Slit2/Robo1参与骨癌痛形成的机制研究
摘要
研究背景
骨癌痛（Bone cancer pain,BCP）是原发性或者转移性骨肿瘤引起的慢性疼痛。据世界卫生组织(WHO)统计,全世界每年新增1000万癌症患者，约三分之一的恶性肿瘤会发生骨转移。绝大部分骨肿瘤患者会出现不同程度的慢性疼痛。由于目前对骨癌痛机制的认识不足以及临床现有治疗措施的局限性，约50%的骨癌患者疼痛未得到有效控制。长期慢性疼痛给患者及其家人带来巨大痛苦，严重影响患者生活质量，甚至使患者丧失生活自理能力。所以，对骨癌痛机制的深入研究，以期为癌痛治疗提供有效的治疗措施，是目前亟待解决的重大问题。
基于对多种骨癌痛动物模型的研究，目前对骨癌痛的独特机制有了初步了解，认为骨癌痛与其它慢性疼痛不同，涉及多种炎症介质、细胞因子、多种受体和离子通道，既有炎性痛，又有神经病理性疼痛的成分。要破解骨癌痛发生这个错综复杂的分子网络，沿用传统的研究方法，从单一基因、或单个因素入手是无法解释和阐明这些现象的本质及其相互之间的因果关系并取得突破性进展的。
利用基因组学为平台，应用基因芯片分析技术，可以从细胞、组织、器官和生物体整体水平上研究结构和功能各异的各种分子及其相互关系，能够定量描述和预测生物功能、表型和行为，能客观真实地反映机体的整体变化。因此，本实验拟应用寡核苷酸微阵列芯片及生物信息学技术，研究骨癌痛模型与神经病理性痛模型、炎性痛模型大鼠脊髓水平基因表达谱差异，筛选出―脊髓组织骨癌痛特异性基因‖，进而利用计算生物学方法，采用统计学中聚类分析，探讨骨癌痛发生发展过程中脊髓组织分子水平的改变与疼痛行为学的关系，发掘在骨癌痛发病机制中起关键作用的特异基因表型。
中枢敏化是指脊髓和脊髓以上部位对疼痛信号传导的放大过程,是目前解释神经病理性疼痛，炎性痛等多种慢性疼痛出现痛觉过敏和痛觉超敏的主要理论。临床资料和基础实验研究提示，脊髓水平的中枢敏化是骨癌痛产生和维持的重要机制。在脊髓水平,背角是疼痛的调制主要部位，也伤害性信息的初级整合中枢。外周伤害性信息经初级传入纤维传入背角后，在此中继、整合,再由投射神经元向上位脑结构传递，最终产生痛觉。因此，目前对疼痛形成中脊髓敏化的研究也多集中在背角部位。
突触传导的可塑性改变是疼痛产生过程中脊髓敏化的重要机制之一。突触可塑性是指突触效能在某些因素的作用下出现不同程度的增强或减弱的特性。突触的可塑性改变包括两方面，一个是对现有突触结构的修饰，改变突触递质的释放而增强或减弱突触的传导性能，另一个是长时程突触可塑性调节机制，是通过增加或减少突触的数量来调节突触的传递效能。有研究报道，突触重塑导致脊髓敏化是骨癌痛发生的重要因素，但突触重塑的方式及其具体机制都不清楚。由于骨癌痛是一个长期慢性进展的过程，突触重塑过程也将是一个长期的持续变化，因此我们假设骨癌痛脊髓敏化是通过增加兴奋性突触数量来实现突触传递增强的。但是，新的突触的形成需要神经元轴突或树突延伸以及有导航作用的分子引导新生突起精确对接才能完成，而这个过程的分子机制也不清楚。
Slit homolog2protein(Slit2)是一种神经轴突的导向分子，在神经系统发育过程有重要作用。能够刺激神经元轴突生长和延伸以及侧支的形成[28-30]，调控神经元迁移，并且通过对生长锥的导向而指导突触的形成和功能性神经网络的建立。Roundabouthomolog1(Robo1)属于细胞粘附分子免疫球蛋白超家族成员，是slit2的受体，通过与slit2结合对神经元和生长锥产生排斥作用，参与调控发育中的中枢神经轴突定向延伸，促进轴突生长和调控神经纤维束化、定位以及突触的形成。研究显示Slit2和Robo1可能在病理状态下参与神经系统结构性重塑过程。因此我们假设，在骨癌痛形成过程中，Slit2和Robo1结合后，发挥了分子导向作用，促进轴突分支靶向性迁移而引导突触形成。为了证实Slit2和Robo1结合导致脊髓中新突触的形成而诱发骨癌痛脊髓敏化这一假说，本实验拟建立大鼠骨癌痛模型，利用RNA干扰技术等分子生物学技术，论证Slit2和Robo1在骨癌痛形成中的作用以及对神经元轴突延伸和突触形成的影响。以期从一个全新的角度揭示骨癌痛发生的机制，为骨癌痛是治疗寻找新的靶点
研究方法与结果
1.骨癌痛大鼠脊髓特异性基因表达谱的筛选
方法选择雌性wistar大鼠，随机分为4组：正常组，骨癌痛组，神经病理性疼痛组和炎性痛组。分别制作乳腺癌细胞胫骨种植的骨癌痛模型，L5脊神经结扎的神经病理性疼痛模型和弗氏完全佐剂（CFA）足底注射的炎性痛模型。模型建立后，进行痛阈测定。在疼痛发展的不同时期分别取各组大鼠脊髓腰L4-6进行基因芯片检测。采用统计分析筛选表达差异的基因谱，结合聚类分析和分子注释功能数据库分析特异基因之间的相互作用，模拟疼痛的分子路线图。
结果基因芯片检测结果提示在炎性痛早期，有2245个基因表达上调，其中包括479个基因仅在早期表达上调，515个基因持续上调至疼痛进展期，有1093个基因则在整个炎性痛发生发展过程中是持续上调。在疼痛的进展期共有2089个基因表达上调，其中263个基因仅在这个时间段上调，另外有118个基因从疼痛进展期出现上调，并持续至后期。在炎性痛的后期有1679个基因表达上调，其中有310个基因仅在后期出现表达上调。
神经病理性疼痛大鼠脊髓组织基因芯片检测结果提示在疼痛早期，有1570个基因表达上调，其中包括227个基因仅在早期表达上调，301个基因上调持续至疼痛进展期，有884个基因则在整个神经病理性疼痛发生发展过程中持续上调。在疼痛的进展期共有1560个基因表达上调，其中265个基因仅在进展期上调，另外有110个基因从疼痛进展期开始出现持续表达上调。在神经病理性疼痛的维持期有1476个基因表达上调，其中有342个基因在疼痛维持期才出现表达上调。
骨癌痛基因芯片的结果提示在骨癌痛早期，有1383个基因表达上调，其中包括381个基因仅在早期表达上调，155个基因上调持续至疼痛进展期，有691个基因则是在骨癌痛发生发展过程中是持续上调。在疼痛的进展期共有1740个基因表达上调，其中414个基因仅在这个时间段上调达，另外有480个基因从疼痛进展期开始出现持续表达上调。在骨癌痛的维持期则有1908个基因表达上调，其中有581个基因在疼痛的维持期出现表达上调
