N-甲基-D天冬氨酸受体2B亚基镇痛疫苗的构建及其镇痛效应的研究
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摘要
研究背景
神经病理性疼痛是神经系统原发性损伤或功能异常所导致的痛觉异常或痛觉过敏。创伤、缺血性损伤、感染或炎症、肿瘤、药物和压迫等原因均可以引起神经病理性疼痛。非甾体类抗炎药和阿片类镇痛药治疗神经病理性疼痛的效果差,临床上常使用三环类抗抑郁药和抗癫痫药物进行治疗,但疗效差,副作用大,能够有效缓解50%以上疼痛症状的患者不足50%。因此寻找一种安全有效、作用持久的镇痛方法已成为神经病理性疼痛研究的重要方向。神经病理性痛的受体机制研究表明,神经元和神经胶质细胞膜上的N-甲基-D-天冬氨酸受体(N-methyl-D-aspartate receptor,NMDAR)表达上调或过度激活可引起中枢敏感化,在神经病理性痛的产生如自发性痛,触诱发痛及痛觉过敏等起重要作用。NMDA受体拮抗机如氯胺酮、MK-801等对神经病理性痛具有良好的镇痛作用,但由于这些药物具有如认知功能障碍、神经空泡样改变等副作用等限制了它们的临床应用。近期的研究表明,NMDA受体的亚型——NR2B型NMDA受体(NR2B)在体内呈选择性分布如主要分布于脊髓背角、前脑等与痛传递和痛情绪有关的区域,与痛感觉和痛情绪的形成密切相关,这提示NR2B可能是一个潜在的镇痛治疗的靶点。现在疫苗技术的发展不仅针对外源性抗原开发出各种疫苗,而且能对机体自身抗原制备特异性疫苗,如最近利用肿瘤细胞表面的特异性位点研制的疫苗已被证实能有效地预防和治疗癌症地发生发展。During等构建的NMDAR1表位疫苗为防治中风和癫痫后脑损伤提供了新的治疗策略。据此,我们的假设:以NR2B为抗原制备疫苗诱导机体产生NR2B抗体,该抗体与神经元或胶质细胞膜上的NR2B结合,下调NR2B的表达或阻断NR2B的激活,发挥镇痛作用。因此,本研究旨在首先观察NR2B多肽疫苗对神经病理性痛大鼠的免疫效应及镇痛效应,然后构建NR2B重组腺病毒(Recombinant adenovirus serotype 5 encoding NR2B gene, rAd5/NR2B)镇痛疫苗,并通过口服免疫大鼠,以评价其对大鼠神经病理性痛痛感觉和痛情绪的影响。
研究方法与结果
1、NR2B多肽疫苗对神经病理性痛大鼠的免疫效应及超前镇痛效应
方法:选取NR2B有免疫原性的一段氨基酸序列合成多肽,并与大分子蛋白载体匙孔虫戚血蓝蛋白( Keyhole LimpetHemocyanin , KLH)交联制备NR2B多肽疫苗即NR2B-KLH。健康雌性SD大鼠随机分为三组:PBS组、KLH组及NR2B组。分别用PBS+福氏佐剂, KLH+福氏佐剂和NR2B-KLH+福氏佐剂皮下注射三次,每次注射后两周以间接ELISA法检测受免大鼠血清NR2B特异性抗体的滴度,并以免疫组化染色法检测大鼠免疫血清与脊髓背角NR2B蛋白的免疫结合反应。于第三次免疫后一周结扎坐骨神经的胫神经和腓总神经分支,保留腓肠神经分支,建立SD大鼠保留神经损伤性神经病理性痛模型(Spared nerve injury, SNI)。术后隔日以up-down法检测大鼠术侧后爪50%机械缩爪阈值的变化及热痛缩爪时间的变化。分别于术前一天和术后7天经枕骨大孔取脑脊液(cerebrospinal fluid, CSF)10～20μl,以间接ELISA法检测CSF中NR2B抗体滴度。术后7天处死大鼠,取腰段脊髓(L4~6),以免疫组化染色法检测脊髓背角c-Fos阳性神经元数目,并采用Western blotting法检测脊髓背角NR2B蛋白的改变。
结果:第二次注射后, NR2B组9只大鼠有6只大鼠出现NR2B抗体(>0.5μg/ml)。第三次注射后9只大鼠均检测到NR2B抗体(>2.2μg/ml),且滴度较第一次明显升高。在第三次注射后14天抗体滴度最高(7.2μg/ml),随后抗体滴度逐渐降低,但初次注射后70天血清仍可检测到NR2B抗体。而PBS组和KLH组未检测到NR2B抗体。免疫组化染色法检测发现NR2B大鼠血清可与大鼠脊髓背角结合呈阳性着色,而其余两组未见着色。与术前比较,术后第7天PBS、KLH及NR2B各组50%缩爪阈值均明显降低(P<0.05);但各组间比较,NR2B组明显高于其余两对照组(P<0.05)。各组术后7天热痛缩爪时间与术前比较均有显著升高(P<0.05),但各组间比较,NR2B组明显低于其余两对照组(P<0.05)。术后7天脊髓背角c-Fos蛋白阳性神经元的数目NR2B组明显低于两对照组(P<0.05)(NR2B组:170±22,PBS组:301±36,KLH组:280±25)。术前NR2B组大鼠脑脊液中NR2B抗体呈阴性,而术后7天呈阳性,而PBS组和KLH组这两个时间点均为阴性。Western blotting法检测发现术后7天NR2B组脊髓背角NR2B蛋白的表达较其余两组明显降低(P<0.05)。
2、NR2B多肽疫苗对已发生的大鼠神经病理性痛的镇痛作用
方法:选取雌性SD大鼠根据第一部分的方法制作SNI模型,术后每天检测术侧后爪50%机械缩爪阈值的改变。术后7天选取50%机械缩爪阈值与术前比较显著降低的大鼠18只随机分为三组:PBS组、KLH组及NR2B组,按照第一部分的免疫方法皮下免疫三次。第三次免疫后14天比较各组大鼠50%机械缩爪阈值、热痛缩爪时间等改变;处死大鼠取血清,以间接ELISA方法检测血清NR2B抗体滴度,取腰段脊髓以免疫组化方法分析脊髓背角GFAP蛋白的表达及星形胶质细胞的激活状况。提取大鼠腰段脊髓总蛋白以western blotting检测NR2B蛋白表达的改变。
结果:经过三次免疫,在第三次免疫后14天NR2B组大鼠血清NR2B抗体的滴度为6.9±2.0μg/ml,而PBS组及KLH组未检测到NR2B抗体。SNI术后第7天,PBS、KLH及NR2B各组大鼠50%缩爪阈值分别为2.5±1.4、2.4±0.9及2.0±0.6( g),而术前分别为13.0±1.9、11.7±2.2及12.6±2.7 (g),较术前明显降低(P<0.05),但各组间比较无统计学差异(P>0.05)。在第三次免疫后14天,NR2B组大鼠50%缩爪阈值为6.8±1.5 (g),而PBS组为3.2±1.3 (g),KLH组为3.7±1.4 (g),NR2B组明显高于两对照组(P<0.05)。术后第7天, PBS、KLH、NR2B各组大鼠热痛缩爪时间分别为9.4±1.9、7.8±2.4和8.2±1.9 ( S),而术前各组分别为2.4±1.0、3.4±1.2、2.2±0.9 (S),较术前明显升高(P<0.05),但各组间比较无统计学差异(P>0.05)。在第三次免疫后14天,各组热痛缩爪时间三组分别为7.9±1.6、7.3±1.9、4.6±1.2 (S),NR2B组明显低于对照组(P<0.05)。第三次免疫后14天NR2B组脊髓背角Ⅰ～Ⅱ星形胶质细胞激活状况明显低于对照组(P<0.05)。PBS、KLH及NR2B各组大鼠经过三次免疫,脊髓背角NR2B蛋白与β-actin相对表达丰度分别为74±19 (%)、55±11 (%)、26±4 (%),NR2B组明显低于对照组(P<0.05)。
3、NR2B重组腺病毒镇痛疫苗的构建
方法:以限制性内切酶EcoRⅠ和BamHⅠ双酶切质粒pcDNA3.1-NR2B获得目的基因NR2B;将NR2B定向克隆入腺病毒穿梭载体pDC515,并行酶切及DNA测序鉴定。在脂质体介导下将pDC515-NR2B转染293细胞,并以RT-PCR及Western blotting方法分别从转录水平和翻译水平检测目的基因NR2B的表达。以携带NR2B基因的腺病毒穿梭质粒p515-NR2B和腺病毒骨架质粒pBHGfrt(del)E1,3FLP共转染腺病毒包装细胞293,连续观察细胞的变化(CPE及病毒空斑的出现)。病毒空斑出现后,挑取病毒空斑再感染293细胞提取病毒DNA、RNA及蛋白,分别以PCR、RT-PCR及western blotting从基因水平、转录水平和蛋白水平对病毒空斑进行鉴定和筛选;经鉴定正确的病毒空斑进行大量扩增和纯化并测定其滴度及纯度,为体内研究做准备。
结果:质粒pDC515-NR2B经EcoRⅠ和BamHⅠ酶切后得到4.9kb和3.9kb两条DNA条带,分别与目的片段NR2B和载体片段DC515大小一致,而且进一步行测序鉴定,结果表明NR2B基因正确插入穿梭载体。提取被质粒pDC515-NR2B转染的293细胞的RNA进行RT-PCR反应,RT-PCR产物经1%琼脂糖凝胶电泳分析,可见大小600bp的特异性DNA条带,而未转染的293细胞未见此条带;提取被转染的293细胞的总蛋白经Western blotting检测可见分子量180kDa大小的蛋白条带,而未转染细胞未见此蛋白条带,这表明NR2B基因可在293细胞表达。以穿梭质粒pDC515-NR2B和骨架质粒pBHGfrt(del)E1,3FLP共转染293细胞,共转染后12天可见293细胞变圆并逐渐从壁上脱落,细胞核占据细胞的大部分即所谓CPE改变;15天后较多出现CPE的细胞聚集在一起形成肉眼可见的病毒空斑。被感染的293细胞经三次冻融、离心,提取NR2B重组腺病毒(recombinant adenovirus type 5 encoding NR2B, rAd5/NR2B)上清。分别以rAd5/NR2B原液、10倍、100倍,100倍稀释液进行PCR反应,PCR产物经1%琼脂糖凝胶电泳分析,可见大小600bp的DNA条带,与阳性对照pDC515-NR2B的结果相同,而阴性对照(未用病毒感染的293细胞上清)未见到相应DNA条带。提取被rAd5/NR2B感染的293细胞的RNA行RT- PCR扩增反应,可扩增出600 bp大小的DNA条带,而未感染病毒rAd5/NR2B的293细胞未扩增出相应条带。提取被腺病毒感染的293细胞的蛋白并行Western blotting检测分析,结果感染病毒rAd5/NR2B的293细胞有180kDa的蛋白条带,而未感染该病毒的293细胞无此蛋白条带。rAd5/NR2B经大量扩增及纯化,病毒滴度为5×1011 (V.P./ml),经HPLC法分析纯度为100%。
4、NR2B重组腺病毒疫苗口服免疫对大鼠神经病理性痛的超前镇痛效应
方法: rAd5/NR2B(1×108 PFU)口服免疫大鼠,2周后以相同剂量加强免疫一次,空白组和rAd5/EGFP组大鼠分别以PBS和rAd5/EGFP以同样的剂量和同样的方法免疫作为对照,每组16只。初次免疫后1周取4只大鼠处死,取大鼠近段小肠集合淋巴结组织,提取该组织RNA以RT-PCR方法检测NR2B的表达。分别于免疫后2、4、6、8、10、15周截尾法取血0.5～1.0ml离心取血清检测免疫血清NR2B抗体的滴度。免疫组化染色法检测各组血清与大鼠脊髓背角NR2B蛋白的免疫结合反应。加强免疫后2周,结扎大鼠左侧坐骨神经的胫神经和腓总神经分支,保留腓肠神经,建立SNI模型,术后隔日检测大鼠术侧后爪机械痛阈和热痛缩爪时间的改变。术后一周取其中四只大鼠处死,取大鼠腰段脊髓(L4~6)),以免疫组化法检测脊髓背角c-Fos表达的变化。分别于SNI术前一天和SNI术后1周和6周经枕骨大孔取CSF 20～50μl,检测CSF中NR2B抗体滴度。SNI术后6周取4只处死大鼠取腰段脊髓蛋白,检测免疫大鼠脊髓背角NR2B蛋白的表达水平。
结果:大鼠经疫苗rAd5/NR2B口服免疫1周后,提取近段小肠集合淋巴结组织RNA,经RT-PCR反应可见600bp大小的NR2B特异性DNA条带,而空白组和rAd5/EGFP组无此条带。
初次免疫后2周rAd5/NR2B组大鼠血清NR2B抗体平均滴度为5.5±1.9μg/ml;经加强免疫后2周,NR2B抗体滴度进一步升高为13.4±3.2μg/ml;在免疫后10周内血清NR2B抗体滴度虽略有降低,但仍保持较高水平,免疫后15周血清仍可检测到NR2B抗体存在。而在对应的时间点空白组和rAd5/EGFP组大鼠血清未检测到NR2B抗体存在。免疫组化染色发现,rAd5/NR2B组血清可与脊髓背角NR2B蛋白发生免疫结合反应,呈阳性着色,而对照组未见阳性着色。
SNI术后1周受免大鼠脊髓背角c-Fos阳性神经元的数目rAd5/NR2B组显著低于对照组(P<0.05)。自SNI术后1周至SNI术后6周,rAd5/NR2B组50%机械缩爪阈值明显高于对照组(P<0.05),而热痛缩爪时间明显低于对照组(P<0.05)。CSF中NR2B抗体在SNI术前一天各组均为阴性,SNI术后1周和6周天rAd5/NR2B组CSF中NR2B抗体转为阳性,而对照组为阴性。Western blotting结果显示,初次免疫后10周rAd5/NR2B组大鼠脊髓背角NR2B蛋白的表达水平明显低于对照组(P<0.05)。
5、NR2B重组腺病毒疫苗口服免疫对神经病理性大鼠痛情绪反应的影响
方法:通过位置偏爱实验,选取24只无位置偏爱的SD大鼠,随机分为三组:空白对照组、rAd5/EGF组以及rAd5/NR2B组,每组8只。rAd5/NR2B口服免疫,剂量1×108PFU,2周后以同样剂量加强免疫一次,此为rAd5/NR2B组。而对照组分别以PBS和rAd5/EGFP,按照同样的剂量和方法免疫。加强免疫后2周,截尾法取血0.5～1.0ml并离心取血清,ELISA法检测血清NR2B抗体的滴度。结扎胫神经和腓总神经,保留腓肠神经,制作SNI模型。SNI术后7天将大鼠放入位置回避箱内进行SNI条件性位置回避(SNI conditioned place avoidance,SNI-CPA)实验。该实验分为预处理期1天,训练期4天和测试期1天,三个阶段,观察并记录大鼠在位置回避箱内停留时间。分别于SNI术前一天和实验结束时经侧脑室取CSF20μl左右,分别以ELISA法和western blotting检测其中NR2B抗体滴度和CSF中NR2B抗体与前脑ACC神经元膜蛋白的自身免疫反应。实验结束处死大鼠取前脑ACC组织,分别以免疫组化方法和western blotting检测前脑ACC组织NR2B蛋白的表达水平。
结果:加强免疫后2周,rAd5/NR2B组免疫血清NR2B抗体滴度为13.5±2.3μg/ml,而空白对照组和rAd5/EGFP组血清未检测到NR2B抗体。SNI条件性位置回避时间(预处理期免疫大鼠在条件性位置回避箱停留时间与测试期停留时间的差值为条件性位置回避时间,此值越低提示痛情绪反应越弱)空白组、rAd5/EGFP组以及rAd5/NR2B组分别为198±49,237±40和138±29 (S), rAd5/NR2B组与对照组比较明显降低(P<0.05)。CSF中NR2B抗体滴度检测显示, rAd5/NR2B组在SNI前为阴性,而SNI术后为抗体阳性,而对照组为阴性。western blotting结果表明rAd5/NR2B组CSF可与前脑ACC神经元膜蛋白提取物呈特异性结合反应,而空白对照组和rAd5/EGFP组未见特异性结合反应。ACC组织免疫组化及western blotting结果表明,rAd5/NR2B组大鼠前脑ACC神经元NR2B蛋白的表达较空白对照组和rAd5/EGFP组明显降低(P<0.05)。
6、统计学处理
采用SPSS12.0统计软件进行处理。计量资料以均数±标准差(±S)表示,组内、组间比较采用单因素方差分析;计数资料以率表示,组间比较采用卡方检验,P<0.05为差异有统计学意义。
研究结论
NR2B多肽疫苗皮下三次免疫可诱导机体产生针对NR2B的体液性免疫性应答,血清内检测到NR2B抗体。在神经病理性痛时,该抗体可进入脊髓背角与NR2B蛋白结合,下调NR2B蛋白的表达,对神经病理性痛产生超前镇痛作用。对已存在的神经病理性痛,该疫苗皮下免疫也可减轻痛觉过敏。
将NR2B基因插入腺病毒载体,成功构建NR2B重组腺病毒疫苗rAd5/NR2B。该疫苗口服免疫可诱导产生高滴度的NR2B抗体,其滴度和持续时间优于多肽疫苗。rAd5/NR2B疫苗口服免疫不但对神经病理性痛痛感觉产生超前镇痛作用,而且对神经病理性痛伴随的痛情绪反应也有抑制作用。
研究总结
本研究首先采用NR2B多肽疫苗皮下免疫的方式证实,NR2B疫苗不仅可诱导机体产生针对NR2B的体液性免疫应答,而且在神经病理性痛时NR2B抗体可进入脊髓与脊髓背角NR2B结合,产生镇痛作用。针对亚单位疫苗的不足,本研究又将NR2B基因插入腺病毒载体构建NR2B重组腺病毒疫苗rAd5/NR2B,而且分别从痛感觉和痛情绪两方面观察了该疫苗的镇痛效应,为神经病理性痛基因疫苗的治疗提供了理论依据。
Background
