their binding interface may be targeted for treating neuronal injuries.

Nogo-A and Nogo-A receptor expression in periventricular white matter of experimental autoimmune encephalomyelitis rats

BACKGROUND：Previous studies have focused on the correlation between Nogo-A expression and multiple sclerosis or between Nogo-A receptor （NgR） expression and multiple sclerosis in the central nervous system. Expression patterns of Nogo-A and NgR remain poorly understood in rat models of experimental autoimmune encephalomyelitis （EAE）.OBJECTIVE：To observe dynamic changes in Nogo-A and NgR protein expression, and to verify the correlation between Nogo-A and NgR protein, as well as expression patterns at various time points, in periventricular tissue of EAE rats.DESIGN, TIME AND SETrlNG：A neuroimmunological, randomized, controlled experiment was performed at the Clinical Institute of Hunan People＇s Hospital of China from September to November 2008.MATERIALS：Immunohistochemistry （streptavidin-biotin-peroxidase complex method） kit was purchased from Boster, China.METHODS：A total of 60 female, Wistar rats, aged 6-8 weeks, ware randomly assigned to EAE and control groups （n = 30, respectively）. Guinea pig spinal cord homogenate, self-made complete Freund＇s adjuvant （0.2 mL/100 g）, and pertussis vaccine （0.2 mL） were subcutaneously injected into the hindlimb foot pad of rats from the EAE group to create rat models of EAE. Complete Freund＇s adjuvant （0.2 mL） was infused into rats from the control group.MAIN OUTCOME MEASURES：Nogo-A and NgR protein expression was determined in periventricular white matter using immunohistochemical methods. Neurological scores ware determined in all rats.RESULTS：Rats from the EAE group developed acute-onset EAE following immunization. The pathogenetic symptoms reached a peak on day 15, and neurological scores ware also greatest at this time point. Neurological scores decreased with recovery of the illness. Nogo-A was shown to be expressed in neuronal cells and oligodendrocytes, and expression increased 11 days after immunization （P 〈 0.01）, decreased by day 13 （P 〈 0.01）, and then increased again by day 15. Nogo-A expression remained greater in the EAE group compared with the control group at day 30 （P 〈 0.01）. In the EAE group, NgR protein was primarily expressed on the surface of neuronal bodies and axons. NgR expression increased 13-18 days after immunization （P 〈 0.01 or P 〈 0.05）.CONCLUSION：Nogo-A and NgR protein expression altered with disease course in periventdcular white matter of EAE rats. Results suggested that Nogo-A and NgR were involved in EAE occurrence.

Transplantation of Nogo-66 receptor gene-silenced cells in a poly(D,L-lactic-co-glycolic acid) scaffold for the treatment of spinal cord injury

Inhibition of neurite growth, which is in large part mediated by the Nogo-66 receptor, affects neural regeneration following bone marrow mesenchymal stem cell transplantation. The tissue engineering scaffold poly（D,L-lactide-co-glycolic acid） has good histocompatibility and can promote the growth of regenerating nerve fibers. The present study used small interfering RNA to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells and Schwann cells, which were subsequently transplanted with poly（D,L-lactide-co-glycolic acid） into the spinal cord lesion regions in rats. Simultaneously, rats treated with scaffold only were taken as the control group. Hematoxylin-eosin staining and immunohistochemistry revealed that at 4 weeks after transplantation, rats had good motor function of the hind limb after treatment with Nogo-66 receptor gene-silenced ceils prus the poly（O,L-lactide-co-glycolic acid） scaffold compared with rats treated with scaffold only, and the number of bone marrow mesenchymal stem cells and neuron-like cells was also increased. At 8 weeks after transplantation, horseradish peroxidase tracing and transmission electron microscopy showed a large number of unmyelinated and myelinated nerve fibers, as well as intact regenerating axonal myelin sheath following spinal cord hemisection injury. These experimental findings indicate that transplantation of Nogo-66 receptor gene-silenced bone marrow mesenchymal stem cells and Schwann cells plus a poly（D,L-lactide-co-glycolic acid） scaffold can significantly enhance axonal regeneration of spinal cord neurons and improve motor function of the extremities in rats following spinal cord injury.