综合分析三种大鼠疼痛模型基因芯片分析的结果显示，有352个基因仅在炎性痛发生发展中持续表达上调，196个基因仅在神经病理性疼痛的进展过程中持续表达上调，而骨癌痛中有216个基因持续表达上调。有344个基因在三种慢性疼痛进展中均持续表达上调。
2. Slit2/Robo1促进兴奋性突触形成诱发骨癌痛
方法选择雌性wistar大鼠，随机分为5组：假手术组(Sham)，骨癌痛组(BCP)，对照病毒组(BCP+NC-LV)，Slit2干扰病毒组（BCP+siSlit2-LV）和Robo1干扰病毒组（BCP+siRobo1-LV）。建立骨癌痛模型并进行脊髓内病毒注射。模型建立成功后进行痛阈测定。3W后处死动物进行取出，用HE染色观察肿瘤细胞侵犯胫骨情况。用免疫组化多重标记法结合RT-PCR和Western blot检测大鼠脊髓腰膨大部分Slit2，Robo1，Rhoa的表达；用免疫共沉淀检测Slit2和Robo1之间的直接作用；用电镜检测脊髓背角突触形成情况；用免疫组化双标结合RT-PCR和Western blot检测背角兴奋性突触的形成及相关标记物表达情况。
结果HE染色结果显示乳腺癌细胞接种的大鼠胫骨骨髓腔中有大量的癌细胞，癌细胞向骨质扩散，突破骨髓腔，随着病程延长，癌细胞骨质侵犯进行性加重，后期可出现病理性骨折。机械痛阈测定结果显示，癌细胞接种后第9天，大鼠接种癌细胞侧的后肢出现痛阈下降，随病程进展疼痛进行性加剧，癌细胞接种对侧后肢痛阈没有改变。siSlit2-LV明显缓解大鼠骨癌痛的症状，而Robo1则显著加剧疼痛。免疫组化结果提示Slit2，Robo1和Rhoa均广泛共表达在脊髓背角的神经元上。定量实验分析显示在骨癌痛大鼠脊髓背角中Slit2表达上调，Robo1和Rhoa表达下调；RNA干扰慢病毒成功的下调大鼠脊髓背角中Slit2和Robo1的表达。而且siSlit2-LV下调Slit2表达的同时导致Robo1和Rhoa的表达上调，siRobo1-LV显著下调Robo1和Rhoa的表达，但不影响Slit2的表达。电镜检测突触数量和免疫组化双标检测突触标记蛋白表达均提示骨癌痛大鼠脊髓背角兴奋性突触数量明显增多，siSlit2-LV可减少骨癌痛大鼠脊髓背角兴奋性突触的形成，而Robo1则明显增加兴奋性突触的形成。RT-PCR和Westernblot对突触标记蛋白的定量研究结果与免于组化一致。
3. Slit2/Robo1促进轴突延伸诱导兴奋性突触形成
方法原代培养大鼠皮质神经元。将细胞随机分为4组：正常组（Normal）,对照病毒组(NC-LV)，Slit2干扰病毒组（siSlit2-LV）和Robo1干扰病毒组（siRobo1-LV）。在细胞培养液中加入慢病毒转染5天后，用荧光显微镜观察病毒转染情况，用免疫组化多重标记法结合RT-PCR和Western blot检测神经元上Slit2，Robo1，Rhoa的表达；利用免疫组化观察神经元形态变化；用免疫组化双标结合RT-PCR和Western blot检测神经元兴奋性突触形成及相关蛋白的表达情况。
结果免疫组化结果提示Slit2，Robo1和Rhoa共表神经元上。定量检测结果显示干扰慢病毒成功的下调神经元中Slit2和Robo1的表达。siSlit2-LV下调Slit2表达的同时导致Robo1和Rhoa的表达上调，siRobo1-LV显著下调Robo1和Rhoa的表达，但不影响Slit2的表达。siSlit2-LV明显减少神经元轴突的长度和分支数量，而Robo1则显著增加轴突长度和促进轴突分支形成。siSlit2-LV可减少神经元兴奋性突触的形成，而Robo1则明显增加兴奋性突触的形成。
4.统计学处理
统计学分析采用SPSS18.0统计软件，计量资料以均数±标准差(x±s)表示，组间比较采用单因素方差分析（AnalysisofVariance,ANOVA），P<0.05为差异有统计学意义。
研究总结
一、主要研究结果
1.利用基因芯片分析了骨癌痛，神经病理性痛疼和炎性痛发生发展过程中相关的脊髓基因表达谱。并筛选出与骨癌痛形成有关的脊髓特异性基因表达谱。利用分子功能关联分析，模拟与骨癌痛形成分子网络图。
2.骨癌痛大鼠脊髓中Slit2表达上调，Robo1和Rhoa表达下调；RNAi慢病毒下调Slit2表达则明显缓解骨癌痛，而下调Robo1表达则显著加剧骨癌痛。
3.骨癌痛大鼠脊髓中兴奋性突触数量明显增多；RNAi慢病毒下调Slit2表达则明显减少突触数量，而下调Robo1表达则显著进一步增加突触数量。
4. RNAi慢病毒下调Slit2表达则减少原代培养的神经元轴突长度和分支数量，而下调Robo1表达则显著增加突长度和分支数量。
5. RNAi慢病毒下调原代培养神经元中Slit2表达则明显减少兴奋性突触形成，而下调Robo1表达则显著增加突触形成。
二、研究结论
1.骨癌痛和炎性痛以及神经病理性疼痛三者的发生机制既有共同之处，又有各自的特性。其调控过程不仅是一个复杂的分子网络式整合过程，同时也是一个动态进展的过程，在不同时期有不同基因调控网络。
2.骨癌痛大鼠脊髓中Slit2表达上调，通过与Robo1结合，抑制Robo1和Rhoa的表达，参与骨癌痛的形成。
3. Slit2通过与Robo1直接结合，抑制Robo1和Rhoa的表达，促进大鼠脊髓背角兴奋性突触的形成而导致骨癌痛发生。
4. Slit2通过与Robo1结合，抑制Robo1和Rhoa的表达，通过促进神经元轴突的延伸和分支形成，以及Slit2介导的轴突导向作用诱导兴奋性突触的形成。
三、创新之处
1.本研究首次分析了分别与骨癌痛、炎性痛和神经病理性疼痛发生发展密切相关的特有的基因表达谱以及慢性疼痛形成共同的基因表达谱。拟合了骨癌痛形成过程中的细胞分子线路图。
2.本研究首次证实了Slit2抑制Robo1和Rhoa的表达，促进神经元轴突的延伸和分支形成，介导轴突导向作用促进兴奋性突触的形成，导致骨癌痛大鼠脊髓敏化，揭示了新的骨癌痛形成的分子机制。
四、展望
1.与慢性疼痛相关的基因表达谱的研究，系统全面揭示了慢性疼痛相关细胞分子线路图，为疼痛的机制研究以及治疗靶点的选择提供了依据，但是相关基因的数量庞大，相互关系复杂，涉及了多方面的细胞分子功能。因此，这些分子在疼痛中的确切作用及其相互关系还有待进一步研究。