Neuropathic pain is defined as pain initiated or caused by a primary lesion or dysfunction in the nervous system. The reasons responsible for neuropathic pain include trauma, ischaemic injury, infection/inflammation, cancer, drugs and compression etc. Patients with neuropathic pain do not respond to non-steriodal anti-inflammatory drugs and have resistance or insensitivity to opiates. The current pharmacological mainstays of clinical management are tricyclic anti-depressants and certain anticonvulsants, but these have only achieved clinical significant pain relief in less than 50% of patients and are associated with sub-optimal side effect profiles. Therefore, an important issue of pain research is to find a novel method of analgesia that is safe and effective. Peripheral nerve injury may result in a persistent pain due to abnormal activity in peripheral afferents and a sustained input to the spinal cord that leads to alterations in the activity and excitability of spinal cord neurons. These spinal changes involve central sensitization of dorsal horn neurons. Spinal N-methyl-D-aspartate (NMDA) receptor plays an important role in the facilitation and maintenance of central sensitization of spinal dorsal horn neurons. NMDA receptor antagonists such as ketamine, dextromethorphan and MK-801 have been reported to produce symptomatic relief in a number of neuropathies including postherpetic neuralgia, central pain caused by spinal cord injury and phantom limb pain. However, these agents also induce unacceptable side effects at analgesic doses including hallucinations, dysphoria and disturbances of cognitive and motor function, which exclude their widespread use.The NMDA receptor complex comprises multiple protein subunits, of which there are at least one NMDAR1 (NR1) subunit and at least one of a family of NR2 subunits (NR2A-NR2D). NR2B subunit is distributed discretely in the CNS and it is possible to support a reduced side effect profile of approach that act selectively at this site. The development of the modern vaccine technology is not only to develop vaccines aiming directly at ectogenic antigen, but also to endogenous antigen. The vaccine made from the tumour cell epitope has been proved to prevent and treat efficiently the tumour. The NMDAR1 epitope vaccine desigened by During et al provided a novel strategy for prevention and cure of stroke and epilepsy. Therefore, as an alternative approach to treat neuropathic pain, we hypothesise that a humoral autoimmune response targeting the NR2B subunit of NMDA receptor would relieve pain like behaviours produced by peripheral injury.
Methods and Results
1. The immunized and preemptive analgesic effect of NR2B peptide vaccine on rats with neuropathic pain
Methods NR2B peptide ( Sequence: TNGKHGKKINGTWNGMIGEY) is derived from C-terminal region of NMDA receptors 2B subunit. NR2B peptide is incomplete immunogens but is made fully immunogenic by coupling them to a suitable carrier protein KLH. Female SD rats were divided into three groups randomly: group PBS (vaccined with PBS), group KLH (vaccined with KLH) and group NR2B (vaccined with NR2B-KLH). Rats were immunized subcutaneouly three times at a two-weekly intervals. NR2B-KLH was emulsified in Freund’s Complete Adjuvent or Freund’s Incomplete Adjuvent (1:1, v/v) just before using. NR2B-specific Ig G titers in sera were determined by ELISA. By day 7 after the third immunization, surgical procedures were performed under 10% Chloral Hydrate anesthesia. After skin and muscle incision, 2 of the 3 terminal branches of the sciatic nerve ( tibial, and common peroneal nerves) with 9-0 silk suture were tightly ligated. Behavioral testing (50% paw withdraw thresholds and the duration of the withdrawal after the heat stimulation ) took place on every other day after surgery, till 7 days after surgery. CSF (10~20μl) was drawn from the cisterna magna using a 27-gauge needle on day 1 before SNI and on day 7 after SNI, respectively. NR2B-specific Ig G titers in CSF were determined by ELISA . On day 7 after the surgery, the L4~6 segment of the spinal cord was harvested. The number of c-Fos positive neurons in spinal cord dorsal horn was determined using immunohistochemistry. The expression of NR2B protein in spinal cord were determined using western blotting.The reaction of sera from rats with NR2B subuits in the spinal cord horn were tested using immunohistochemistry.
Results After the second vaccination with NR2B peptide , NR2B-specific IgG were detected in six of nine rats(>0.5μg/ml). After the third vaccination with NR2B peptide, all of the rats had more than 2.2μg/ml NR2B-spicific antibody titer. Titers of NR2B-specific IgG peaked 42 days post initial immunization and persisted over the 70 days evaluated. No NR2B-specific antibody was detected in sera from rats of group PBS or KLH. On day 7 after surgery, the 50% paw withdraw thresholds of group NR2B were significantly higher than that of group PBS or KLH (P<0.05). The paw withdrawal duration to radiant heat stimulation in group NR2B were less than that of group PBS or KLH (P<0.05). The number of c-Fos positive neurons in spinal cord dorsal horn of NR2B group were less than that in PBS or KLH group (P<0.05). NR2B-specific IgG of NR2B group was positive on day 7 after SNI. There is a significant reduction for NR2B protein level in the lumbar spinal cord in group NR2B compared with that in group PBS or KLH (P< 0.01). Sera from rats immunized with NR2B-KLH react with NR2B protein in spinal horn.
2. Evaluation of NR2B peptide as subunit vaccines against neuropathic pain being existed previously
Methods After skin and muscle incision, 2 of the 3 terminal branches of the sciatic nerve (tibial, and common peroneal nerves) with 9-0 silk suture were tightly ligated. Behavioral testing (50% paw withdraw thresholds and the duration of the withdrawal after the heat stimulation ) took place on every day after surgery, till 7days after surgery. Female SD rats developed hyperalgesia were divided into three groups randomly: PBS、KLH and NR2B. Rats were immunized with PBS、KLH or NR2B, respectively, three times subcutaneously at a two-weekly intervals, in combination with Complete Freund’s adjuvant (CFA) or Incomplete Freund’s adjuvant (ICFA).By day 14 after the third immunization, 50% paw withdraw thresholds and the paw withdraw duration to radiant heat stimulation were assessed and then sera were harvested for detection of titers of NR2B antibody. The L4~6 segment of the spinal cord was harvested. The astrocytic activation was determined using immunohistochemistry with antibodies of GFAP (an astrocyte marker). The expressive level of NR2B protein in spinal cord horn were detected using western bloting.
Results The titers of NR2B antibody in NR2B group was 6.94±2.04μg/ml on day 14 after the third immunization and there was no detection of NR2B antibody in PBS or KLH groups. All rats have developed a marked hyperalgesia 7 days after SNI, however, three times immunization later, mechanical hypersensitivity and heat hyperalgesia in NR2B groups were relieved, compared with PBS or KLH groups. The expression of GFAP indicated that astrocytic in group NR2B became less intense responses in the ipsilateralⅠ~Ⅳlemanie of dorsal horn than control group(PBS or KLH). Western blotting analysis showed that NR2B peptide vaccine decreased the expressive level of NR2B protein in spinal cord horn after the third immunization.
