【作者】 王萍；

【导师】 田玉科；

【作者基本信息】 华中科技大学 ， 麻醉学， 2011， 博士

【摘要】 神经病理性疼痛(Neuropathic pain)是神经系统损伤引起的一种难以治疗的慢性状态,国际疼痛研究协会(IASP,1994)称之为“由于外周或中枢神经系统的直接损伤或功能紊乱引起的疼痛”。感染、创伤、代谢性疾病、肿瘤化疗、手术、射线、神经毒性药物、神经受压缺血、炎症和肿瘤的侵袭都可引起神经病理性(中枢性和周围性)疼痛。急性疼痛对机体具有警示和“保护”作用,而神经病理性疼痛与之不同,它可以持续存在,对机体无益,甚至会严重影响生活质量。神经病理性疼痛的产生机制十分复杂,从神经损伤(伤害性刺激)到疼痛产生,会在神经系统发生一系列的变化。脊髓是疼痛信息传递和整合的初级中枢,脊髓背角汇聚着来自外周的不同传入神经与来自脑干和大脑皮质的下行投射神经,加上背角局部中间神经元,组成十分复杂的神经网络,并含有非常丰富的生物活性物质,因此,脊髓在神经病理性疼痛的发生发展中起到重要的作用。传统观点认为神经元是中枢神经系统中重要的信号细胞,而胶质细胞仅仅是起绝缘、营养与支持作用,因此,以往在对疼痛的研究中大量的实验关注于神经元的功能。但近年来发现,胶质细胞在疼痛的产生与发展过程中起重要的作用。Colburn等首次提出小胶质细胞的活化出现在疼痛的起始阶段,而星型胶质细胞在疼痛的发展和持续阶段有很强的活化反应。外周神经损伤后初级传入神经纤维中枢端释放P物质(substance P, SP)、兴奋性氨基酸(Excitatory amino acids, EAAs)、三磷酸腺苷(Adenosine triphosphate,ATP)等,作用于脊髓胶质细胞的受体,胶质细胞激活后产生和释放大量疼痛相关介质,如细胞因子、炎性介质和神经活性物质等。过量的这些物质积聚又能敏化神经元和胶质细胞,最终造成疼痛信号的产生和扩大,使病理性疼痛进一步发展和持续。近年来,在中枢神经系统发现了一类兼具神经元与胶质细胞特性的细胞,这类细胞表面特异性表达硫酸软骨素蛋白多糖NG2,被称为NG2细胞。新近的研究认为它是继少突胶质细胞、星形胶质细胞、小胶质细胞后的又一类“新型”胶质细胞系,广泛分布于中枢神经系统的灰质与白质中。在对NG2细胞的研究中发现,在多种神经系统损伤与疾病过程中可出现NG2细胞的活化,主要表现为胞体增大,突起增多,NG2分子表达增多。在海马区NG2细胞与兴奋性神经元或抑制性神经元均可形成突触联系,NG2细胞膜表面的AMPA受体和GABAA受体参与了由神经元到NG2细胞的信息传导。在神经系统疾病过程中NG2细胞可产生和释放多种神经兴奋性物质。此外,研究发现脂多糖刺激小胶质细胞活化时可诱导小胶质细胞表达NG2分子及炎性介质,RNA干扰下调NG2分子的表达后,小胶质细胞活化表达的炎性介质下调。上述研究提示NG2细胞在神经活动(包括疼痛的发生发展)中可能占据某种重要的地位,但由于对该细胞了解较晚,相关研究还处在初级阶段。基于以上理论及研究基础,本研究拟通过以下三部分的研究,来观察慢性神经病理性疼痛大鼠脊髓NG2细胞的变化及其参与疼痛的相关机制：1：制作大鼠慢性神经病理性疼痛模型,观察大鼠疼痛行为学的改变(包括机械刺激50%缩爪阈值和辐射热刺激缩爪持续时间)及大鼠脊髓NG2细胞及NG2分子表达变化,初步探讨NG2细胞在慢性神经病理性疼痛中的的作用；2：体外培养大鼠NG2细胞,观察其膜表面AMPA受体激活后NG2分子与疼痛相关介质表达的变化；构建大鼠NG2基因的shRNA慢病毒载体,观察RNA干扰下调NG2分子表达后,NG2细胞AMPA受体活化后疼痛相关介质表达的改变；3：将大鼠NG2基因的shRNA慢病毒载体注射到慢性神经病理性疼痛模型大鼠蛛网膜下腔,观察脊髓NG2细胞疼痛相关介质表达的变化及其对疼痛的影响。1.慢性神经病理性疼痛大鼠脊髓NG2细胞及NG2分子表达的变化方法：选择成年雌性Sprague-Dawlye大鼠96只,体重220-250g,随机分为三组,对照组(Naive组,n=24),不做任何处理；假手术组(Sham组,n=24),于大鼠背部切开皮肤,分离肌肉直至L5横突,咬开横突,暴露L5脊神经后缝合肌肉与皮肤；神经病理性疼痛模型组(Spinal nerve ligation,SNL组,n=48),于大鼠背部切开皮肤,分离肌肉直至L5横突,咬开横突,暴露L5脊神经,用丝线结扎并剪断L5脊神经,缝合肌肉与皮肤。观察术前、术后3d、7d、14d、21d、28d的术侧后趾对机械刺激的50%缩爪阈值和辐射热刺激的缩爪持续时间；于相同时点取脊髓腰膨大组织(每个时点随机选择4只),制作冰冻切片后作免疫荧光染色以观察脊髓腰膨大背角组织中NG2细胞的改变；同时于相同时点提取SNL组大鼠术侧脊髓腰膨大组织(每个时点随机选择4只)的蛋白质,Western blot分析以检测NG2分子的表达变化。结果：术前各组大鼠术侧后肢机械刺激50%缩爪阈值与辐射热刺激缩爪持续时间差异无统计学意义(P>0.05)；与术前相比,Naive组与Sham组大鼠各时点术侧后肢机械刺激50%缩爪阈值与辐射热刺激缩爪持续时间差异无统计学意义(P>0.05),SNL组大鼠术后3天开始各时点术侧后肢机械刺激50%缩爪阈值明显降低,辐射热刺激缩爪持续时间明显增高,差异有统计学意义(P<0.05)；与Sham组相比,SNL组大鼠术后3天开始各时点术侧后肢机械刺激50%缩爪阈值明显降低,辐射热刺激缩爪持续时间明显增高,差异有统计学意义(P<0.05)。免疫荧光染色结果显示,SNL组大鼠术后第7d、14d、21d术侧脊髓腰膨大背角组织中NG2细胞明显活化,可见胞体增大,突起增多。NG2细胞数目稍增多,但与术前相比,差异无统计学意义(P>0.05)；Western blot检测结果显示,SNL组大鼠术后第7d、14d、21d术侧脊髓腰膨大组织中NG2的表达量明显增加,与术前相比,差异有统计学意义(P<0.05)。2.体外培养NG2细胞表面AMPA受体活化后疼痛相关介质表达的改变方法：使用percoll不连续梯度离心法从Sprague-Dawlye大鼠乳鼠海马中分离培养NG2细胞,免疫荧光染色鉴定细胞性质,流式细胞技术检测细胞纯度。构建大鼠NG2基因的shRNA慢病毒载体LV-NG2-RNAi及对照慢病毒载体LV-Con-RNAi,并检验慢病毒载体的干扰效能。体外培养NG2细胞接种于6孔板中,分为三组：对照组(n=12)：不添加任何试剂；AMAP组(n=12)：加入AMPA受体激动剂AMPA0.5mM;AMPA+CNQX组(n=12)：同时加入AMPA受体激动剂AMPA0.5mM与AMPA受体拮抗剂CNQX50μMo于加药后1h、6h、12h、24h(每时间点3孔)使用Real-time PCR检测细胞NG2分子、神经营养因子BDNF、NGF与细胞因子IL-1β、TNF-α表达的变化。体外培养NG2细胞接种于6孔板中,分为两组：对照组(n=3)：加入LV-Con-RNAi 4d后加入AMPA受体激动剂AMPA0.5mM;实验组(n=3)：加入LV-NG2-RNAi 4d后加入AMPA受体激动剂AMPA0.5mM。