Nogo-A蛋白及调控轴突再生作用研究进展

中枢神经系统损伤产生的抑制性微环境是神经再生困难的重要原因,其中Nogo-A蛋白是抑制轴突再生的关键因子,其结构域以片段形式通过不同途径释放到细胞外,与受体NgR1、PirB、S1PR2或HSPG相结合,激活多种信号通路调控细胞凋亡、运动以及细胞骨架蛋白聚合,引起细胞骨架解聚、生长锥塌陷,抑制神经元轴突生长。本文对Nogo-A蛋白的结构、受体和抑制轴突再生作用机制的研究进展进行综述。

Axonal growth inhibitors and their receptors in spinal cord injury:from biology to clinical translation

Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelinassociated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19(that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the Rho A/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.

siRNA干扰大鼠神经元Nogo受体mRNA表达的时程研究

目的:观察大鼠Nogo受体(NgR)特异性小干扰RNA(smallinterferingRNA,siRNA)在原代神经元干扰其mRNA表达的时程效果。方法:体外培养大鼠原代皮层和海马细胞,应用阳离子脂质体转染试剂转染针对2个基因片段(199和964位点)的大鼠NgR特异性siRNA和对照siRNA,分别于转染后24h、48h、72h和96h,应用实时荧光定量PCR检测NgRmRNA表达情况。结果:2对siRNA(siNgR199和siNgR964)均能够下调靶基因mRNA的表达水平,24h、48h、72h和96h,NgRmRNA表达分别为对照siRNA组的38.12%、12.47%、3.96%、18.4%(siNgR199)和49.54%、24.25%、13.17%、37.93%(siNgR964),与对照siRNA组比较有统计学意义(P<0.05)。结论:NgR特异性siRNA能够在原代皮层和海马细胞下调靶基因mRNA的表达水平,且基因沉默效果在转染后3d最显著。

Nogo及其受体在脊髓损伤修复中的作用机制

成体哺乳动物中枢神经系统(CNS)髓磷脂可影响神经的可塑性并抑制神经纤维的再生。Nogo-A被认为是中枢神经系统中抑制轴突生长最关键的一种髓磷脂抑制分子。在脊髓损伤(SCI)动物模型中,抑制Nogo-A的活性可明显促进轴突再生及功能改善。Nogo-A及其信号转导机制的研究日益成为SCI修复过程中的研究热点;Nogo-A及其信号转导分子特别是Nogo-66受体(NgR)、p75神经营养素受体(p75NTR)和LINGO-1成为损伤后促进轴突再生、抑制生长锥塌陷的主要治疗靶点。抑制Nogo-A及其受体NgR/p75NTR/LINGO-1可能有助于SCI的修复,促进患者功能的恢复。

李氏5号方对半乳糖老化小鼠海马神经元褪黑素受体和Nogo受体表达的影响

为了观察李氏5号方对半乳糖老化小鼠海马神经元褪黑素受体(MTR)和Nogo受体(NogoR)表达的影响。我们将昆明小鼠随机分为五组:正常对照组(C组)、D半乳糖模型组(D组)、李氏5号方大剂量药物治疗组(L组)、中剂量药物治疗组(M组)和小剂量药物治疗组(S组);D、L、M、S四组每日皮下注射半乳糖65mg/kg体重,持续三个月。应用免疫组织化学染色方法检测小鼠海马神经元MTR和NogoR的表达水平。NogoR免疫组化染色结果:D组海马阳性反应细胞着色浅,少有突起,细胞数目少,仅为正常小鼠的20%,三个剂量李氏5号方治疗组染色结果与C组相似。结果提示:半乳糖老化小鼠海马神经元MTR免疫反应阳性神经元数的上调可能是老化小鼠的一种代偿性反应;NogoR表达下调提示老化小鼠可能存在脑白质的损害,出于机体自我保护的需要,NogoR表达下调以避免神经元修复机制对大脑的进一步损害;不同剂量的李氏5号方水提取液对两种受体的调节作用极为明显,说明这两种受体的表达可受到外源性药物的干预。

Nogo与Nogo受体在正常小鼠大脑的分布

NOgo与NOgo受体对抑制中枢神经系统的再生有着重要的作用。本研究用本室制备的抗Nogo和抗Nogo受体的多克隆抗体进行免疫组织化学染色,在低倍镜下计数阳性细胞、观察Nogo与Nogo受体在正常小鼠脑内的分布。结果显示:Nogo样和Nogo受体样免疫阳性结构在大脑皮层、海马、下丘脑、苍白球、尾壳核和黑质等处的神经元细胞核中有较强的表达,而在胞浆和突起内表达较弱。由此我们得出结论,Nogo和Nogo受体蛋白在成年小鼠脑内分布广泛。