3. Construction of recombinant adenovirus analgesic vaccine encoding NR2B gene
Methods NR2B gene was obtained by digesting the plasmids pcDNA3.1-NR2B with EcoRⅠand BamHⅠand was cloned into the adenovirus shuttle vector pDC515. Then the vector was transferred into 293 cell and the expression of NR2B gene in 293 cell was identified by RT-PCR and western blotting. The shuttle plasmid pDC515-NR2B and the genomic plasmid pBHGfrt(del)E1,3FLP were cotransfected into the package 293 cells using lipofectamine TM 2000. The contransfected 293 cells were observed persistently for the production of CPE and the viral plaques. The viral plaques were picked and were put into infecting the passage cell 293. The recombinant Ad5/ NR2B were identified by PCR、RT-PCR and western blotting. The amplification and purification of the recombinant Ad5/NR2B were made and the titer and purity of the virus were assayed
Results First, transgene NR2B was cloned into the small adenovirus shuttle vector pDC515. The presence and oritention of the insert were confirmed using restriction endonuclease analysis and then the plasmid was termed as pDC515-NR2B. Second, pDC515-NR2B are cotransfected with an Ad genomic plasmid pBHGfrt△E1,3FLP into E1 complementing cell line 293 cells. The recombination between the two contransfected plsmids was mediated by frt site specific recombinase, FLP. The formation of viral plaques begins with the infection of one cell by one virus followed by multiple cycles of complete infection when released viruses infect surrounding cells. By day 15 following contransfection, well-defined plaques can be observed under the microscope . In order to build up working stocks of virus from plaque isolates before extensive experimentation, the presence and oritention of transgene NR2B in the recombinant adenovirus vector were confirmed using NR2B-specific PCR. The viral stocks of rAd5/NR2B were generated by the transient transfection of 293 cells and purified using CsCl ultracentrifugation gradients. The final titer was 5×1011 (Plaque Forming Unit, PFU)/ml and the purity is 100% using identification of HPLC methods.
4. rAd5/NR2B vaccine induces protective immunity against neuropathic pain
Methods The rAd5/NR2B (1×108 PFU) was administered via an orogastric tube into the stomachs of a group of rats (n=16), with a control group (n=16) receiving a similar dose of a recombinant Ad5 virus expressing EGFP or PBS, and followed by a booster immunization with the same dose two weeks after the initial immunization. A week later, expression of NR2B gene in the intestine M cells was assessed by RT-PCR. NR2B-specific Ig G titers in sera were determined by ELISA at a two-weekly intervals. Two weeks after the boost immunization, SNI model was established through the ligation of tibial nerve and peroneal nerve. A week after the SNI procedure, the number of c-Fos positive neurons in the spinal dorasl horn were assessed using immunohistochemistry. 50% paw withdraw thresholds of mechanical stimulation were assessed using the up-down paradigm every other day after SNI and withdrawal duration of heat response were also assessed at the same timepoint. Serial spinal sections from rats were stained with sera from rats treated with PBS , rAd5/EGFP or rAd5/NR2B. CSF (20~50μl) was drawn from the cisterna magna using a 27-gauge needle on day 1 before SNI, on day 7 after SNI and on day 42 after SNI, respectively. Six weeks after SNI, the expression of NR2B gene in the lumbar spinal cord were assessed using western blotting.
Results Following the primary immunization and the booster immunization, NR2B-spicific IgG in sera were 5.5.±1.9μg/ml and 13.4±3.2μg/ml,respectively. In contrast, NR2B-spicific IgG were not detected in sera from the control groups (na?ve and rAd5/EGFP).By day 7 after the primary vaccination with rAd5/NR2B, the expression of NR2B protein were detected in the intestine of rats immunized with rAd5/NR2B. By day 7 after surgery, the 50% paw withdraw thresholds of group rAd5/NR2B were significantly higher than control groups (na?ve and rAd5/EGFP) (P<0.05). The paw withdrawal duration to radiant heat stimulation in grouprAd5/NR2B were less than the control groups (na?ve and rAd5/EGFP) (P<0.05). The significant difference of 50% paw withdraw thresholds and paw withdrawal duration to radiant heat stimulation among the three groups remains till 42 days after the surgery. NR2B-spicific IgG in CSF was only detected on day 7 and 42 post the initial immunization in rAd5/NR2B group. And only sera from rats immunized with rAd5/NR2B reacted with NR2B protein in the lumbar spinal cord dorsal horn. Western botting analysis showed that immunization with rAd5/NR2B decreased the expression of NR2B protein in the spinal dorsal horn.
5. rAd5/NR2B vaccine induces protective immunity against the affective component relating to neuropathic pain
Methods SD rats without preference one compartment to the others before conditioning were selected and were divided into three groups randomly: na?ve, rAd5/EGFP and rAd5/NR2B. The rAd5/NR2B (1×108 PFU) was administered via an orogastric tube into the stomach of a group of rats, with a control group receiving a similar dose of a recombinant Ad5 virus expressing EGFP or PBS. Two weeks later, re-immunization were performed by the same dose of vaccine. Titers of NR2B antibody in sera were determined using ELISA at week 2 following the booost immunization and at the same time, SNI model was established through the ligation of tibial nerve and peroneal nerve. From the day 7 after SNI on, conditioned place avoidance (CPA) test were carried out. CSF (20μl) was drawn from the lateral ventricle using a 27-gauge needle on day 1 before SNI and on the day of SNI-CPA completion ,respectively. Anti-NR2B antibody titer in CSF were detected using ELISA and CSF was screened against ACC membrane extract by immunoblot analysis. The NR2B protein expression in ACC were assessed using western blotting and immunohistochemistry.
Results Two weeks following the booster immunization, titers of NR2B antibody in rAd5/NR2B groups was up to 13.5±2.3μg/ml. CPA scores in rAd5/NR2B group was significantly less than that in na?ve or rAd5/EGFP group(P<0.05). ( rAd5/NR2B group: 138.1±29.2S, na?ve group: 198.1±49.5S,rAd5/EGFP group: 236.8±40.2S). By day 13 after SNI, NR2B antibody in CSF of rAd5/NR2B group could be detected and react with NR2B protein of ACC membrane extracts. The expressive level of NR2B protein in ACC of rAd5/NR2B-i mmunized rats was decreased significantly compared with rats in na?ve or rAd5/EGFP group.
6. Statistical Analysis
All of the analyses were performed by SPSS 12.0 software package. Measurement data were presented as means±SD. The differences between multiple individual means were analyzed by one-way ANOVA. Categorical data were presented as rate. The differences between multiple individual rates were analyzed by Chi-square test. P < 0.05 was considered statistically significant in all tests.
Conclusion
NR2B peptide vaccine could induce the humoral immunological response to produce NR2B-specific antibody in sera. NR2B-specific antibody could react with NR2B subunit in spinal dorsal horn to reduce the NR2B protein expression and then have a pre-emptive analgesic effect on neuropathic pain. As for neuropathic pain existed previously, NR2B peptide vaccine could also relieve hyperalgesia. NR2B gene was inserted into the adenovirus shuttle vector pDC515 and then pDC515-NR2B was constructed successfully. The vaccine rAd5/NR2B was constructed successfully using cotransfection with pDC515-NR2B and pBHGfrt(del)E1,3FLP, which induced the higher titer of NR2B-specific antibody than NR2B peptide vaccine. The oral immunization with rAd5/NR2B could not only produce the analgesic effect against pain-related sensory but also alleviate the pain-related affective response.
Summary
The present study firstly demonstrated that NR2B peptide vaccine not only induced the humoral immunological response but also NR2B-spicific antibody could react with NR2B protein in the spinal dorsal horn to fight against neuropathic pain. In order to improve the immunological effect, rAd5/NR2B vaccine could construct.The present study made a research about the analgesic effect of rAd5/NR2B vaccine on both sensory and affective component of neuropathic pain, providing a novel direction for the treatment of neuropathic pain.
神经病理性疼痛是神经系统原发性损伤或功能异常所导致的痛觉异常或痛觉过敏。创伤、缺血性损伤、感染或炎症、肿瘤、药物和压迫等原因均可以引起神经病理性疼痛。非甾体类抗炎药和阿片类镇痛药治疗神经病理性疼痛的效果差,临床上常使用三环类抗抑郁药和抗癫痫药物进行治疗,但疗效差,副作用大,能够有效缓解50%以上疼痛症状的患者不足50%。因此寻找一种安全有效、作用持久的镇痛方法已成为神经病理性疼痛研究的重要方向。神经病理性痛的受体机制研究表明,神经元和神经胶质细胞膜上的N-甲基-D-天冬氨酸受体(N-methyl-D-aspartate receptor,NMDAR)表达上调或过度激活可引起中枢敏感化,在神经病理性痛的产生如自发性痛,触诱发痛及痛觉过敏等起重要作用。NMDA受体拮抗机如氯胺酮、MK-801等对神经病理性痛具有良好的镇痛作用,但由于这些药物具有如认知功能障碍、神经空泡样改变等副作用等限制了它们的临床应用。近期的研究表明,NMDA受体的亚型——NR2B型NMDA受体(NR2B)在体内呈选择性分布如主要分布于脊髓背角、前脑等与痛传递和痛情绪有关的区域,与痛感觉和痛情绪的形成密切相关,这提示NR2B可能是一个潜在的镇痛治疗的靶点。现在疫苗技术的发展不仅针对外源性抗原开发出各种疫苗,而且能对机体自身抗原制备特异性疫苗,如最近利用肿瘤细胞表面的特异性位点研制的疫苗已被证实能有效地预防和治疗癌症地发生发展。During等构建的NMDAR1表位疫苗为防治中风和癫痫后脑损伤提供了新的治疗策略。据此,我们的假设:以NR2B为抗原制备疫苗诱导机体产生NR2B抗体,该抗体与神经元或胶质细胞膜上的NR2B结合,下调NR2B的表达或阻断NR2B的激活,发挥镇痛作用。因此,本研究旨在首先观察NR2B多肽疫苗对神经病理性痛大鼠的免疫效应及镇痛效应,然后构建NR2B重组腺病毒(Recombinant adenovirus serotype 5 encoding NR2B gene, rAd5/NR2B)镇痛疫苗,并通过口服免疫大鼠,以评价其对大鼠神经病理性痛痛感觉和痛情绪的影响。