于加药后12h使用Real-time PCR检测细胞NG2分子、神经营养因子BDNF、NGF与细胞因子IL-1β、TNF-α表达的变化。结果：成功体外分离培养了大鼠NG2细胞,细胞纯度可达80%以上。构建的慢病毒载体LV-NG2-RNAi的滴度可达8×108TU/ml,干扰效率可达80%以上。Real-time PCR检测结果显示,NG2细胞表面AMPA受体活化后NG2、BDNF、NGF在给药后1h、6h、12h、24h表达增高,与对照组相比,差异有统计学意义(P<0.05)；IL-1β在给药后6h、12h、24h表达增高,与对照组相比,差异有统计学意义(P<0.05)；TNF-α在给药后1h、6h、12h表达增高,与对照组相比,差异有统计学意义(P<0.05)。RNA干扰下调NG2分子表达后,Real-time PCR检测结果显示,NG2细胞表面AMPA受体活化后NG2表达降低,与对照组相比,差异有统计学意义(P<0.05)；BDNF、NGF、IL-1β、TNF-α表达降低,与对照组相比,差异有统计学意义(P<0.05)3.鞘内注射NG2-shRNA慢病毒载体对大鼠神经病理性疼痛的影响方法：选择成年雌性Sprague-Dawlye大鼠48只,体重220-250g,随机分为四组：对照组(Naive组,n=12)；神经病理性疼痛模型组(SNL组,n=12)；鞘内注射LV-Con-RNAi后7d制备神经病理性疼痛模型组(SNL+Con组,n=12)；鞘内注射LV-NG2-RNAi后7d制备神经病理性疼痛模型组(SNL+NG2组,n=12)。术后7d使用western blot检测大鼠脊髓腰膨大组织NG2蛋白的表达；同时提取大鼠脊髓腰膨大组织中NG2细胞,使用流式细胞技术检测NG2细胞神经营养因子BDNF.NGF与细胞因子IL-1β、TNF-α表达的变化；于术前、术后3d、7d、14d、21d、28d观察术侧后趾对机械刺激的50%缩爪阈值和辐射热刺激的缩爪持续时间。结果：western blot检测结果显示,与Naive组相比,SNL组和SNL+Con组大鼠脊髓腰膨大组织中的NG2蛋白表达量增高(P<0.05),SNL+NG2组大鼠脊髓腰膨大组织中的NG2蛋白表达量无明显改变(P>0.05)。流式细胞技术检测结果显示,与Naive组比较,SNL组和SNL+Con组大鼠脊髓腰膨大组织NG2细胞BDNF.NGF表达增多,但SNL+NG2组大鼠脊髓腰膨大NG2细胞BDNF、NGF表达无明显改变；与Naive组比较,SNL组、SNL+Con组和SNL+NG2组大鼠脊髓腰膨大组织NG2细胞IL-1β、TNF-α表达均增多。行为学观察结果显示,术前各组大鼠术侧后肢机械刺激50%缩爪阈值与辐射热刺激缩爪持续时间差异无统计学意义(P>0.05)；SNL组与SNL+Con组大鼠从术后3天开始各时点术侧后肢机械刺激50%缩爪阈值降低,与Naive组比较差异有统计学意义(P<0.05)；SNL+NG2组大鼠从术后14天开始各时点术侧后肢机械刺激50%缩爪阈值降低,与Naive组比较差异有统计学意义(P<0.05)。SNL组与SNL+Con组大鼠从术后3天开始各时点术侧后肢辐射热刺激缩爪持续时间增高,与Naive组比较差异有统计学意义(P<0.05)；SNL+NG2组大鼠从术后7天开始各时点术侧后肢辐射热刺激缩爪持续时间增高,与Naive组比较差异有统计学意义(P<0.05)4.统计学分析采用SPSS 13.0统计软件包分析数据,计量资料以均数±标准差(x±s)表示,重复测量数据采用重复测量设计的方差分析,其他数据采用单因素方差分析。P<0.05为差异有统计学意义。研究总结一、主要研究结果1.本研究通过疼痛行为学观察确定成功构建了大鼠神经病理性疼痛模型。免疫荧光染色显示,大鼠神经病理性疼痛模型术侧脊髓腰膨大背角组织中NG2细胞活化,表现为胞体增大,突起增多；Western Blot检测显示,大鼠神经病理性疼痛模型术侧脊髓腰膨大组织NG2分子表达增加。2.体外成功培养了大鼠NG2细胞；大鼠NG2细胞表面AMPA受体活化后NG2分子、神经营养因子BDNF、NGF与细胞因子IL-1β、TNF-α表达增高；成功构建了大鼠NG2基因的shRNA慢病毒载体；RNA干扰下调NG2分子表达后,NG2细胞表面AMPA受体活化后BDNF、NGF、IL-1β、TNF-α表达均降低。3.鞘内注射NG2-shRNA慢病毒载体可以有效下调神经病理性疼痛大鼠脊髓腰膨大组织中的NG2分子；RNA干扰下调NG2分子表达后神经病理性疼痛大鼠脊髓腰膨大NG2细胞的BDNF和NGF表达降低；延迟了神经病理性疼痛大鼠术侧后肢机械刺激50%缩爪阈值的下降和辐射热刺激缩爪持续时间的增高。二、研究结论1.神经病理性疼痛大鼠术侧脊髓腰膨大背角组织中NG2细胞活化,参与了疼痛的过程。2.体外培养大鼠NG2细胞表面AMPA受体活化后NG2分子及疼痛相关介质表达上调；NG2分子参与了大鼠NG2细胞表面AMPA受体活化后的信号传导及基因表达的调控过程,影响疼痛相关介质的表达。3.下调神经病理性疼痛大鼠脊髓腰膨大NG2细胞的NG2分子表达可以下调NG2细胞BDNF和NGF的表达,延迟疼痛的发生。三、创新之处1.近年来研究发现NG2细胞参与多种神经系统损伤与疾病过程。本实验首次观察了大鼠L5脊神经结扎模型中脊髓腰膨大NG2细胞与NG2分子的改变,初步证实NG2细胞参与了疼痛的过程。2.文献报道NG2细胞表面AMPA受体活化后,细胞内钙离子浓度的增加,但并没有相关实验观察其后的生物效能(包括基因表达变化)。本实验观察了AMPA受体活化后神经营养因子和细胞因子表达的改变,并且观察了NG2分子表达与神经营养因子和细胞因子表达之间的关系。四、展望1.本研究发现大鼠神经病理性疼痛的过程中脊髓NG2细胞活化,NG2分子参与了NG2细胞表面AMPA受体活化后疼痛相关介质表达的过程,但是NG2分子与疼痛相关介质之间的信号通路及其调节机制仍需要进一步深入研究。2.近年来研究发现NG2细胞与神经元之间存在着广泛的突触联系。伤害性信息经突触联系将信息传递到NG2细胞后,激活NG2细胞,使其释放疼痛相关介质,这些介质可能进一步促进脊髓背角神经元的敏化。因此,脊髓NG2细胞与神经元之间的相互作用及其机制也需要展开研究。更多还原