2.实验论证了骨癌痛形成过程中Slit2、Robo1和Rhoa之间的交互作用以及在兴奋性突触的形成和骨癌痛脊髓敏化中的作用，揭示了新的骨癌痛形成的分子机制，但是Slit2、Robo1和Rhoa只骨癌痛形成过程的一个重要分子环节，RNA干扰技术成功调控其表达和功能后也只是部分逆转是骨癌痛。而且Slit2、Robo1和Rhoa生物功能复杂，以此为靶点治疗骨癌痛的安全性和有效性还需深入研究。
Background
The majority of patients with metastatic bone cancer will experience moderate tosevere pain. Bone pain is one of the most common types of chronic pain in thesepatients.Bone cancer pain is usually progressiveas the disease advances, and is particularlydifficult to treat. The mechanisms responsible for bone pain are poorly understood, butbone cancer pain seems to be enhanced by a state of spinal sensitization. Previous studiesindicate that spinal sensitization of bone cancer pain appears unique when compared tochanges that occur in persistent inflammatory or neuropathic pain states. It has beenhypothesized that one such sensitization mechanism is the regulation of gene expression.Thus, identification of unique gene expression profiles in the spinal cord from animals withbone cancer pain may provide insight into the widespread factors that drive spinal cordplasticity. This, in turn, will contribute to the development of more effective treatments forbone cancer pain.Genechip of full gene probe sets instead of traditional approach andcoulddescribe all of the potential genes that may contribute to generation and maintenanceof chronic pain.In the present study, whole-genome mRNA expression profiles in spinal cords of rats with bone cancer pain, inflammatory pain and neuropathic pain wereexamined to identify target genes that may contribute to spinal sensitization of chronic pain.The mRNA expression was examined using Affymetrix Rat Genome230.2microarrays thatinclude30,000probe sets. The function and cross-effect of the pain-related genes wereanalysised with cluster and molecule annotation system to reveal the molecule mechanismin development of chronic pain.
Spinal sensitization is the main mechanism of chronic pain.Plasticity changes ofsynapseincluding modifications of existing synapses and formation or loss of synapticconnections contribute to sensitization. Previousstudies demonstrated that bone cancer painresulted from an augmentation of excitatory synaptic transmission between neurons over awide area of spinal lumbar segments, but whether the synaptic plasticity is due tomodifications of existing synapses or the formation of new synaptic connections is stillunknown. Here we have shown that sarcoma implantation induced excitabilitysynaptogenesis which drives the development of bone cancer pain. But the mechanisms ofsynapse formation induced by sarcoma inoculation are still unknown.