研究方法与结果
1、NR2B多肽疫苗对神经病理性痛大鼠的免疫效应及超前镇痛效应
方法:选取NR2B有免疫原性的一段氨基酸序列合成多肽,并与大分子蛋白载体匙孔虫戚血蓝蛋白( Keyhole LimpetHemocyanin , KLH)交联制备NR2B多肽疫苗即NR2B-KLH。健康雌性SD大鼠随机分为三组:PBS组、KLH组及NR2B组。分别用PBS+福氏佐剂, KLH+福氏佐剂和NR2B-KLH+福氏佐剂皮下注射三次,每次注射后两周以间接ELISA法检测受免大鼠血清NR2B特异性抗体的滴度,并以免疫组化染色法检测大鼠免疫血清与脊髓背角NR2B蛋白的免疫结合反应。于第三次免疫后一周结扎坐骨神经的胫神经和腓总神经分支,保留腓肠神经分支,建立SD大鼠保留神经损伤性神经病理性痛模型(Spared nerve injury, SNI)。术后隔日以up-down法检测大鼠术侧后爪50%机械缩爪阈值的变化及热痛缩爪时间的变化。分别于术前一天和术后7天经枕骨大孔取脑脊液(cerebrospinal fluid, CSF)10～20μl,以间接ELISA法检测CSF中NR2B抗体滴度。术后7天处死大鼠,取腰段脊髓(L4~6),以免疫组化染色法检测脊髓背角c-Fos阳性神经元数目,并采用Western blotting法检测脊髓背角NR2B蛋白的改变。
结果:第二次注射后, NR2B组9只大鼠有6只大鼠出现NR2B抗体(>0.5μg/ml)。第三次注射后9只大鼠均检测到NR2B抗体(>2.2μg/ml),且滴度较第一次明显升高。在第三次注射后14天抗体滴度最高(7.2μg/ml),随后抗体滴度逐渐降低,但初次注射后70天血清仍可检测到NR2B抗体。而PBS组和KLH组未检测到NR2B抗体。免疫组化染色法检测发现NR2B大鼠血清可与大鼠脊髓背角结合呈阳性着色,而其余两组未见着色。与术前比较,术后第7天PBS、KLH及NR2B各组50%缩爪阈值均明显降低(P<0.05);但各组间比较,NR2B组明显高于其余两对照组(P<0.05)。各组术后7天热痛缩爪时间与术前比较均有显著升高(P<0.05),但各组间比较,NR2B组明显低于其余两对照组(P<0.05)。术后7天脊髓背角c-Fos蛋白阳性神经元的数目NR2B组明显低于两对照组(P<0.05)(NR2B组:170±22,PBS组:301±36,KLH组:280±25)。术前NR2B组大鼠脑脊液中NR2B抗体呈阴性,而术后7天呈阳性,而PBS组和KLH组这两个时间点均为阴性。Western blotting法检测发现术后7天NR2B组脊髓背角NR2B蛋白的表达较其余两组明显降低(P<0.05)。
2、NR2B多肽疫苗对已发生的大鼠神经病理性痛的镇痛作用
方法:选取雌性SD大鼠根据第一部分的方法制作SNI模型,术后每天检测术侧后爪50%机械缩爪阈值的改变。术后7天选取50%机械缩爪阈值与术前比较显著降低的大鼠18只随机分为三组:PBS组、KLH组及NR2B组,按照第一部分的免疫方法皮下免疫三次。第三次免疫后14天比较各组大鼠50%机械缩爪阈值、热痛缩爪时间等改变;处死大鼠取血清,以间接ELISA方法检测血清NR2B抗体滴度,取腰段脊髓以免疫组化方法分析脊髓背角GFAP蛋白的表达及星形胶质细胞的激活状况。提取大鼠腰段脊髓总蛋白以western blotting检测NR2B蛋白表达的改变。
结果:经过三次免疫,在第三次免疫后14天NR2B组大鼠血清NR2B抗体的滴度为6.9±2.0μg/ml,而PBS组及KLH组未检测到NR2B抗体。SNI术后第7天,PBS、KLH及NR2B各组大鼠50%缩爪阈值分别为2.5±1.4、2.4±0.9及2.0±0.6( g),而术前分别为13.0±1.9、11.7±2.2及12.6±2.7 (g),较术前明显降低(P<0.05),但各组间比较无统计学差异(P>0.05)。在第三次免疫后14天,NR2B组大鼠50%缩爪阈值为6.8±1.5 (g),而PBS组为3.2±1.3 (g),KLH组为3.7±1.4 (g),NR2B组明显高于两对照组(P<0.05)。术后第7天, PBS、KLH、NR2B各组大鼠热痛缩爪时间分别为9.4±1.9、7.8±2.4和8.2±1.9 ( S),而术前各组分别为2.4±1.0、3.4±1.2、2.2±0.9 (S),较术前明显升高(P<0.05),但各组间比较无统计学差异(P>0.05)。在第三次免疫后14天,各组热痛缩爪时间三组分别为7.9±1.6、7.3±1.9、4.6±1.2 (S),NR2B组明显低于对照组(P<0.05)。第三次免疫后14天NR2B组脊髓背角Ⅰ～Ⅱ星形胶质细胞激活状况明显低于对照组(P<0.05)。PBS、KLH及NR2B各组大鼠经过三次免疫,脊髓背角NR2B蛋白与β-actin相对表达丰度分别为74±19 (%)、55±11 (%)、26±4 (%),NR2B组明显低于对照组(P<0.05)。
3、NR2B重组腺病毒镇痛疫苗的构建
方法:以限制性内切酶EcoRⅠ和BamHⅠ双酶切质粒pcDNA3.1-NR2B获得目的基因NR2B;将NR2B定向克隆入腺病毒穿梭载体pDC515,并行酶切及DNA测序鉴定。在脂质体介导下将pDC515-NR2B转染293细胞,并以RT-PCR及Western blotting方法分别从转录水平和翻译水平检测目的基因NR2B的表达。以携带NR2B基因的腺病毒穿梭质粒p515-NR2B和腺病毒骨架质粒pBHGfrt(del)E1,3FLP共转染腺病毒包装细胞293,连续观察细胞的变化(CPE及病毒空斑的出现)。病毒空斑出现后,挑取病毒空斑再感染293细胞提取病毒DNA、RNA及蛋白,分别以PCR、RT-PCR及western blotting从基因水平、转录水平和蛋白水平对病毒空斑进行鉴定和筛选;经鉴定正确的病毒空斑进行大量扩增和纯化并测定其滴度及纯度,为体内研究做准备。
结果:质粒pDC515-NR2B经EcoRⅠ和BamHⅠ酶切后得到4.9kb和3.9kb两条DNA条带,分别与目的片段NR2B和载体片段DC515大小一致,而且进一步行测序鉴定,结果表明NR2B基因正确插入穿梭载体。提取被质粒pDC515-NR2B转染的293细胞的RNA进行RT-PCR反应,RT-PCR产物经1%琼脂糖凝胶电泳分析,可见大小600bp的特异性DNA条带,而未转染的293细胞未见此条带;提取被转染的293细胞的总蛋白经Western blotting检测可见分子量180kDa大小的蛋白条带,而未转染细胞未见此蛋白条带,这表明NR2B基因可在293细胞表达。以穿梭质粒pDC515-NR2B和骨架质粒pBHGfrt(del)E1,3FLP共转染293细胞,共转染后12天可见293细胞变圆并逐渐从壁上脱落,细胞核占据细胞的大部分即所谓CPE改变;15天后较多出现CPE的细胞聚集在一起形成肉眼可见的病毒空斑。被感染的293细胞经三次冻融、离心,提取NR2B重组腺病毒(recombinant adenovirus type 5 encoding NR2B, rAd5/NR2B)上清。分别以rAd5/NR2B原液、10倍、100倍,100倍稀释液进行PCR反应,PCR产物经1%琼脂糖凝胶电泳分析,可见大小600bp的DNA条带,与阳性对照pDC515-NR2B的结果相同,而阴性对照(未用病毒感染的293细胞上清)未见到相应DNA条带。提取被rAd5/NR2B感染的293细胞的RNA行RT- PCR扩增反应,可扩增出600 bp大小的DNA条带,而未感染病毒rAd5/NR2B的293细胞未扩增出相应条带。提取被腺病毒感染的293细胞的蛋白并行Western blotting检测分析,结果感染病毒rAd5/NR2B的293细胞有180kDa的蛋白条带,而未感染该病毒的293细胞无此蛋白条带。rAd5/NR2B经大量扩增及纯化,病毒滴度为5×1011 (V.P./ml),经HPLC法分析纯度为100%。
4、NR2B重组腺病毒疫苗口服免疫对大鼠神经病理性痛的超前镇痛效应
方法: rAd5/NR2B(1×108 PFU)口服免疫大鼠,2周后以相同剂量加强免疫一次,空白组和rAd5/EGFP组大鼠分别以PBS和rAd5/EGFP以同样的剂量和同样的方法免疫作为对照,每组16只。初次免疫后1周取4只大鼠处死,取大鼠近段小肠集合淋巴结组织,提取该组织RNA以RT-PCR方法检测NR2B的表达。分别于免疫后2、4、6、8、10、15周截尾法取血0.5～1.0ml离心取血清检测免疫血清NR2B抗体的滴度。免疫组化染色法检测各组血清与大鼠脊髓背角NR2B蛋白的免疫结合反应。加强免疫后2周,结扎大鼠左侧坐骨神经的胫神经和腓总神经分支,保留腓肠神经,建立SNI模型,术后隔日检测大鼠术侧后爪机械痛阈和热痛缩爪时间的改变。术后一周取其中四只大鼠处死,取大鼠腰段脊髓(L4~6)),以免疫组化法检测脊髓背角c-Fos表达的变化。分别于SNI术前一天和SNI术后1周和6周经枕骨大孔取CSF 20～50μl,检测CSF中NR2B抗体滴度。SNI术后6周取4只处死大鼠取腰段脊髓蛋白,检测免疫大鼠脊髓背角NR2B蛋白的表达水平。
结果:大鼠经疫苗rAd5/NR2B口服免疫1周后,提取近段小肠集合淋巴结组织RNA,经RT-PCR反应可见600bp大小的NR2B特异性DNA条带,而空白组和rAd5/EGFP组无此条带。
初次免疫后2周rAd5/NR2B组大鼠血清NR2B抗体平均滴度为5.5±1.9μg/ml;经加强免疫后2周,NR2B抗体滴度进一步升高为13.4±3.2μg/ml;在免疫后10周内血清NR2B抗体滴度虽略有降低,但仍保持较高水平,免疫后15周血清仍可检测到NR2B抗体存在。而在对应的时间点空白组和rAd5/EGFP组大鼠血清未检测到NR2B抗体存在。免疫组化染色发现,rAd5/NR2B组血清可与脊髓背角NR2B蛋白发生免疫结合反应,呈阳性着色,而对照组未见阳性着色。
SNI术后1周受免大鼠脊髓背角c-Fos阳性神经元的数目rAd5/NR2B组显著低于对照组(P<0.05)。自SNI术后1周至SNI术后6周,rAd5/NR2B组50%机械缩爪阈值明显高于对照组(P<0.05),而热痛缩爪时间明显低于对照组(P<0.05)。CSF中NR2B抗体在SNI术前一天各组均为阴性,SNI术后1周和6周天rAd5/NR2B组CSF中NR2B抗体转为阳性,而对照组为阴性。Western blotting结果显示,初次免疫后10周rAd5/NR2B组大鼠脊髓背角NR2B蛋白的表达水平明显低于对照组(P<0.05)。
5、NR2B重组腺病毒疫苗口服免疫对神经病理性大鼠痛情绪反应的影响
方法:通过位置偏爱实验,选取24只无位置偏爱的SD大鼠,随机分为三组:空白对照组、rAd5/EGF组以及rAd5/NR2B组,每组8只。rAd5/NR2B口服免疫,剂量1×108PFU,2周后以同样剂量加强免疫一次,此为rAd5/NR2B组。而对照组分别以PBS和rAd5/EGFP,按照同样的剂量和方法免疫。加强免疫后2周,截尾法取血0.5～1.0ml并离心取血清,ELISA法检测血清NR2B抗体的滴度。结扎胫神经和腓总神经,保留腓肠神经,制作SNI模型。SNI术后7天将大鼠放入位置回避箱内进行SNI条件性位置回避(SNI conditioned place avoidance,SNI-CPA)实验。该实验分为预处理期1天,训练期4天和测试期1天,三个阶段,观察并记录大鼠在位置回避箱内停留时间。分别于SNI术前一天和实验结束时经侧脑室取CSF20μl左右,分别以ELISA法和western blotting检测其中NR2B抗体滴度和CSF中NR2B抗体与前脑ACC神经元膜蛋白的自身免疫反应。实验结束处死大鼠取前脑ACC组织,分别以免疫组化方法和western blotting检测前脑ACC组织NR2B蛋白的表达水平。
结果:加强免疫后2周,rAd5/NR2B组免疫血清NR2B抗体滴度为13.5±2.3μg/ml,而空白对照组和rAd5/EGFP组血清未检测到NR2B抗体。SNI条件性位置回避时间(预处理期免疫大鼠在条件性位置回避箱停留时间与测试期停留时间的差值为条件性位置回避时间,此值越低提示痛情绪反应越弱)空白组、rAd5/EGFP组以及rAd5/NR2B组分别为198±49,237±40和138±29 (S), rAd5/NR2B组与对照组比较明显降低(P<0.05)。CSF中NR2B抗体滴度检测显示, rAd5/NR2B组在SNI前为阴性,而SNI术后为抗体阳性,而对照组为阴性。western blotting结果表明rAd5/NR2B组CSF可与前脑ACC神经元膜蛋白提取物呈特异性结合反应,而空白对照组和rAd5/EGFP组未见特异性结合反应。ACC组织免疫组化及western blotting结果表明,rAd5/NR2B组大鼠前脑ACC神经元NR2B蛋白的表达较空白对照组和rAd5/EGFP组明显降低(P<0.05)。
6、统计学处理
采用SPSS12.0统计软件进行处理。计量资料以均数±标准差(±S)表示,组内、组间比较采用单因素方差分析;计数资料以率表示,组间比较采用卡方检验,P<0.05为差异有统计学意义。
研究结论
NR2B多肽疫苗皮下三次免疫可诱导机体产生针对NR2B的体液性免疫性应答,血清内检测到NR2B抗体。在神经病理性痛时,该抗体可进入脊髓背角与NR2B蛋白结合,下调NR2B蛋白的表达,对神经病理性痛产生超前镇痛作用。对已存在的神经病理性痛,该疫苗皮下免疫也可减轻痛觉过敏。
将NR2B基因插入腺病毒载体,成功构建NR2B重组腺病毒疫苗rAd5/NR2B。该疫苗口服免疫可诱导产生高滴度的NR2B抗体,其滴度和持续时间优于多肽疫苗。rAd5/NR2B疫苗口服免疫不但对神经病理性痛痛感觉产生超前镇痛作用,而且对神经病理性痛伴随的痛情绪反应也有抑制作用。
研究总结
本研究首先采用NR2B多肽疫苗皮下免疫的方式证实,NR2B疫苗不仅可诱导机体产生针对NR2B的体液性免疫应答,而且在神经病理性痛时NR2B抗体可进入脊髓与脊髓背角NR2B结合,产生镇痛作用。针对亚单位疫苗的不足,本研究又将NR2B基因插入腺病毒载体构建NR2B重组腺病毒疫苗rAd5/NR2B,而且分别从痛感觉和痛情绪两方面观察了该疫苗的镇痛效应,为神经病理性痛基因疫苗的治疗提供了理论依据。
Background
Neuropathic pain is defined as pain initiated or caused by a primary lesion or dysfunction in the nervous system. The reasons responsible for neuropathic pain include trauma, ischaemic injury, infection/inflammation, cancer, drugs and compression etc. Patients with neuropathic pain do not respond to non-steriodal anti-inflammatory drugs and have resistance or insensitivity to opiates. The current pharmacological mainstays of clinical management are tricyclic anti-depressants and certain anticonvulsants, but these have only achieved clinical significant pain relief in less than 50% of patients and are associated with sub-optimal side effect profiles. Therefore, an important issue of pain research is to find a novel method of analgesia that is safe and effective. Peripheral nerve injury may result in a persistent pain due to abnormal activity in peripheral afferents and a sustained input to the spinal cord that leads to alterations in the activity and excitability of spinal cord neurons. These spinal changes involve central sensitization of dorsal horn neurons. Spinal N-methyl-D-aspartate (NMDA) receptor plays an important role in the facilitation and maintenance of central sensitization of spinal dorsal horn neurons. NMDA receptor antagonists such as ketamine, dextromethorphan and MK-801 have been reported to produce symptomatic relief in a number of neuropathies including postherpetic neuralgia, central pain caused by spinal cord injury and phantom limb pain. However, these agents also induce unacceptable side effects at analgesic doses including hallucinations, dysphoria and disturbances of cognitive and motor function, which exclude their widespread use.The NMDA receptor complex comprises multiple protein subunits, of which there are at least one NMDAR1 (NR1) subunit and at least one of a family of NR2 subunits (NR2A-NR2D). NR2B subunit is distributed discretely in the CNS and it is possible to support a reduced side effect profile of approach that act selectively at this site. The development of the modern vaccine technology is not only to develop vaccines aiming directly at ectogenic antigen, but also to endogenous antigen. The vaccine made from the tumour cell epitope has been proved to prevent and treat efficiently the tumour. The NMDAR1 epitope vaccine desigened by During et al provided a novel strategy for prevention and cure of stroke and epilepsy. Therefore, as an alternative approach to treat neuropathic pain, we hypothesise that a humoral autoimmune response targeting the NR2B subunit of NMDA receptor would relieve pain like behaviours produced by peripheral injury.