【Abstract】 BackgroundNeuropathic pain refers to chronic pain that originates from pathology of the nervous system. The International Association for the Study of Pain (IASP,1994) defines neuropathic pain as "pain initiated or caused by a primary lesion or dysfunction of the nervous system". Infection (herpes zoster), nerve trauma, metabolic diseases, cancer chemotherapy, surgery, radiation, neurotoxicity drugs, nerve compression, inflammation and tumor invasion are examples of diseases that may cause neuropathic pain. Acute pain plays a warning and "protective" role. By contrast, neuropathic pain can persist and serves no known defensive, or other helpful, function. And it might even affect the quality of life seriously. The mechanism of neuropathic pain is very complex. There are a series of complex changes in the nervous system from nerve damage (nociceptive stimulus) to pain production. The spinal cord is the primary center of the pain information delivery and integration. The spinal dorsal horn includes different peripheral afferent nerves, descending projection fibers from the brain stem and cerebral cortex and local interneurons. These compose a very complex neural network, and it is very rich in bio-active substances. Therefore, the spinal cord plays an important role in the development of neuropathic pain. Traditional view is that neurons are important signal cells in the central nervous system, and the role of glial cells is only insulation, nutrition and support. Therefore, a large number of experiments in pain research focused on the function of neurons in the past. But in recent years, we find that glial cells play an important role in the process of pain. Colburn proposed that microglia are more important for the initiation, while astrocytes are more important for the maintenance of neuropathic pain. Injury to a peripheral nerve initiates increased release of neurotransmitters such as substance P, glutamate and possibly ATP from the central terminals of primary afferents. These neurotransmitters can activate glial cells in the spinal cord through binding to their receptors. These triggers cause production and release of a large number of pain-related mediators from glial cells, such as cytokines, inflammatory mediators and neurotrophins. These agents are then capable of further enhancing neurons and glial cells activation, eventually leading to the generation and expansion of pain signals, thereby contributing to neuropathic pain.Recent studies suggest that there is a class of cells with both neuronal and glial properties in central nervous system. These cells show specific expression of NG2 surface chondroition sulphate proteoglycan on their surface, and are known as NG2 cells. The latest results indicate that NG2 cells are a population of CNS glial cells that are distinct from mature oligodendrocytes, astrocytes and microglia. These cells widely distribute in the gray matter and white matter. The most common phenomena indicating NG2 cells response to stimulation are up-regulation of chondroitin sulfate proteoglycan expression, increase in cell size (hypertrophy) and increase in the number of cellular processes in a variety of nervous system injury and disease. The discovery of expressions of glutamate and y-aminobutyric acid (GABA) receptors in NG2 cells and the formation of direct synaptic junctions between NG2 cells