New synapse establishment requires an interaction between axons and dendrites,accompanied by the appositional organization of pre-and postsynaptic specializations.Thus, the axon guidance cues are required to help establish specific connections between aneuron and its multiple cellular targets. Slit is a largeextracellular matrix protein that wasidentified and implicated in axonal guidance and branching during the development ofnervous system. Roundabout (Robo) was found to encode a protein which is atransmembrane receptor of slit and a member of the immunoglobulin superfamily. Slits actthrough receptors of the Robo in axon chemorepulsion and guidance cues during thedevelopment of the central nervous system. The small GTPase RhoA, a signaling proteinregulating neuronal morphogenesis, plays a critical role in regulationof axonalre-generation.In our chip array experiments, we have identified tSlit2upregulation andRobo1and RhoA downregulation in rats with bone cancer pain. Thus, it seems reasonable to hypothesize that Slit2/Robo1via RhoA mediate the synaptogenesis and contribute to thespinal sensitization of bone cancer pain. To test this notion, siRNA was used to knock downSlit2and Robo1in vivo and in vitro, a carcinoma tibia implantation rat model was used totest the bone cancer-related pain behaviors, and synaptogenesis was examined in vivo andvitro. We have shown thatcarcinoma inoculation induces excitatory synaptogenesis andbone cancer pain behaviors which are reversed by Slit2knockdown but aggravated byRobo1knockdown. In vitro experiments, neurite outgrowth and synaptogenesis of culturedneurons are inhibited by knockdown Slit2, but enhanced by Robo1knockdown. And thatcarcinoma implantation induces an increase of Slit2and decrease Robo1andRhoA.whileSlit2knockdown results in increase of Robo1and RhoA via N-terminal of Slit2directly bounding to Robo1and Robo1knockdown decreased RhoA with Slit2uneffected.These results indicate thatSlit2inhibiting Robo1and then RhoA promotessynaptogenesisand contributes to bone cancer pain. Our findingssuggest a new spinal sensitizationmechanism underlying the development of bone cancer pain.
Methods and results
1. Unique gene expression profiles in spinal cord of rats with bone cancer painMethods To identify altered genes that might contribute to spinal sensitization of chronicpain, we conducted a time-course mRNA profiling experiment on carcinoma tibiaimplantation, L5spinal nerve ligation (SNL), and intraplantar complete Freund’s adjuvant(CFA) injection rat models. The mRNA expression in lumbar spinal cord of respectiveanimals was examined using Affymetrix Rat Genome230.2microarrays that include30,000probe sets.The gene expression profiles in the chronic pain rats were compared withthe corresponding data of sham.
Results We identified2245,1989and1679probe sets that were upregulated respectivelyon days3,7,14post-CFA injection,1570,1560and1494probe sets that were upregulated respectively on days7,14,21post-SNL, and1381,1740and1908probe sets that wereupregulated respectively on days7,14,21post-carcinoma implantation.These genes mayinvolve the onset or maintenance of chronic pain. By comparing the microarray resultsamong the three rat models, we were able to identify a large set of genes whose expressionswere upregulated at each time point as a unique result of CFA injection, SNL andcarcinoma implantation (352,196and216probe sets identified respectively). These genesmay be involved in the maintenance of inflammatory, neuropathic and bone cancer pain,respectively. When the genes whose expressions were upregulated were pooled together,1093,884and344genes overlapped among three time points and344gene sets wereoverlap among three models,.This suggests that these three types of pain on the one handshared a similar mechanisms, but on the other hand have a special gene expression profilethemselves.
2. Slit2/Robo1promotes synaptogenesis and contributes to bone cancer painMethods A Local injection of carcinoma cells directly into rat tibia were used to mimicclinical bone cancer pain and the siRNA lentivirus were injected into the ipsilateral dorsalhorn to specially knock down Slit2or Robo1expression. Coimmunoprecipitation assayusing L3-5spinal cord of carcinoma implantation rats and cultured neurons was used to testwhether functions of Slit2in bone cancer pain was mediated through direct interaction withRobo1. Immunoprecipitation was performed using antibody to Slit2and Robo1, Slit2weredetectable by western blotting using antibodies to Robo1, Slit2. Paw withdrawal thresholdwas measurement of mechanical allodynia.Tthe subcellular distribution and expression ofSlit2, Robo1and RhoA were tested by mmunofluorescent stain, western blot and RT-PCR.Synapse was examined with Transmission Electron Microscopy and excitatory synapse wasexamined using double immunofluorescent labeling with the anti-Synaptophysin (Syn), apresynaptic vesicle protein and anti-PSD95antibody, a major scaffolding protein in theexcitatory postsynaptic density (PSD).