Methods and Results
1. The immunized and preemptive analgesic effect of NR2B peptide vaccine on rats with neuropathic pain
Methods NR2B peptide ( Sequence: TNGKHGKKINGTWNGMIGEY) is derived from C-terminal region of NMDA receptors 2B subunit. NR2B peptide is incomplete immunogens but is made fully immunogenic by coupling them to a suitable carrier protein KLH. Female SD rats were divided into three groups randomly: group PBS (vaccined with PBS), group KLH (vaccined with KLH) and group NR2B (vaccined with NR2B-KLH). Rats were immunized subcutaneouly three times at a two-weekly intervals. NR2B-KLH was emulsified in Freund’s Complete Adjuvent or Freund’s Incomplete Adjuvent (1:1, v/v) just before using. NR2B-specific Ig G titers in sera were determined by ELISA. By day 7 after the third immunization, surgical procedures were performed under 10% Chloral Hydrate anesthesia. After skin and muscle incision, 2 of the 3 terminal branches of the sciatic nerve ( tibial, and common peroneal nerves) with 9-0 silk suture were tightly ligated. Behavioral testing (50% paw withdraw thresholds and the duration of the withdrawal after the heat stimulation ) took place on every other day after surgery, till 7 days after surgery. CSF (10~20μl) was drawn from the cisterna magna using a 27-gauge needle on day 1 before SNI and on day 7 after SNI, respectively. NR2B-specific Ig G titers in CSF were determined by ELISA . On day 7 after the surgery, the L4~6 segment of the spinal cord was harvested. The number of c-Fos positive neurons in spinal cord dorsal horn was determined using immunohistochemistry. The expression of NR2B protein in spinal cord were determined using western blotting.The reaction of sera from rats with NR2B subuits in the spinal cord horn were tested using immunohistochemistry.
Results After the second vaccination with NR2B peptide , NR2B-specific IgG were detected in six of nine rats(>0.5μg/ml). After the third vaccination with NR2B peptide, all of the rats had more than 2.2μg/ml NR2B-spicific antibody titer. Titers of NR2B-specific IgG peaked 42 days post initial immunization and persisted over the 70 days evaluated. No NR2B-specific antibody was detected in sera from rats of group PBS or KLH. On day 7 after surgery, the 50% paw withdraw thresholds of group NR2B were significantly higher than that of group PBS or KLH (P<0.05). The paw withdrawal duration to radiant heat stimulation in group NR2B were less than that of group PBS or KLH (P<0.05). The number of c-Fos positive neurons in spinal cord dorsal horn of NR2B group were less than that in PBS or KLH group (P<0.05). NR2B-specific IgG of NR2B group was positive on day 7 after SNI. There is a significant reduction for NR2B protein level in the lumbar spinal cord in group NR2B compared with that in group PBS or KLH (P< 0.01). Sera from rats immunized with NR2B-KLH react with NR2B protein in spinal horn.
2. Evaluation of NR2B peptide as subunit vaccines against neuropathic pain being existed previously
Methods After skin and muscle incision, 2 of the 3 terminal branches of the sciatic nerve (tibial, and common peroneal nerves) with 9-0 silk suture were tightly ligated. Behavioral testing (50% paw withdraw thresholds and the duration of the withdrawal after the heat stimulation ) took place on every day after surgery, till 7days after surgery. Female SD rats developed hyperalgesia were divided into three groups randomly: PBS、KLH and NR2B. Rats were immunized with PBS、KLH or NR2B, respectively, three times subcutaneously at a two-weekly intervals, in combination with Complete Freund’s adjuvant (CFA) or Incomplete Freund’s adjuvant (ICFA).By day 14 after the third immunization, 50% paw withdraw thresholds and the paw withdraw duration to radiant heat stimulation were assessed and then sera were harvested for detection of titers of NR2B antibody. The L4~6 segment of the spinal cord was harvested. The astrocytic activation was determined using immunohistochemistry with antibodies of GFAP (an astrocyte marker). The expressive level of NR2B protein in spinal cord horn were detected using western bloting.
Results The titers of NR2B antibody in NR2B group was 6.94±2.04μg/ml on day 14 after the third immunization and there was no detection of NR2B antibody in PBS or KLH groups. All rats have developed a marked hyperalgesia 7 days after SNI, however, three times immunization later, mechanical hypersensitivity and heat hyperalgesia in NR2B groups were relieved, compared with PBS or KLH groups. The expression of GFAP indicated that astrocytic in group NR2B became less intense responses in the ipsilateralⅠ~Ⅳlemanie of dorsal horn than control group(PBS or KLH). Western blotting analysis showed that NR2B peptide vaccine decreased the expressive level of NR2B protein in spinal cord horn after the third immunization.
3. Construction of recombinant adenovirus analgesic vaccine encoding NR2B gene
Methods NR2B gene was obtained by digesting the plasmids pcDNA3.1-NR2B with EcoRⅠand BamHⅠand was cloned into the adenovirus shuttle vector pDC515. Then the vector was transferred into 293 cell and the expression of NR2B gene in 293 cell was identified by RT-PCR and western blotting. The shuttle plasmid pDC515-NR2B and the genomic plasmid pBHGfrt(del)E1,3FLP were cotransfected into the package 293 cells using lipofectamine TM 2000. The contransfected 293 cells were observed persistently for the production of CPE and the viral plaques. The viral plaques were picked and were put into infecting the passage cell 293. The recombinant Ad5/ NR2B were identified by PCR、RT-PCR and western blotting. The amplification and purification of the recombinant Ad5/NR2B were made and the titer and purity of the virus were assayed
Results First, transgene NR2B was cloned into the small adenovirus shuttle vector pDC515. The presence and oritention of the insert were confirmed using restriction endonuclease analysis and then the plasmid was termed as pDC515-NR2B. Second, pDC515-NR2B are cotransfected with an Ad genomic plasmid pBHGfrt△E1,3FLP into E1 complementing cell line 293 cells. The recombination between the two contransfected plsmids was mediated by frt site specific recombinase, FLP. The formation of viral plaques begins with the infection of one cell by one virus followed by multiple cycles of complete infection when released viruses infect surrounding cells. By day 15 following contransfection, well-defined plaques can be observed under the microscope . In order to build up working stocks of virus from plaque isolates before extensive experimentation, the presence and oritention of transgene NR2B in the recombinant adenovirus vector were confirmed using NR2B-specific PCR. The viral stocks of rAd5/NR2B were generated by the transient transfection of 293 cells and purified using CsCl ultracentrifugation gradients. The final titer was 5×1011 (Plaque Forming Unit, PFU)/ml and the purity is 100% using identification of HPLC methods.
4. rAd5/NR2B vaccine induces protective immunity against neuropathic pain
Methods The rAd5/NR2B (1×108 PFU) was administered via an orogastric tube into the stomachs of a group of rats (n=16), with a control group (n=16) receiving a similar dose of a recombinant Ad5 virus expressing EGFP or PBS, and followed by a booster immunization with the same dose two weeks after the initial immunization. A week later, expression of NR2B gene in the intestine M cells was assessed by RT-PCR. NR2B-specific Ig G titers in sera were determined by ELISA at a two-weekly intervals. Two weeks after the boost immunization, SNI model was established through the ligation of tibial nerve and peroneal nerve. A week after the SNI procedure, the number of c-Fos positive neurons in the spinal dorasl horn were assessed using immunohistochemistry. 50% paw withdraw thresholds of mechanical stimulation were assessed using the up-down paradigm every other day after SNI and withdrawal duration of heat response were also assessed at the same timepoint. Serial spinal sections from rats were stained with sera from rats treated with PBS , rAd5/EGFP or rAd5/NR2B. CSF (20~50μl) was drawn from the cisterna magna using a 27-gauge needle on day 1 before SNI, on day 7 after SNI and on day 42 after SNI, respectively. Six weeks after SNI, the expression of NR2B gene in the lumbar spinal cord were assessed using western blotting.
Results Following the primary immunization and the booster immunization, NR2B-spicific IgG in sera were 5.5.±1.9μg/ml and 13.4±3.2μg/ml,respectively. In contrast, NR2B-spicific IgG were not detected in sera from the control groups (na?ve and rAd5/EGFP).By day 7 after the primary vaccination with rAd5/NR2B, the expression of NR2B protein were detected in the intestine of rats immunized with rAd5/NR2B. By day 7 after surgery, the 50% paw withdraw thresholds of group rAd5/NR2B were significantly higher than control groups (na?ve and rAd5/EGFP) (P<0.05). The paw withdrawal duration to radiant heat stimulation in grouprAd5/NR2B were less than the control groups (na?ve and rAd5/EGFP) (P<0.05). The significant difference of 50% paw withdraw thresholds and paw withdrawal duration to radiant heat stimulation among the three groups remains till 42 days after the surgery. NR2B-spicific IgG in CSF was only detected on day 7 and 42 post the initial immunization in rAd5/NR2B group. And only sera from rats immunized with rAd5/NR2B reacted with NR2B protein in the lumbar spinal cord dorsal horn. Western botting analysis showed that immunization with rAd5/NR2B decreased the expression of NR2B protein in the spinal dorsal horn.
5. rAd5/NR2B vaccine induces protective immunity against the affective component relating to neuropathic pain
Methods SD rats without preference one compartment to the others before conditioning were selected and were divided into three groups randomly: na?ve, rAd5/EGFP and rAd5/NR2B. The rAd5/NR2B (1×108 PFU) was administered via an orogastric tube into the stomach of a group of rats, with a control group receiving a similar dose of a recombinant Ad5 virus expressing EGFP or PBS. Two weeks later, re-immunization were performed by the same dose of vaccine. Titers of NR2B antibody in sera were determined using ELISA at week 2 following the booost immunization and at the same time, SNI model was established through the ligation of tibial nerve and peroneal nerve. From the day 7 after SNI on, conditioned place avoidance (CPA) test were carried out. CSF (20μl) was drawn from the lateral ventricle using a 27-gauge needle on day 1 before SNI and on the day of SNI-CPA completion ,respectively. Anti-NR2B antibody titer in CSF were detected using ELISA and CSF was screened against ACC membrane extract by immunoblot analysis. The NR2B protein expression in ACC were assessed using western blotting and immunohistochemistry.
Results Two weeks following the booster immunization, titers of NR2B antibody in rAd5/NR2B groups was up to 13.5±2.3μg/ml. CPA scores in rAd5/NR2B group was significantly less than that in na?ve or rAd5/EGFP group(P<0.05). ( rAd5/NR2B group: 138.1±29.2S, na?ve group: 198.1±49.5S,rAd5/EGFP group: 236.8±40.2S). By day 13 after SNI, NR2B antibody in CSF of rAd5/NR2B group could be detected and react with NR2B protein of ACC membrane extracts. The expressive level of NR2B protein in ACC of rAd5/NR2B-i mmunized rats was decreased significantly compared with rats in na?ve or rAd5/EGFP group.