and glutamatergic and GABAergic neurons in the hippocampus implied they are integrated in the neural network. NG2 cells can produce and release a variety of neural excitatory substances in the course of nervous system diseases. In addition, lipopolysaccharide (LPS) induced the expression of NG2 and cytokines in microglial cells. However in this study, LPS did not induce the mRNA expression of cytokines in microglial cells transfected with NG2 siRNA. These studies suggest that NG2 cells might be involved in neuropathology, including the development of pain. However, we are still far from a complete understanding of the functional roles they play in the CNS.Based on the above theory and research base, our objective is to study the effect of NG2 cells in spinal cord in a rat model of neuropathic pain through the following three-part: 1. After the rat models of spinal nerve ligation (SNL) for neuropathic pain were made, the changes of nociceptive behaviors, including mechanical withdrawal threshold (MWT) and thermal withdrawal duration (TWD), were observed. And then, the changes of NG2 cells and the expression of NG2 molecule in spinal cord were detected.2. The rat NG2 cells were cultured in vitro. The expression of NG2 and pain-related molecules were detected after activation of the membrane surface AMPA receptor. Lentiviral vector of shRNA based on rat NG2 gene sequence was constructed and its interference effect was identified. The changes of the expression of pain-related molecules were detected after activation of the membrane surface AMPA receptor when NG2 molecule was down-regulated.3.The lentiviral vectors for RNA interference of the rat NG2 gene were injected intrathecally, the change of the expression of pain-related molecules in NG2 cells were detected and the nociceptive behaviors were observed. Methods and Results1:The change of NG2 cells in spinal cord of nuropathic pain in ratsMethods:Ninety-six female Sprague-Dawlye rats were selected and randomly divided into three groups:Naive group (not received any surgery, n=24); Sham group (the rat L5 spinal never was exposed, n=24) and SNL group (the rat L5 spinal never was exposed and ligated, n=48). Mechanical witndrawal threshold and thermal withdrawal duration were used to measure the response of operative hind paw to mechanical and radiant heat stimulation before operation and 3d,7d,14d,21d,28d after operation. At the same time, the rats were perfused with PBS followed by 2% paraformaldehyde (PFA) in PBS (4 rats every time point). The lumbar 4-6 spinal cord frozen slices were made and the changes of NG2 cells were observed through immunohistochemistry. Meanwhile the western blot analysis method was also used to detect the expression of NG2 molecule in spinal cord of the rats.Results:The mechanical witndrawal threshold and thermal withdrawal duration had not any difference among all groups before operation (P>0.05). The mechanical witndrawal threshold and thermal withdrawal duration in sham group and naive group