大鼠Nogo受体基因RNA干扰慢病毒载体的构建与鉴定

目的:构建大鼠Nogo受体(Nogoreceptor,NgR)基因RNA干扰慢病毒表达载体,并观察其对293T细胞感染效率。方法:应用基因工程技术,首先构建携带目的基因siNgR199的慢病毒穿梭质粒表达载体,然后使用慢病毒包装质粒混合物和构建好的慢病毒穿梭质粒共转染293T细胞,转染48h后收集上清离心过滤后冰浴保存,最后进行病毒滴度测定,与标准病毒液分别感染293T细胞,按MOI值分为5个梯度:1、3、5、10、20,以确定病毒滴度及适合感染的MOI值。其中采用PCR方法对重组载体进行鉴定,利用绿色荧光蛋白作为报告基因,对病毒滴度和感染效率进行检测。结果:酶切鉴定及PCR结果与病毒载体的预期结果一致,病毒滴度达1×108ifu/m1,适合感染的MOI值为3。结论:应用基因工程技术,成功构建了大鼠siNgR199慢病毒表达载体,为应用于治疗脊髓损伤后轴突再生创造了条件。

Nogo-66受体在大鼠神经组织中的表达

目的观察Nogo-66受体(NgR)蛋白在脑、脊髓、视网膜、视神经及坐骨神经的表达。方法采用免疫荧光组织化学-激光共聚焦显微镜观察9只正常SD大鼠脑、脊髓、视网膜、视神经及坐骨神经中NgR蛋白表达。结果9只大鼠脑、脊髓、视网膜、视神经切片中NgR蛋白均表达阳性,荧光染色位于神经元及其突起。9只大鼠坐骨神经切片中NgR蛋白均表达阴性。结论在大鼠中枢神经系统神经元中NgR广泛分布,而在周围神经坐骨神经则无NgR表达,提示NgR的阳性表达与中枢神经系统再生能力低下密切相关。

Nogo与Nogo受体研究

nogo是新近发现的一种基因 ,编码 3种蛋白质 :Nogo A、Nogo B和Nogo C .迄今为止 ,已证明它有抑制成熟中枢神经系统 (CNS)神经元轴突再生及诱导细胞凋亡的作用 .Nogo受体是一种糖基醇磷脂结合蛋白 .对Nogo和Nogo受体的研究 ,对于CNS再生障碍及肿瘤的认识和治疗有重要意义 .

Nogo-66受体在神经元生长锥中的表达

为探讨Nogo-66受体在神经元生长锥中的定位及机能意义,本研究采用免疫荧光染色法,激光共聚焦显微镜下观察在原代培养小脑颗粒细胞生长锥的表达及其定位特征。结果发现,Nogo-66受体密集分布在细胞质和胞膜、神经突起内,延伸至生长锥体部C区和P区内,P区密度更高。Nogo-66受体定位于神经元生长锥中,提示Nogo-66受体可能参与轴突延伸和寻路的活动。

编码大鼠Nogo受体mRNA的shRNA重组腺病毒载体的构建和鉴定

目的:构建Nogo受体(NgR)特异性shRNA重组腺病毒载体,为应用基因沉默技术从转录后水平进行缺血性脑损伤的基因治疗研究奠定基础.方法:将前期构建的大鼠Nogo受体mRNA的shRNA特异性真核表达载体pGenesil-1-shRNA的表达启动子U6连同shRNA亚克隆至穿梭质粒pAdTrack,酶切及DNA测序鉴定后,将含NgR基因的重组穿梭质粒pAdTrack-U6-shRNA经PmeI线性化后转化入pAdEasy-1感受态细菌.将pAd-U6-shRNA质粒经PacI线性化后转染293细胞,包装重组腺病毒Adeno-U6-shRNA,并进行PCR鉴定、PCR产物测序及病毒滴度测定.结果:结果证实pAdTrack-U6-shRNA及pAd-U6-shRNA质粒构建正确,收获病毒后的PCR及DNA测序结果证明Adeno-U6-shRNA包被成功.结论:成功构建了大鼠Nogo受体mRNA的shRNA重组腺病毒载体Adeno-U6-shRNA,将为应用基因沉默技术研究NgR在缺血性脑损伤后神经再生中的作用奠定基础.