6. Statistical Analysis
All of the analyses were performed by SPSS 12.0 software package. Measurement data were presented as means±SD. The differences between multiple individual means were analyzed by one-way ANOVA. Categorical data were presented as rate. The differences between multiple individual rates were analyzed by Chi-square test. P < 0.05 was considered statistically significant in all tests.
Conclusion
NR2B peptide vaccine could induce the humoral immunological response to produce NR2B-specific antibody in sera. NR2B-specific antibody could react with NR2B subunit in spinal dorsal horn to reduce the NR2B protein expression and then have a pre-emptive analgesic effect on neuropathic pain. As for neuropathic pain existed previously, NR2B peptide vaccine could also relieve hyperalgesia. NR2B gene was inserted into the adenovirus shuttle vector pDC515 and then pDC515-NR2B was constructed successfully. The vaccine rAd5/NR2B was constructed successfully using cotransfection with pDC515-NR2B and pBHGfrt(del)E1,3FLP, which induced the higher titer of NR2B-specific antibody than NR2B peptide vaccine. The oral immunization with rAd5/NR2B could not only produce the analgesic effect against pain-related sensory but also alleviate the pain-related affective response.
Summary
The present study firstly demonstrated that NR2B peptide vaccine not only induced the humoral immunological response but also NR2B-spicific antibody could react with NR2B protein in the spinal dorsal horn to fight against neuropathic pain. In order to improve the immunological effect, rAd5/NR2B vaccine could construct.The present study made a research about the analgesic effect of rAd5/NR2B vaccine on both sensory and affective component of neuropathic pain, providing a novel direction for the treatment of neuropathic pain.
引文
1. Schenk D, Barbour R, Dunn W, et al. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature ,1999,400:173-177
2. Matthew J. During, Charles W. Symes, Patricia A. Lawlor. An Oral Vaccine AgainstNMDAR1 with Efficacy in Experimental Stroke and Epilepsy. Science, 2000,287(25), 1453-1460
3. Helmuth L. New stroke treatment strategy explored. Science, 2000, 287(5457): 1379~1381
4.王公明,田玉科,戴体俊. NMDA受体2B亚基:一个潜在的镇痛治疗靶点.国际麻醉学与复苏杂志,2006,27(5):309-11.
5. P-H Tan, L-C Yang, H-C Shin et al. Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat. Gene Therapy , 2005, 12(1): 59~66
6. Boyce, S., Wyatt, A., Webb, J. K., et al. Selective NMDA NR2B antagonists induce antinociception without motor dysfunction: correlation with restricted localisation of NR2B subunit in dorsal horn. Neuropharmacology, 1999, 38(5): 611– 623
7. Herin GA, Aizenman E. Amino terminal domain regulation of NMDA receptor function. Eur J Pharmacol.2004,500(1-3): 101-11
8.徐宝钢.β-淀粉样多肽1 - 42 (Aβ42)多克隆抗体的制备及其鉴定.放射免疫学杂志2003年第16卷第6期: 322-325
9. Wong CP, Levy R. Recombinant adenovirus vaccine encoding a chimeric T-cell antigen Receptor induce protective immunity against a T-cell lymphoma. Cancer research, 2000, 60(10): 2689-2695
10. Decosterd I, Woolf CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 2000; 87: 149-158.
11. Dixon WJ. Efficient analysis of experimental observations. Ann Rev PharmacolToxicol 1980; 20: 441-462.
12. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994; 53: 55-63.
13.鲍建芳.沈建根编著,免疫学实验技术,浙江科学技术出版社, 2002年
14. Kim SH, Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat.Pain. 1992; 50(3): 355-363.
15. Hua XY, Moore A, Malkmus S, et al. Inhibition of spinal protein kinase Cαexpression by an antisense oligonucleotide attenuates morphine infusion-induced tolerance. Neuroscience. 2002; 113: 99-107
16.邵传森、朱圣禾主编,临床疾病与免疫,科学出版社2002年1月
17. Darnell RB, Posner JB. Paraneoplastic syndromes affecting the nervous system. Semin oncol 2006; 33: 270-298.
18. Huber JD, Witt KA, Hom S, et al. Inflammatory pain alters blood-brain barriers permeability and tight junctional protein expression. Am J Physiol Heart Circ Physiol, 2001, 280(3): H1241~1248.
19.万丽,罗爱林,田玉科.吗啡对非伤害性刺激后CCI大鼠脊髓C-fos表达的影响.临床麻醉学杂志,2004,20(5):286-289.
20. Yamazaki Y, Maeda T, Someya G, et al. Temporal and spatial distribution of Fos protein in the lumber spinal dorsal horn neurons in the rat with chronic constriction injury to the sciatic nerve. Brain Res. 2001; 914:106-114.
21. Hong-Duck Kim, Fan-Kun Kong, Yunpeng Cao, Terry L.Lewis, et al. Immunization of Alzheimer model mice with adenovirus vectors encoding amyloidβ-protein and GM-CSF reduces amyloid load in the brain. Neuroscience, 2004, 370: 218~223.
1. Janean E, Holden , Julie A, Pizzi. The challenge of pain. Advanced Drug Delivery Reviews, 2003, 55: 935–948.
2. Raymond D, Hubbard BS, Beth A, Winkelstein. Transient cervical nerve root compression in the rat induces bilateral forepaw allodynia and spina glial activation: mechanical factors in painful neck injuries. Spine, 2005,30(17): 1924-1932
3. Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensationlike those seen in man.Pain. 1988,33:87-107.
4. Kim SH, Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat.Pain. 1992,50:355-363.
5. Decosterd I, Woolf CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain.Pain. 2000,87:149-158.
6. Watkins LR, Milligan ED, Maier SF. Glial activation: a driving force for pathological pain.Trends Neurosci,2001,24:450-455.
7. Woolf CJ, Salter MW. Neuronal plasticity: increasing the gain in pain. Science,2000,288:1765-1769.
8. P-H Tan, L-C Yang, H-C Shin et al. Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat. Gene Therapy , 2005, 12(1): 59~66.
9. TOVAR KR, WESTBROOK GL. Mobile NMDA receptor at hippocampal synapses [J]. Neuron, 2002, 34(2) : 255~264.
10. BARRIA A, MALINOW R. Subunit-specific NMDA receptor trafficking to synapses [J]. Neuron, 2002, 35: 345~354.
11. TOVAR KR, WESTBROOK GL. The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro [J]. J Neurosci, 1999, 19(10): 4180~4188.
1. Adam DC, Jean DB, David BW. Modulating the immune response to genetic immunization. FASEB Journal, 1998, 12(15): 1611-1626.
2.姜勋平主编.基因免疫的原理和方法.北京:科学出版社,2004.
3. Ali M, Lemoine NR, Ring CJ. The use of DNA viruses as vectors for gene therapy. Gene Ther, 1994 , 1(6):367-84.
4. Tritel M, Stoddard AM, Flynn BJ et al. Prime-boost vaccination with HIV-1 Gag protein and cytosine phosphate guanosine oligodeoxynucleotide, followed by adenovirus, induces sustained and robust humoral and cellular immune responses. J Immunol. 2003 , 171(5): 2538-47.
5. Petrenko AB, Yamakura T, Baba H, et al. The role of N-methyl-D-aspartate (NMDA) receptors in pain: a review. Anesth Analg, 2003, 97(4): 1108～1116.
6. Kovacs G, Kocsis P, Tarnawa I, et al. NR2B containing NMDA receptor dependent windup of single spinal neurons. Neuropharmacology, 2004, 46(1): 23~30.
7. Parson CG.. NMDA receptors as targets for drug action in neuropathic pain. Eur J Pharmacol, 2001, 429(1-3): 71～78.
8.唐兵,黎军,何国祥等.血管紧张素Ⅱ2型受体基因重组腺病毒简便快速的构建方法.医学研究生学报,2005,18(5):402～403.
9. Graham FL, Smiley J, Russell WC, Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol, 1977, 36: 59-74.
10. Ng P, Parks RJ, Cummings DT, et al. A high-efficiency Cre/loxP-based system for construction of adenoviral vectors. Hum Gene Ther, 1999, 10(16): 266～72.
11. Xiang, Z. Q., Yang, Y., Wilson, J. M., and Ertl, H. C. A replication-defective human adenovirus recombinant serves as a highly efficacious vaccine carrier. Virology, 1996,19: 220– 227.
12. Saito, I., Oya, Y., Yamamoto, K., Yuasa, T., and Shimojo, H. (1985). Construction ofnondefective adenovirus type 5 bearing a 2.8-kilobase hepatitis B virus DNA near the right end of its genome. J. Virol. 54: 711– 719.
13. Zhao, J., et al. (2003). Improved protection of rhesus macaques against intrarectal simian immunodeficiency virus SIV (mac251) challenge by a replication-competent Ad5hr-SIVenv/rev and Ad5hr-SIVgag recombinant priming/gp120 boosting regimen. J. Virol. 77: 8354– 8365.
14.孙树汉主编.基因工程的原理与方法,第一版,人民军医出版社. 2001: 125～126.
15. Massie B, Mosser DD, Koutroumanis M et al. New adenovirus vectors for protein production and gene transfer. Cytotechnology, 1998B, 28: 53-64.
16. Haj-Ahmad, Y., and Graham, F. L. Development of a helper-independent human adenovirus vector and its use in the transfer of the herpes simplex virus thymidine kinase gene. J. Virol. 1986, 57: 267– 274.
17. Elaine G. Rodrigues , Fidel Zavala, Ruth S. Nussenzweig ,et al. Efficient induction of protective antimalria immunity by recombinant adenovirus. Vaccine,1998, 16(19):1812-1817.
1.姜勋平,主编.基因免疫的原理和方法:北京科学出版社,2004.
2. Ribas A, Butterfield LH, Glaspy JA, et al. Current developments in cancer vaccines and cellular immunotherapy. J Clin Oncol, 2003, 21(12):2415~2432.
3. During MJ, Symes CW, Lawlor PA, et al. An oral vaccine against NMDAR1 with efficacy in experimental stroke and epilepsy. Science, 2000, 287(5457): 1453~1460.
4. Matsui M, Moriya O, Akatsuka T. Enhanced induction of hepatitis C virus-specific cytotoxic T lymphocytes and protective efficacy in mice by DNA vaccination followed by adenovirus boosting in combination with the interleukin-12 expression plasmid. Vaccine, 21: 1629~1639.
5. John D. Shanley, Carol A. Wu. Intranasal immunization with a replication-deficient adenovirus vector expressing glycoprotein H of murine cytomegalovirus induces mucosal and systemic immunity. Vaccine, 2005, 23: 996~1003.
6. P-H Tan, L-C Yang, H-C Shin et al. Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat. Gene Therapy , 2005, 12(1): 59~66.
7. Huber JD, Witt KA, Hom S, et al. Inflammatory pain alters blood-brain barriers permeability and tight junctional protein expression. Am J Physiol Heart Circ Physiol, 2001, 280(3): H1241~1248.
8. Decosterd I, Woolf CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 2000; 87: 149-158.
9. Yamazaki Y, Maeda T, Someya G, et al. Temporal and spatial distribution of Fos protein in the lumber spinal dorsal horn neurons in the rat with chronic constriction injury to the sciatic nerve. Brain Res. 2001; 914:106-114.
10. Woolf CJ. Evidence for a central component of post-injury pain hypersensitivity. Nature, 1993, 306: 686-688.
11. Nagy GG, Watanabe M, Fukaya M, et al. Synaptic distribution of the NR1, NR2A and NR2B subunits of the N-methyl-d-aspartate receptor in the rat lumbar spinal cord revealed with an antigen-unmasking technique. Eur J Neurosci, 2004 , 20 (12): 3301-3312.
1. Price DD. Psychological and neural mechanisms of the affective dimension of pain. Science, 2000, 288: 1769~1772
2.张玉秋。痛情绪和相关记忆产生的神经机制。自然科学通报,2005,15(12):1409～1415
3. Feng Wei, Guo-Du Wang Geoffrey A, et al. Genetic enhancement of inflammatory pain by forebrain NR2B overexpression. Nature neuroscience, 2001, 4(1):164-169
4. Lei L G, et al. NMDA receptors in the anterior cingulated cortex mediate pain-related aversion. Exp Neurol, 2004, 189: 413~421
5. Johansen J , Fields H L. Glutamatergic activation of anterior cingulated cortex produces an aversive teaching sigal. Nat Neurosci, 2004, 7: 398~403.