Results Immunofluorescence stain indicated that Slit2, Robo1and RhoA were colocalizedin neuron of dorsal spinal cord. Carcinoma implantation resulted in an increaseof Slit2, but decrease of Robo1and RhoA in ipsilateral dorsal horn. Transfection of dorsalhorn neurons with siRNAto slit2reduced the level of endogenous slit2protein, butincreased Robo1and RhoA, while knockdown of Robo1decreased RhoA and had no effecton Slit2. These Immunohistochemical studies were confirmed by RT-PCR and immunoblotanalysis. The results of immunoprecipitation suggested that Slit2could bind to Robo1directly in vivo and vitro.Taken together, carcinoma implantation upregulated Slit2whichinhibited the expression of Robo1and subsequent RhoA via Slit2binding to Robo1. Wehad showed that carcinoma inoculation resulted in significant bone cancer-related painbehaviors on the ipsilateral paw, but not on the contralateral side. Bone cancer pain wereattenuated by Slit2knockdown but aggravated by Robo1knockdown. These resultsindicated that upregulation of Slit2and thereby downregulation of Robo1and then RhoAwere necessary, but not sufficient for the development of bone cancer pain. Quantitativeanalysis synapse by Transmission Electron Microscopyshowed that synapse numberbroadly increased in cancer bearing rats and knockdown of slit2resulted in a significantreduction in the synapse number.Moreover, the opposite effect was observed upon Robo1knockdown, which resulted in a significant increase in the synapse number. Together withthe quantitative experiments of slit2, Robo1and RhoA expression, the results suggestedthat upregulation of slit2induced by carcinoma implantation resulted in an increase ofsynapse in dorsal horn by inhibition of Robo1and RhoA. Using confocal microscopy, weshowed that both the PSD95and Syn expression increased in ipsilateral dorsal hornbut notin contralateral of cancer bearing rats. Quantified analysis of the magnified images showedthat synapse number was significantly higher in cancer bearing rats compared with sham.Moreover, injection of siSlit2-LV into dorsal horn resulted in decreasing synaptic formation,but siRobo1-LV led to an opposite effects.RT-PCR and immunoblot analysis revealed thatcarcinoma implantation induced a prominent increase both in the levels of PSD95andSynwhich were reversed by knockdown of Slit2, but further increased by Robo1 knockdown. These results indicated that upregulation of Slit2inhibited Robo1andpromoted the excitatory synaptogenesis in dorsal horn post-carcinoma implantation.
3. Slit2/Robo1promotesneurite outgrowth and contributes to synaptogenesisMethods To test whether upregulation of Slit2promote excitability synapse formationthrough inhibiting Robo1and subsequent removing the RhoA-inhibiting effect on theprocesses of axon enlongation and arborization, we first investigate the subcellulardistribution and interaction among Slit2, Robo1and RhoA. Cortial neurons from embryos(E17–E19) of pregnant rats were cultured, transfectedat5day in vitro (DIV)with a plasmidencoding green fluorescent protein(GFP) together with anRNAi to Slit2or Robo1or acontrol RNAi, and examined at10DIV. To unambiguously visualize multiple branchesfrom a single neuronal cell body, neurons were cultured at low density. We usedimmunocytochemistry to investigate the subcellular distribution of Slit2, Robo1and RhoA.Neuronal branches were examined using immunohistochemistrical staining ofmicrotubule-associated protein2(MAP2), a neuronal marker. We next examinedtheexcitability synapse formation of cortial neurons cocultured with astroglia from embryosof pregnant rats. The siRNA lentivires transfected cells at5DIV and and fixed10days laterfor staining with antibodies that recognize Synand PSD95and Syn.To quantify the numberof synapses formed on the normal or transfected neuron, we counted the number of apposedSyn/PSD95puncta along dendrites of normal or GFP-expressing neurons.
Results We used immunocytochemistry to investigate the subcellular distribution of Slit2,Robo1and RhoA and found that they were broadly co-expressed onneuronal soma anddendritesas well as axons. Knockdown Slit2inhibited Robo1and RhoA, while knockdownRobo1resulted in decrease of RhoA but not Slit2. The results were further confirmed byRT-PCR and western blot. Together with immunoprecipitation study descripted above usingcultured-neurons, these results indicated that Slit2bound to and inhibited Robo1, which inturn inhibited RhoA in vitro. These results were agreed with those of animal experimentupon. Using immunohistochemistrical staining of microtubule-associated protein2(MAP2), we showed that Slit2knockdown strongly reduced neurite number and length, whileknockdown of Robo1enhanced neurite outgrowth. Together with quantitative analysis ofSlit2, Robo1and RhoA expression in neuron, the results indicated that Slit2promoted axonelongationand branching probably due to inhibiting Robo1and sequent blockingRhoA-inhibition on neurite extension. Thus we next examined the synapse formation ofneurons cocultured with astroglia from embryos of pregnant rats. Quantify the number ofsynapses showed that knockdown of Slit2resulted in a significant decrease inexcitatorysynaptic numbe. Conversely, knockdown of Robo1induced an increase of excitatorysynapse.
4. Statistical analysis
All data are presented as means±SD. The statistical significance of difference betweenvalues was determined by analysis of variance (ANOVA). For all analyses, significancewas set at P <0.05.
Conclusion
1. We have identified hundreds of altered genes that may mediate the onset ormaintenance of inflammatory, neuropathy and bone cancer pain respectively. When thegenes whose expressions were upregulated were pooled together, lots of gene sets wereoverlap among three models.Thissuggests that these three types of pain on the one handshared a similarmechanisms, but on the other hand have a special gene expression profilethemselves.
2. Slit2mediates axon guidance and branch and promotes excitatory synaptogenesis viainhibition of Robo1and subsequent RhoA–inhibition of axon remodeling and contributesbone cancer pain.
Significance
In the present study, we have identified the unique gene expression profiles in the spinalcord from animals with bone cancer pain may provide insight into the widespread factorsthat drive spinal cord sensitivity of pain. This, in turn, will contribute to the development ofmore effective treatments for bone cancer pain.Synaptic plasticity is fundamental to spinal sensitivity of bone cancer pain. Here we haveshown that excitatory synaptogenesis contributes to bone cancer pain and described thatSlit2, Robo1and RhoA act as such cues that promote neurite outgrowth and guides theaxon for synapse formation. These results havedemonstrated a molecular mechanism ofsynaptogenesis in bone cancer pain.This maybe lead to a new treatment target for bonecancer pain.