had not significantly difference between before and after operation (P>0.05). The mechanical witndrawal threshold was decreased and thermal withdrawal duration was increased in SNL group at 3d,7d,14d,21d,28d after operation compare to before operation (P<0.05). The mechanical witndrawal threshold was decreased and thermal withdrawal duration was increased in SNL group at 3d,7d,14d,21d,28d after operation compare to sham group (P<0.05). The immunohistochemistry result displayed that the cell size of NG2 cells and the number of cellular processes were increased at 7d,14d and 21d after operation. The number of NG2 cells in the lumbar 4-6 spinal dorsal horn was no difference between before and after operation. Meanwhile the western blot result revealed that the expression of NG2 molecule in lumbar 4-6 spinal cord was significantly increased at 7d,14d and 21d after operation in SNL group (P<0.05).2:The change of expression of pain-related molecules in NG2 cells after activation of AMPA receptor in vitroMethods:NG2 cells were isolated from neonatal rat hippocampus by Percoll gradient methods and cultured. NG2 cells were identified by immunocytochemistry and the purity of cells was detected by fluorescence-activated cell sorting. Lentiviral vector of shRNA based on rat NG2 gene sequence was constructed and its interference effect was identified. NG2 cells were cultured in 6-well plates and divided into three groups:control group (treated without any agents, n=12), AMAP group (treated with AMPA receptor agonist AMPA 0.5mM, n=12), AMPA+CNQX group (treated with AMPA receptor agonists AMPA 0.5mM and AMPA receptor antagonist CNQX 50μM, n=12). The expression of NG2 molecule, neurotrophic factor BDNF, NGF and cytokines IL-1(3, TNF-a were detected by Real-time PCR method at 1h,6h,12h,24h after treated (3 wells per time point). NG2 cells were cultured in 6-well plates and divided into two groups:control group (treated with AMPA receptor agonist AMPA 0.5mM at 4d after with LV-Con-RNAi treatment, n=3), experimental group (treated with AMPA receptor agonist AMPA 0.5mM at 4d after with LV-NG2-RNAi treatment, n=3). The change of the expression of NG2 molecule, neurotrophic factor BDNF, NGF and cytokines IL-1(3, TNF-a were detected by Real-time PCR method at 12h after treated. Results:Rat NG2 cells were cultured sucssesfully in vitro.Lentiviral vector LV-NG2-RNAi was constructed and the titer was 8×108TU/ml.The interference efficiency was up to 80% when C6 cells were infected with MOI=20.Real-time PCR results showed that the expression of NG2,BDNF and NGF were increased at 1h,6h,12h,24h after activation of AMPA receptor, there was significant defference between control and AMPA group (P<0.05); the expression of IL-1βwere increased at 6h,12h,24h after activation of AMPA receptor, there was significant defference between control and AMPA group (P< 0.05);the expression of TNF-a was increased at 1h,6h,12h after activation of AMPA receptor, there was significant defference between control and AMPA