6.刘闯。条件性位置偏爱实验。中国药物滥用防治杂志1996,4:37-39。
7. Joshua P. Johansen, Howard L. Fields, and Barton H. Manning. The affective component of pain in rodents: Direct evidence for a contribution of the anterior cingulated cortex. Proc Natl Acad Sci, 2001, 98(14): 8077~8082。
8.诸葛启钏。大鼠脑立体定位图谱,第三版,人民卫生出版社。
9.严相默,主编。临床疼痛学,修订版。延吉:延吉人民出版社,237,1996
10.刘国凯,罗爱伦。神经病理性痛的治疗进展。中华麻醉学杂志,2003,23(2):157～159
11. Harkins SW, Price DD. Brainth J. Effects of extravesion and neuroticism on experimental pain , clinical pain, and illness behavior. Pain, 1989, 36: 209~218。
12. Koyama T, Tanaka YZ, Mikami A. Nociceptive neurons in the macaque anterior cingulated activate during anticipation of pain. NeuroReport, 1998,9: 2663~2667
13. Ploghaus A, Tracey I , Gati J S. et al. Dissociating pain from its anticipation in the human brain. Science, 1999, 284: 1979~1981
14. Rainville P, Duncan G H, Price DD et al. Pain affect encoded in human anteriorcingulate but not somatosensory cortex. Science, 1997, 277: 968~971.
15. Pastoriza LN, et al. Medial frontal cortex lesions selectively attenuate the hot plate response: possible nocifensive apraxia in the rat. Pain, 1996, 64: 11~17.
16. Kung J C, Su NM, Fan RJ, et al. Contribution of the anterior cingulate cortex to laser pain conditioning in rats. Brain Res, 2003, 970: 58~72
17. Gao Y J, Ren W H, Zhang Y Q, et al. Contributions of the anterior cingulate cortex and amygdala ot pain fear conditioned place avoidance in rats. Pain, 2004, 110: 343~353。
18. BARRIA A, MALINOW R. Subunit-specific NMDA receptor trafficking to synapses [J]. Neuron, 2002, 35: 345~354.
19. TOVAR KR, WESTBROOK GL. The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro [J]. J Neurosci, 1999, 19(10): 4180~4188.
1 LoGrasso P, Mckelvy J. Advances in pain therapeutics. Curr Opin Chem Biol, 2003, 7(4): 452-456.
2 Loftis JM, Janowsky A. The N-methyl-D-aspartate receptor subunit NR2B: localization, functional properties, regulation, and clinical implications. Pharmacol Ther, 2003, 97(1):55-85.
3 Blaise ML, Sowdhamini R, Rao MR, et al. Evolutionary trace analysis of ionotropic glutamate receptor sequences and modeling the interactions of agonists with different NMDA receptor subunits. J Mol Model (Online). 2004, 10(5-6):305-316
4 Chung HJ, Huang YH, Lau LF, et al. Regulation of the NMDA receptor complex and trafficking by activity-dependent phosphorylation of the NR2B subunit PDZ ligand. J Neurosci. 2004,10; 24(45):10248-10259.
5 Liu Y, Zhang J. Recent development in NMDA receptors. Chin Med J (Engl). 2000 ,113(10):948-956.
6 Petrenko AB, Yamakura T, Baba H, et al. The role of N-methyl-D-aspartate (NMDA) receptors in pain: a review. Anesth Analg. 2003 ,97(4):1108-1116
7 Monyer, H., Nurmashev N, Laurie DJ, et al. Developmental and regional expression in the rat brain and functionl properties of four NMDA receptors. Neuron, 1994, 12(3): 529-540.
8 Boyce, S., Wyatt, A., Webb, J. K., et al. Selective NMDA NR2B antagonists induce antinociception without motor dysfunction: correlation with restricted localisation of NR2B subunit in dorsal horn. Neuropharmacology, 1999, 38(5): 611– 623
9 Nagy GG, Watanabe M, Fukaya M, et al. Synaptic distribution of the NR1, NR2A and NR2B subunits of the N-methyl-d-aspartate receptor in the rat lumbar spinal cord revealed with an antigen-unmasking technique. Eur J Neurosci, 2004 ,20(12):3301-3312.
10 Luque, J.M., Bleuel, Z., Malherbe, P., et al. Alternatively spliced isoforms of theN-methyl-D-aspartate receptor subunit 1 are differentially distributed within the rat spinal cord. Neuroscience , 1994, 63(90): 629–635.
11 Q-P MA and R. J. HARGREAVES. Localization of N-methyl-aspartate NR2Bsubunits on primary sensory neurons that give rise to small-caliber sciatic nerve fibers in rats. Neuroscience, 2000, 101(9),699-707
12 Ishii, T., Moriyoshi, K., Sugihara, H., et al. Molecular characterization of the family of the N-methyl-D-aspartate receptor subunits. J Biol Chem, 1993, 268(4): 2836–2843.
13 Goebel, D. J., & Poosch, M. S. NMDA receptor subunit gene expression in the rat brain: a quantitative analysis of endogenous mRNA levels of NR1Com, NR2A, NR2B, NR2C, NR2D and NR3A. Brain Res Mol Brain Res, 1999, 69(2): 164– 170
14 Laurie, D. J., Bartke, I., Schoepfer, R., et al. Regional, developmental and interspecies expression of the four NMDAR2 subunits, examined using monoclonal antibodies. Brain Res Mol Brain Res , 1997,51(1～2): 23– 32.
15 Min Zhuo. Glutamate receptors and persistent pain: targeting forebrain NR2B subunits. DDT, 2002, 7(4): 259~267.
16 Petralia R.S. Wang Y. X. and Wenthold R.J.. The NMDA receptor subunits NR2A and NR2B show histological and ultrastructural localization patterns similar to those of NR1. J. Neurosci. , 1994, 14(10): 6102-612.
17 Liu H, Wang H, Sheng M, et al. Evidence for presynaptic N-methyl-D-aspartate autoreceptors in the spinal cord dorsal horn. Proc naln. Acad. Sci. USA, 1991, 91(5): 9383-9387.
18 P-H Tan, L-C Yang, H-C Shin et al. Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat. Gene Therapy , 2005, 12(1): 59~66.
19 Annika B. Malmberg, Heather Gilbert. Powerful antinociceptive effects of the cone snail venom-derived subtype-selective NMDA receptor antagonists conantokins G and T. Pain , 2003,101(2) :109–116.
20 Woolf CJ. Evidence for a central component of post-injury pain hypersensitivity. Nature, 1983, 306(5944): 686~688.
21 Feng Wei, Guo-Du Wang Geoffrey A, et al. Genetic enhancement of inflammatory pain by forebrain NR2B overexpression. Nature neuroscience, 2001, 4(1):164-169.
22 Hestrin, S. Developmental regulation of NMDAreceptor-mediated synaptic currents at a central synapse. Nature, 1992,357(6380): 686-689.
23 Wei, F. and Zhuo, M.. Potentiation of synaptic responses in the anterior cingulate cortex following digital amputation in rat. J. Physiol, 2001,532(7) : 823–833
24 Toyoda H, Zhao MG, Zhuo M, et al. Roles of NMDA receptor NR2A and NR2B subtypes for long-term depression in the anterior cingulate cortex. Eur J Neurosci, 2005, 22(2): 485-494
25 Kana Taniguchi, Katsuhiro Shinjo, et al. Antinoceptive activity of CP-101,606 an NMDA receptor NR2B subunit antagonist. British Journal of Pharmacology, 1997, 122(9), 809-812.
26 Boris A., Chizh, P., Max Headley and Thomas M,et al. NMDA receptor antagonists as analgesics: focus on the NR2B subtype. TRENDS in Pharmacological Sciences, 2001, 22 (12): 636~642.
2. Matthew J. During, Charles W. Symes, Patricia A. Lawlor. An Oral Vaccine AgainstNMDAR1 with Efficacy in Experimental Stroke and Epilepsy. Science, 2000,287(25), 1453-1460
3. Helmuth L. New stroke treatment strategy explored. Science, 2000, 287(5457): 1379~1381
4.王公明,田玉科,戴体俊. NMDA受体2B亚基:一个潜在的镇痛治疗靶点.国际麻醉学与复苏杂志,2006,27(5):309-11.
5. P-H Tan, L-C Yang, H-C Shin et al. Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat. Gene Therapy , 2005, 12(1): 59~66
6. Boyce, S., Wyatt, A., Webb, J. K., et al. Selective NMDA NR2B antagonists induce antinociception without motor dysfunction: correlation with restricted localisation of NR2B subunit in dorsal horn. Neuropharmacology, 1999, 38(5): 611– 623
7. Herin GA, Aizenman E. Amino terminal domain regulation of NMDA receptor function. Eur J Pharmacol.2004,500(1-3): 101-11
8.徐宝钢.β-淀粉样多肽1 - 42 (Aβ42)多克隆抗体的制备及其鉴定.放射免疫学杂志2003年第16卷第6期: 322-325
9. Wong CP, Levy R. Recombinant adenovirus vaccine encoding a chimeric T-cell antigen Receptor induce protective immunity against a T-cell lymphoma. Cancer research, 2000, 60(10): 2689-2695
10. Decosterd I, Woolf CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 2000; 87: 149-158.
11. Dixon WJ. Efficient analysis of experimental observations. Ann Rev PharmacolToxicol 1980; 20: 441-462.
12. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994; 53: 55-63.
13.鲍建芳.沈建根编著,免疫学实验技术,浙江科学技术出版社, 2002年
14. Kim SH, Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat.Pain. 1992; 50(3): 355-363.
15. Hua XY, Moore A, Malkmus S, et al. Inhibition of spinal protein kinase Cαexpression by an antisense oligonucleotide attenuates morphine infusion-induced tolerance. Neuroscience. 2002; 113: 99-107
16.邵传森、朱圣禾主编,临床疾病与免疫,科学出版社2002年1月
17. Darnell RB, Posner JB. Paraneoplastic syndromes affecting the nervous system. Semin oncol 2006; 33: 270-298.
18. Huber JD, Witt KA, Hom S, et al. Inflammatory pain alters blood-brain barriers permeability and tight junctional protein expression. Am J Physiol Heart Circ Physiol, 2001, 280(3): H1241~1248.
19.万丽,罗爱林,田玉科.吗啡对非伤害性刺激后CCI大鼠脊髓C-fos表达的影响.临床麻醉学杂志,2004,20(5):286-289.
20. Yamazaki Y, Maeda T, Someya G, et al. Temporal and spatial distribution of Fos protein in the lumber spinal dorsal horn neurons in the rat with chronic constriction injury to the sciatic nerve. Brain Res. 2001; 914:106-114.
21. Hong-Duck Kim, Fan-Kun Kong, Yunpeng Cao, Terry L.Lewis, et al. Immunization of Alzheimer model mice with adenovirus vectors encoding amyloidβ-protein and GM-CSF reduces amyloid load in the brain. Neuroscience, 2004, 370: 218~223.
1. Janean E, Holden , Julie A, Pizzi. The challenge of pain. Advanced Drug Delivery Reviews, 2003, 55: 935–948.
2. Raymond D, Hubbard BS, Beth A, Winkelstein. Transient cervical nerve root compression in the rat induces bilateral forepaw allodynia and spina glial activation: mechanical factors in painful neck injuries. Spine, 2005,30(17): 1924-1932
3. Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensationlike those seen in man.Pain. 1988,33:87-107.
4. Kim SH, Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat.Pain. 1992,50:355-363.
5. Decosterd I, Woolf CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain.Pain. 2000,87:149-158.
6. Watkins LR, Milligan ED, Maier SF. Glial activation: a driving force for pathological pain.Trends Neurosci,2001,24:450-455.
7. Woolf CJ, Salter MW. Neuronal plasticity: increasing the gain in pain. Science,2000,288:1765-1769.
8. P-H Tan, L-C Yang, H-C Shin et al. Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat. Gene Therapy , 2005, 12(1): 59~66.
9. TOVAR KR, WESTBROOK GL. Mobile NMDA receptor at hippocampal synapses [J]. Neuron, 2002, 34(2) : 255~264.
10. BARRIA A, MALINOW R. Subunit-specific NMDA receptor trafficking to synapses [J]. Neuron, 2002, 35: 345~354.
11. TOVAR KR, WESTBROOK GL. The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro [J]. J Neurosci, 1999, 19(10): 4180~4188.
1. Adam DC, Jean DB, David BW. Modulating the immune response to genetic immunization. FASEB Journal, 1998, 12(15): 1611-1626.
2.姜勋平主编.基因免疫的原理和方法.北京:科学出版社,2004.
3. Ali M, Lemoine NR, Ring CJ. The use of DNA viruses as vectors for gene therapy. Gene Ther, 1994 , 1(6):367-84.
4. Tritel M, Stoddard AM, Flynn BJ et al. Prime-boost vaccination with HIV-1 Gag protein and cytosine phosphate guanosine oligodeoxynucleotide, followed by adenovirus, induces sustained and robust humoral and cellular immune responses. J Immunol. 2003 , 171(5): 2538-47.
5. Petrenko AB, Yamakura T, Baba H, et al. The role of N-methyl-D-aspartate (NMDA) receptors in pain: a review. Anesth Analg, 2003, 97(4): 1108～1116.