骨癌痛（Bone cancer pain,BCP）是原发性或者转移性骨肿瘤引起的慢性疼痛。据世界卫生组织(WHO)统计,全世界每年新增1000万癌症患者，约三分之一的恶性肿瘤会发生骨转移。绝大部分骨肿瘤患者会出现不同程度的慢性疼痛。由于目前对骨癌痛机制的认识不足以及临床现有治疗措施的局限性，约50%的骨癌患者疼痛未得到有效控制。长期慢性疼痛给患者及其家人带来巨大痛苦，严重影响患者生活质量，甚至使患者丧失生活自理能力。所以，对骨癌痛机制的深入研究，以期为癌痛治疗提供有效的治疗措施，是目前亟待解决的重大问题。
基于对多种骨癌痛动物模型的研究，目前对骨癌痛的独特机制有了初步了解，认为骨癌痛与其它慢性疼痛不同，涉及多种炎症介质、细胞因子、多种受体和离子通道，既有炎性痛，又有神经病理性疼痛的成分。要破解骨癌痛发生这个错综复杂的分子网络，沿用传统的研究方法，从单一基因、或单个因素入手是无法解释和阐明这些现象的本质及其相互之间的因果关系并取得突破性进展的。
利用基因组学为平台，应用基因芯片分析技术，可以从细胞、组织、器官和生物体整体水平上研究结构和功能各异的各种分子及其相互关系，能够定量描述和预测生物功能、表型和行为，能客观真实地反映机体的整体变化。因此，本实验拟应用寡核苷酸微阵列芯片及生物信息学技术，研究骨癌痛模型与神经病理性痛模型、炎性痛模型大鼠脊髓水平基因表达谱差异，筛选出―脊髓组织骨癌痛特异性基因‖，进而利用计算生物学方法，采用统计学中聚类分析，探讨骨癌痛发生发展过程中脊髓组织分子水平的改变与疼痛行为学的关系，发掘在骨癌痛发病机制中起关键作用的特异基因表型。
中枢敏化是指脊髓和脊髓以上部位对疼痛信号传导的放大过程,是目前解释神经病理性疼痛，炎性痛等多种慢性疼痛出现痛觉过敏和痛觉超敏的主要理论。临床资料和基础实验研究提示，脊髓水平的中枢敏化是骨癌痛产生和维持的重要机制。在脊髓水平,背角是疼痛的调制主要部位，也伤害性信息的初级整合中枢。外周伤害性信息经初级传入纤维传入背角后，在此中继、整合,再由投射神经元向上位脑结构传递，最终产生痛觉。因此，目前对疼痛形成中脊髓敏化的研究也多集中在背角部位。
突触传导的可塑性改变是疼痛产生过程中脊髓敏化的重要机制之一。突触可塑性是指突触效能在某些因素的作用下出现不同程度的增强或减弱的特性。突触的可塑性改变包括两方面，一个是对现有突触结构的修饰，改变突触递质的释放而增强或减弱突触的传导性能，另一个是长时程突触可塑性调节机制，是通过增加或减少突触的数量来调节突触的传递效能。有研究报道，突触重塑导致脊髓敏化是骨癌痛发生的重要因素，但突触重塑的方式及其具体机制都不清楚。由于骨癌痛是一个长期慢性进展的过程，突触重塑过程也将是一个长期的持续变化，因此我们假设骨癌痛脊髓敏化是通过增加兴奋性突触数量来实现突触传递增强的。但是，新的突触的形成需要神经元轴突或树突延伸以及有导航作用的分子引导新生突起精确对接才能完成，而这个过程的分子机制也不清楚。
Slit homolog2protein(Slit2)是一种神经轴突的导向分子，在神经系统发育过程有重要作用。能够刺激神经元轴突生长和延伸以及侧支的形成[28-30]，调控神经元迁移，并且通过对生长锥的导向而指导突触的形成和功能性神经网络的建立。Roundabouthomolog1(Robo1)属于细胞粘附分子免疫球蛋白超家族成员，是slit2的受体，通过与slit2结合对神经元和生长锥产生排斥作用，参与调控发育中的中枢神经轴突定向延伸，促进轴突生长和调控神经纤维束化、定位以及突触的形成。研究显示Slit2和Robo1可能在病理状态下参与神经系统结构性重塑过程。因此我们假设，在骨癌痛形成过程中，Slit2和Robo1结合后，发挥了分子导向作用，促进轴突分支靶向性迁移而引导突触形成。为了证实Slit2和Robo1结合导致脊髓中新突触的形成而诱发骨癌痛脊髓敏化这一假说，本实验拟建立大鼠骨癌痛模型，利用RNA干扰技术等分子生物学技术，论证Slit2和Robo1在骨癌痛形成中的作用以及对神经元轴突延伸和突触形成的影响。以期从一个全新的角度揭示骨癌痛发生的机制，为骨癌痛是治疗寻找新的靶点
研究方法与结果
1.骨癌痛大鼠脊髓特异性基因表达谱的筛选
方法选择雌性wistar大鼠，随机分为4组：正常组，骨癌痛组，神经病理性疼痛组和炎性痛组。分别制作乳腺癌细胞胫骨种植的骨癌痛模型，L5脊神经结扎的神经病理性疼痛模型和弗氏完全佐剂（CFA）足底注射的炎性痛模型。模型建立后，进行痛阈测定。在疼痛发展的不同时期分别取各组大鼠脊髓腰L4-6进行基因芯片检测。采用统计分析筛选表达差异的基因谱，结合聚类分析和分子注释功能数据库分析特异基因之间的相互作用，模拟疼痛的分子路线图。
结果基因芯片检测结果提示在炎性痛早期，有2245个基因表达上调，其中包括479个基因仅在早期表达上调，515个基因持续上调至疼痛进展期，有1093个基因则在整个炎性痛发生发展过程中是持续上调。在疼痛的进展期共有2089个基因表达上调，其中263个基因仅在这个时间段上调，另外有118个基因从疼痛进展期出现上调，并持续至后期。在炎性痛的后期有1679个基因表达上调，其中有310个基因仅在后期出现表达上调。
神经病理性疼痛大鼠脊髓组织基因芯片检测结果提示在疼痛早期，有1570个基因表达上调，其中包括227个基因仅在早期表达上调，301个基因上调持续至疼痛进展期，有884个基因则在整个神经病理性疼痛发生发展过程中持续上调。在疼痛的进展期共有1560个基因表达上调，其中265个基因仅在进展期上调，另外有110个基因从疼痛进展期开始出现持续表达上调。在神经病理性疼痛的维持期有1476个基因表达上调，其中有342个基因在疼痛维持期才出现表达上调。
骨癌痛基因芯片的结果提示在骨癌痛早期，有1383个基因表达上调，其中包括381个基因仅在早期表达上调，155个基因上调持续至疼痛进展期，有691个基因则是在骨癌痛发生发展过程中是持续上调。在疼痛的进展期共有1740个基因表达上调，其中414个基因仅在这个时间段上调达，另外有480个基因从疼痛进展期开始出现持续表达上调。在骨癌痛的维持期则有1908个基因表达上调，其中有581个基因在疼痛的维持期出现表达上调