group (P<0.05). Real-time PCR results showed that the expression of NG2 molecule was decreased in experimental group, there was significant defference between control and experimental group (P<0.05);the expression of BDNF, NGF, IL-1βand TNF-αwere decreased in experimental group, there was significant defference between control and experimental group (P<0.05).3:The effect of intrathecal injection of NG2-shRNA lentivirus of nuropathic pain in ratsMethods:Forty-eight female Sprague-Dawlye rats were selected and randomly divided into four groups:Naive group (not received any surgery, n=12);SNL group (noly received L5 spinal nerve ligation surgery, n=12);SNL+Con group (received SNL surgery at 7d after intrathecal injection of LV-Con-shRNA lentivirus, n=12);SNL+NG2 group (received SNL surgery at 7d after intrathecal injection of LV-NG2-shRNA lentivirus, n=12).The western blot analysis method was used to detect the expression of NG2 molecule in spinal cord of the rats. Meanwhile the expression of neurotrophic factor BDNF, NGF and cytokines IL-1β, TNF-a in NG2 cells were detected by fluorescence-activated cell sorting method. Mechanical witndrawal threshold and thermal withdrawal duration were used to measure the response of operative hind paw to mechanical and radiant heat stimulation before operation and 3d,7d, 14d,21d,28d after operation.Results:The expression of NG2 molecule in spinal cord of the rats in SNL group and SNL+Con group was increased (P<0.05), but the expression of NG2 molecule in SNL+NG2 group had not defference compare to Naive group (P>0.05). The expression of BDNF and NGF in NG2 cells was increased in SNL group and SNL+Con group, but there was no defference between SNL+NG2 group and Naive group. The expression of IL-1βand TNF-αin NG2 cells was increased in SNL group, SNL+Con group and SNL+NG2 group. The mechanical witndrawal threshold was decreased and thermal withdrawal duration was increased in SNL group and SNL+Con group 3d after operation (P<0.05).The mechanical witndrawal threshold in SNL+NG2 group was significantly decreased 14d after operation (P<0.05). The thermal withdrawal duration in SNL+NG2 group was significantly increased 7d after operation (P<0.05).4:Statistical analysisAll of the analyses were performed by SPSS 13.0 software package. Data are expressed as mean±standard error of the mean (SEM). The data were analyzed by repeated measures analysis of variance or one-way analysis of variance. A P value of<0.05 was accepted as significant. Major Results:1.The rat models of spinal nerve ligation(SNL) for neuropathic pain were made successfully through observing the change of nociceptive behaviors. The immunohistochemistry result displayed that NG2 cells in lumbar 4-6 spinal cord were activated in rat SNL model, the cell size of NG2 cells and the number of cellular processes were increased. Meanwhile the western blot result revealed that the expression of NG2 molecule in spinal cord was significantly increased.2. The rat NG2 cells were cultured