6. Kovacs G, Kocsis P, Tarnawa I, et al. NR2B containing NMDA receptor dependent windup of single spinal neurons. Neuropharmacology, 2004, 46(1): 23~30.
7. Parson CG.. NMDA receptors as targets for drug action in neuropathic pain. Eur J Pharmacol, 2001, 429(1-3): 71～78.
8.唐兵,黎军,何国祥等.血管紧张素Ⅱ2型受体基因重组腺病毒简便快速的构建方法.医学研究生学报,2005,18(5):402～403.
9. Graham FL, Smiley J, Russell WC, Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol, 1977, 36: 59-74.
10. Ng P, Parks RJ, Cummings DT, et al. A high-efficiency Cre/loxP-based system for construction of adenoviral vectors. Hum Gene Ther, 1999, 10(16): 266～72.
11. Xiang, Z. Q., Yang, Y., Wilson, J. M., and Ertl, H. C. A replication-defective human adenovirus recombinant serves as a highly efficacious vaccine carrier. Virology, 1996,19: 220– 227.
12. Saito, I., Oya, Y., Yamamoto, K., Yuasa, T., and Shimojo, H. (1985). Construction ofnondefective adenovirus type 5 bearing a 2.8-kilobase hepatitis B virus DNA near the right end of its genome. J. Virol. 54: 711– 719.
13. Zhao, J., et al. (2003). Improved protection of rhesus macaques against intrarectal simian immunodeficiency virus SIV (mac251) challenge by a replication-competent Ad5hr-SIVenv/rev and Ad5hr-SIVgag recombinant priming/gp120 boosting regimen. J. Virol. 77: 8354– 8365.
14.孙树汉主编.基因工程的原理与方法,第一版,人民军医出版社. 2001: 125～126.
15. Massie B, Mosser DD, Koutroumanis M et al. New adenovirus vectors for protein production and gene transfer. Cytotechnology, 1998B, 28: 53-64.
16. Haj-Ahmad, Y., and Graham, F. L. Development of a helper-independent human adenovirus vector and its use in the transfer of the herpes simplex virus thymidine kinase gene. J. Virol. 1986, 57: 267– 274.
17. Elaine G. Rodrigues , Fidel Zavala, Ruth S. Nussenzweig ,et al. Efficient induction of protective antimalria immunity by recombinant adenovirus. Vaccine,1998, 16(19):1812-1817.
1.姜勋平,主编.基因免疫的原理和方法:北京科学出版社,2004.
2. Ribas A, Butterfield LH, Glaspy JA, et al. Current developments in cancer vaccines and cellular immunotherapy. J Clin Oncol, 2003, 21(12):2415~2432.
3. During MJ, Symes CW, Lawlor PA, et al. An oral vaccine against NMDAR1 with efficacy in experimental stroke and epilepsy. Science, 2000, 287(5457): 1453~1460.
4. Matsui M, Moriya O, Akatsuka T. Enhanced induction of hepatitis C virus-specific cytotoxic T lymphocytes and protective efficacy in mice by DNA vaccination followed by adenovirus boosting in combination with the interleukin-12 expression plasmid. Vaccine, 21: 1629~1639.
5. John D. Shanley, Carol A. Wu. Intranasal immunization with a replication-deficient adenovirus vector expressing glycoprotein H of murine cytomegalovirus induces mucosal and systemic immunity. Vaccine, 2005, 23: 996~1003.
6. P-H Tan, L-C Yang, H-C Shin et al. Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat. Gene Therapy , 2005, 12(1): 59~66.
7. Huber JD, Witt KA, Hom S, et al. Inflammatory pain alters blood-brain barriers permeability and tight junctional protein expression. Am J Physiol Heart Circ Physiol, 2001, 280(3): H1241~1248.
8. Decosterd I, Woolf CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 2000; 87: 149-158.
9. Yamazaki Y, Maeda T, Someya G, et al. Temporal and spatial distribution of Fos protein in the lumber spinal dorsal horn neurons in the rat with chronic constriction injury to the sciatic nerve. Brain Res. 2001; 914:106-114.
10. Woolf CJ. Evidence for a central component of post-injury pain hypersensitivity. Nature, 1993, 306: 686-688.
11. Nagy GG, Watanabe M, Fukaya M, et al. Synaptic distribution of the NR1, NR2A and NR2B subunits of the N-methyl-d-aspartate receptor in the rat lumbar spinal cord revealed with an antigen-unmasking technique. Eur J Neurosci, 2004 , 20 (12): 3301-3312.
1. Price DD. Psychological and neural mechanisms of the affective dimension of pain. Science, 2000, 288: 1769~1772
2.张玉秋。痛情绪和相关记忆产生的神经机制。自然科学通报,2005,15(12):1409～1415
3. Feng Wei, Guo-Du Wang Geoffrey A, et al. Genetic enhancement of inflammatory pain by forebrain NR2B overexpression. Nature neuroscience, 2001, 4(1):164-169
4. Lei L G, et al. NMDA receptors in the anterior cingulated cortex mediate pain-related aversion. Exp Neurol, 2004, 189: 413~421
5. Johansen J , Fields H L. Glutamatergic activation of anterior cingulated cortex produces an aversive teaching sigal. Nat Neurosci, 2004, 7: 398~403.
6.刘闯。条件性位置偏爱实验。中国药物滥用防治杂志1996,4:37-39。
7. Joshua P. Johansen, Howard L. Fields, and Barton H. Manning. The affective component of pain in rodents: Direct evidence for a contribution of the anterior cingulated cortex. Proc Natl Acad Sci, 2001, 98(14): 8077~8082。
8.诸葛启钏。大鼠脑立体定位图谱,第三版,人民卫生出版社。
9.严相默,主编。临床疼痛学,修订版。延吉:延吉人民出版社,237,1996
10.刘国凯,罗爱伦。神经病理性痛的治疗进展。中华麻醉学杂志,2003,23(2):157～159
11. Harkins SW, Price DD. Brainth J. Effects of extravesion and neuroticism on experimental pain , clinical pain, and illness behavior. Pain, 1989, 36: 209~218。
12. Koyama T, Tanaka YZ, Mikami A. Nociceptive neurons in the macaque anterior cingulated activate during anticipation of pain. NeuroReport, 1998,9: 2663~2667
13. Ploghaus A, Tracey I , Gati J S. et al. Dissociating pain from its anticipation in the human brain. Science, 1999, 284: 1979~1981
14. Rainville P, Duncan G H, Price DD et al. Pain affect encoded in human anteriorcingulate but not somatosensory cortex. Science, 1997, 277: 968~971.
15. Pastoriza LN, et al. Medial frontal cortex lesions selectively attenuate the hot plate response: possible nocifensive apraxia in the rat. Pain, 1996, 64: 11~17.
16. Kung J C, Su NM, Fan RJ, et al. Contribution of the anterior cingulate cortex to laser pain conditioning in rats. Brain Res, 2003, 970: 58~72
17. Gao Y J, Ren W H, Zhang Y Q, et al. Contributions of the anterior cingulate cortex and amygdala ot pain fear conditioned place avoidance in rats. Pain, 2004, 110: 343~353。
18. BARRIA A, MALINOW R. Subunit-specific NMDA receptor trafficking to synapses [J]. Neuron, 2002, 35: 345~354.
19. TOVAR KR, WESTBROOK GL. The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro [J]. J Neurosci, 1999, 19(10): 4180~4188.
1 LoGrasso P, Mckelvy J. Advances in pain therapeutics. Curr Opin Chem Biol, 2003, 7(4): 452-456.
2 Loftis JM, Janowsky A. The N-methyl-D-aspartate receptor subunit NR2B: localization, functional properties, regulation, and clinical implications. Pharmacol Ther, 2003, 97(1):55-85.
3 Blaise ML, Sowdhamini R, Rao MR, et al. Evolutionary trace analysis of ionotropic glutamate receptor sequences and modeling the interactions of agonists with different NMDA receptor subunits. J Mol Model (Online). 2004, 10(5-6):305-316
4 Chung HJ, Huang YH, Lau LF, et al. Regulation of the NMDA receptor complex and trafficking by activity-dependent phosphorylation of the NR2B subunit PDZ ligand. J Neurosci. 2004,10; 24(45):10248-10259.
5 Liu Y, Zhang J. Recent development in NMDA receptors. Chin Med J (Engl). 2000 ,113(10):948-956.
6 Petrenko AB, Yamakura T, Baba H, et al. The role of N-methyl-D-aspartate (NMDA) receptors in pain: a review. Anesth Analg. 2003 ,97(4):1108-1116
7 Monyer, H., Nurmashev N, Laurie DJ, et al. Developmental and regional expression in the rat brain and functionl properties of four NMDA receptors. Neuron, 1994, 12(3): 529-540.
8 Boyce, S., Wyatt, A., Webb, J. K., et al. Selective NMDA NR2B antagonists induce antinociception without motor dysfunction: correlation with restricted localisation of NR2B subunit in dorsal horn. Neuropharmacology, 1999, 38(5): 611– 623
9 Nagy GG, Watanabe M, Fukaya M, et al. Synaptic distribution of the NR1, NR2A and NR2B subunits of the N-methyl-d-aspartate receptor in the rat lumbar spinal cord revealed with an antigen-unmasking technique. Eur J Neurosci, 2004 ,20(12):3301-3312.
10 Luque, J.M., Bleuel, Z., Malherbe, P., et al. Alternatively spliced isoforms of theN-methyl-D-aspartate receptor subunit 1 are differentially distributed within the rat spinal cord. Neuroscience , 1994, 63(90): 629–635.
11 Q-P MA and R. J. HARGREAVES. Localization of N-methyl-aspartate NR2Bsubunits on primary sensory neurons that give rise to small-caliber sciatic nerve fibers in rats. Neuroscience, 2000, 101(9),699-707
12 Ishii, T., Moriyoshi, K., Sugihara, H., et al. Molecular characterization of the family of the N-methyl-D-aspartate receptor subunits. J Biol Chem, 1993, 268(4): 2836–2843.
13 Goebel, D. J., & Poosch, M. S. NMDA receptor subunit gene expression in the rat brain: a quantitative analysis of endogenous mRNA levels of NR1Com, NR2A, NR2B, NR2C, NR2D and NR3A. Brain Res Mol Brain Res, 1999, 69(2): 164– 170
14 Laurie, D. J., Bartke, I., Schoepfer, R., et al. Regional, developmental and interspecies expression of the four NMDAR2 subunits, examined using monoclonal antibodies. Brain Res Mol Brain Res , 1997,51(1～2): 23– 32.
15 Min Zhuo. Glutamate receptors and persistent pain: targeting forebrain NR2B subunits. DDT, 2002, 7(4): 259~267.
16 Petralia R.S. Wang Y. X. and Wenthold R.J.. The NMDA receptor subunits NR2A and NR2B show histological and ultrastructural localization patterns similar to those of NR1. J. Neurosci. , 1994, 14(10): 6102-612.
17 Liu H, Wang H, Sheng M, et al. Evidence for presynaptic N-methyl-D-aspartate autoreceptors in the spinal cord dorsal horn. Proc naln. Acad. Sci. USA, 1991, 91(5): 9383-9387.
18 P-H Tan, L-C Yang, H-C Shin et al. Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat. Gene Therapy , 2005, 12(1): 59~66.
19 Annika B. Malmberg, Heather Gilbert. Powerful antinociceptive effects of the cone snail venom-derived subtype-selective NMDA receptor antagonists conantokins G and T. Pain , 2003,101(2) :109–116.
20 Woolf CJ. Evidence for a central component of post-injury pain hypersensitivity. Nature, 1983, 306(5944): 686~688.
21 Feng Wei, Guo-Du Wang Geoffrey A, et al. Genetic enhancement of inflammatory pain by forebrain NR2B overexpression. Nature neuroscience, 2001, 4(1):164-169.
22 Hestrin, S. Developmental regulation of NMDAreceptor-mediated synaptic currents at a central synapse. Nature, 1992,357(6380): 686-689.
23 Wei, F. and Zhuo, M.. Potentiation of synaptic responses in the anterior cingulate cortex following digital amputation in rat. J. Physiol, 2001,532(7) : 823–833
24 Toyoda H, Zhao MG, Zhuo M, et al. Roles of NMDA receptor NR2A and NR2B subtypes for long-term depression in the anterior cingulate cortex. Eur J Neurosci, 2005, 22(2): 485-494
25 Kana Taniguchi, Katsuhiro Shinjo, et al. Antinoceptive activity of CP-101,606 an NMDA receptor NR2B subunit antagonist. British Journal of Pharmacology, 1997, 122(9), 809-812.
26 Boris A., Chizh, P., Max Headley and Thomas M,et al. NMDA receptor antagonists as analgesics: focus on the NR2B subtype. TRENDS in Pharmacological Sciences, 2001, 22 (12): 636~642.
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