综合分析三种大鼠疼痛模型基因芯片分析的结果显示，有352个基因仅在炎性痛发生发展中持续表达上调，196个基因仅在神经病理性疼痛的进展过程中持续表达上调，而骨癌痛中有216个基因持续表达上调。有344个基因在三种慢性疼痛进展中均持续表达上调。
2. Slit2/Robo1促进兴奋性突触形成诱发骨癌痛
方法选择雌性wistar大鼠，随机分为5组：假手术组(Sham)，骨癌痛组(BCP)，对照病毒组(BCP+NC-LV)，Slit2干扰病毒组（BCP+siSlit2-LV）和Robo1干扰病毒组（BCP+siRobo1-LV）。建立骨癌痛模型并进行脊髓内病毒注射。模型建立成功后进行痛阈测定。3W后处死动物进行取出，用HE染色观察肿瘤细胞侵犯胫骨情况。用免疫组化多重标记法结合RT-PCR和Western blot检测大鼠脊髓腰膨大部分Slit2，Robo1，Rhoa的表达；用免疫共沉淀检测Slit2和Robo1之间的直接作用；用电镜检测脊髓背角突触形成情况；用免疫组化双标结合RT-PCR和Western blot检测背角兴奋性突触的形成及相关标记物表达情况。
结果HE染色结果显示乳腺癌细胞接种的大鼠胫骨骨髓腔中有大量的癌细胞，癌细胞向骨质扩散，突破骨髓腔，随着病程延长，癌细胞骨质侵犯进行性加重，后期可出现病理性骨折。机械痛阈测定结果显示，癌细胞接种后第9天，大鼠接种癌细胞侧的后肢出现痛阈下降，随病程进展疼痛进行性加剧，癌细胞接种对侧后肢痛阈没有改变。siSlit2-LV明显缓解大鼠骨癌痛的症状，而Robo1则显著加剧疼痛。免疫组化结果提示Slit2，Robo1和Rhoa均广泛共表达在脊髓背角的神经元上。定量实验分析显示在骨癌痛大鼠脊髓背角中Slit2表达上调，Robo1和Rhoa表达下调；RNA干扰慢病毒成功的下调大鼠脊髓背角中Slit2和Robo1的表达。而且siSlit2-LV下调Slit2表达的同时导致Robo1和Rhoa的表达上调，siRobo1-LV显著下调Robo1和Rhoa的表达，但不影响Slit2的表达。电镜检测突触数量和免疫组化双标检测突触标记蛋白表达均提示骨癌痛大鼠脊髓背角兴奋性突触数量明显增多，siSlit2-LV可减少骨癌痛大鼠脊髓背角兴奋性突触的形成，而Robo1则明显增加兴奋性突触的形成。RT-PCR和Westernblot对突触标记蛋白的定量研究结果与免于组化一致。
3. Slit2/Robo1促进轴突延伸诱导兴奋性突触形成
方法原代培养大鼠皮质神经元。将细胞随机分为4组：正常组（Normal）,对照病毒组(NC-LV)，Slit2干扰病毒组（siSlit2-LV）和Robo1干扰病毒组（siRobo1-LV）。在细胞培养液中加入慢病毒转染5天后，用荧光显微镜观察病毒转染情况，用免疫组化多重标记法结合RT-PCR和Western blot检测神经元上Slit2，Robo1，Rhoa的表达；利用免疫组化观察神经元形态变化；用免疫组化双标结合RT-PCR和Western blot检测神经元兴奋性突触形成及相关蛋白的表达情况。
结果免疫组化结果提示Slit2，Robo1和Rhoa共表神经元上。定量检测结果显示干扰慢病毒成功的下调神经元中Slit2和Robo1的表达。siSlit2-LV下调Slit2表达的同时导致Robo1和Rhoa的表达上调，siRobo1-LV显著下调Robo1和Rhoa的表达，但不影响Slit2的表达。siSlit2-LV明显减少神经元轴突的长度和分支数量，而Robo1则显著增加轴突长度和促进轴突分支形成。siSlit2-LV可减少神经元兴奋性突触的形成，而Robo1则明显增加兴奋性突触的形成。
4.统计学处理
统计学分析采用SPSS18.0统计软件，计量资料以均数±标准差(x±s)表示，组间比较采用单因素方差分析（AnalysisofVariance,ANOVA），P<0.05为差异有统计学意义。
研究总结
一、主要研究结果
1.利用基因芯片分析了骨癌痛，神经病理性痛疼和炎性痛发生发展过程中相关的脊髓基因表达谱。并筛选出与骨癌痛形成有关的脊髓特异性基因表达谱。利用分子功能关联分析，模拟与骨癌痛形成分子网络图。
2.骨癌痛大鼠脊髓中Slit2表达上调，Robo1和Rhoa表达下调；RNAi慢病毒下调Slit2表达则明显缓解骨癌痛，而下调Robo1表达则显著加剧骨癌痛。
3.骨癌痛大鼠脊髓中兴奋性突触数量明显增多；RNAi慢病毒下调Slit2表达则明显减少突触数量，而下调Robo1表达则显著进一步增加突触数量。
4. RNAi慢病毒下调Slit2表达则减少原代培养的神经元轴突长度和分支数量，而下调Robo1表达则显著增加突长度和分支数量。
5. RNAi慢病毒下调原代培养神经元中Slit2表达则明显减少兴奋性突触形成，而下调Robo1表达则显著增加突触形成。
二、研究结论
1.骨癌痛和炎性痛以及神经病理性疼痛三者的发生机制既有共同之处，又有各自的特性。其调控过程不仅是一个复杂的分子网络式整合过程，同时也是一个动态进展的过程，在不同时期有不同基因调控网络。
2.骨癌痛大鼠脊髓中Slit2表达上调，通过与Robo1结合，抑制Robo1和Rhoa的表达，参与骨癌痛的形成。
3. Slit2通过与Robo1直接结合，抑制Robo1和Rhoa的表达，促进大鼠脊髓背角兴奋性突触的形成而导致骨癌痛发生。
4. Slit2通过与Robo1结合，抑制Robo1和Rhoa的表达，通过促进神经元轴突的延伸和分支形成，以及Slit2介导的轴突导向作用诱导兴奋性突触的形成。
三、创新之处
1.本研究首次分析了分别与骨癌痛、炎性痛和神经病理性疼痛发生发展密切相关的特有的基因表达谱以及慢性疼痛形成共同的基因表达谱。拟合了骨癌痛形成过程中的细胞分子线路图。