successfully in vitro. The expression of NG2 and pain-related molecules were increased after activation of membrane surface AMPA receptor. Lentiviral vector of shRNA based on rat NG2 gene sequence was constructed successfully. The expression of pain-related molecules was decreased after activation of membrane surface AMPA receptor when NG2 molecule was down-regulated.3. The expression of NG2 molecule in spinal cord of the rats suffering with neuropathic pain was decreased after intrathecal injection of the lentiviral vector for RNA interference of the rat NG2 gene. The expression of BDNF and NGF in NG2 cells in lumbar 4-6 spinal cord was decreased. The onset of both thermal hyperalgesia and mechanical allodynia was delayed.Major Conclusion:1.NG2 cells in spinal cord are activated in rat SNL model and involved in the processes of pain. 2. NG2 and pain-related molecules are upregulated after activation of the membrane surface AMPA receptor. NG2 molecules are involved in the expression of pain-related molecules in NG2 cells after activation of the membrane surface AMPA receptor.3.The expression of BDNF and NGF in NG2 cells in spinal cord of the rats suffering with neuropathic pain was decreased after intrathecal injection of the lentiviral vector for RNA interference of the rat NG2 gene. And the onset of pain was delayed.Innovation:1.Recent studies have found that NG2 cells are involved in a variety of nervous system injury and disease. In this study, we observed the changes of NG2 cells and the expression of NG2 molecule in spinal cord of the rats suffering with L5 spinal nerve ligation firstly, and confirmed that NG2 cells are involved in the processes of pain.2.Evidence show that NG2 cells have AMPA-type glutamate receptors which are activated by neural activity and result in raised intracellular calcium. But now there are not experiments about the subsequent biological effects, including gene expression in cells. In this study, we observed the expression of NG2 and pain-related molecules after activation of membrane surface AMPA receptor and revealed the relationship between NG2 molecule and these pain-related molecules.Prospect:1.In this study, we found that NG2 cells in spinal cord were activated in rats suffering with neuropathic pain; the expression of NG2 and pain-related molecules were upregulated after activation of membrane surface AMPA receptor; the expression of pain-related molecules were decreased after activation of membrane surface AMPA receptor when NG2 molecules were down- regulated. But the signal pathway between NG2 molecule and pain-related molecules still need further study.2.Recent studies indicated NG2 cells formed direct synaptic junctions with neurons. Nociceptive signals activate NG2 cells through synaptic connections, and activated NG2 cells release pain-related molecules. These molecules might promote the spinal dorsal horn neurons sensitized furtherly. Therefore, the relationship between NG2 cells and neurons in spinal cord need to study.更多还原