2.本研究首次证实了Slit2抑制Robo1和Rhoa的表达，促进神经元轴突的延伸和分支形成，介导轴突导向作用促进兴奋性突触的形成，导致骨癌痛大鼠脊髓敏化，揭示了新的骨癌痛形成的分子机制。
四、展望
1.与慢性疼痛相关的基因表达谱的研究，系统全面揭示了慢性疼痛相关细胞分子线路图，为疼痛的机制研究以及治疗靶点的选择提供了依据，但是相关基因的数量庞大，相互关系复杂，涉及了多方面的细胞分子功能。因此，这些分子在疼痛中的确切作用及其相互关系还有待进一步研究。
2.实验论证了骨癌痛形成过程中Slit2、Robo1和Rhoa之间的交互作用以及在兴奋性突触的形成和骨癌痛脊髓敏化中的作用，揭示了新的骨癌痛形成的分子机制，但是Slit2、Robo1和Rhoa只骨癌痛形成过程的一个重要分子环节，RNA干扰技术成功调控其表达和功能后也只是部分逆转是骨癌痛。而且Slit2、Robo1和Rhoa生物功能复杂，以此为靶点治疗骨癌痛的安全性和有效性还需深入研究。
Background
The majority of patients with metastatic bone cancer will experience moderate tosevere pain. Bone pain is one of the most common types of chronic pain in thesepatients.Bone cancer pain is usually progressiveas the disease advances, and is particularlydifficult to treat. The mechanisms responsible for bone pain are poorly understood, butbone cancer pain seems to be enhanced by a state of spinal sensitization. Previous studiesindicate that spinal sensitization of bone cancer pain appears unique when compared tochanges that occur in persistent inflammatory or neuropathic pain states. It has beenhypothesized that one such sensitization mechanism is the regulation of gene expression.Thus, identification of unique gene expression profiles in the spinal cord from animals withbone cancer pain may provide insight into the widespread factors that drive spinal cordplasticity. This, in turn, will contribute to the development of more effective treatments forbone cancer pain.Genechip of full gene probe sets instead of traditional approach andcoulddescribe all of the potential genes that may contribute to generation and maintenanceof chronic pain.In the present study, whole-genome mRNA expression profiles in spinal cords of rats with bone cancer pain, inflammatory pain and neuropathic pain wereexamined to identify target genes that may contribute to spinal sensitization of chronic pain.The mRNA expression was examined using Affymetrix Rat Genome230.2microarrays thatinclude30,000probe sets. The function and cross-effect of the pain-related genes wereanalysised with cluster and molecule annotation system to reveal the molecule mechanismin development of chronic pain.
Spinal sensitization is the main mechanism of chronic pain.Plasticity changes ofsynapseincluding modifications of existing synapses and formation or loss of synapticconnections contribute to sensitization. Previousstudies demonstrated that bone cancer painresulted from an augmentation of excitatory synaptic transmission between neurons over awide area of spinal lumbar segments, but whether the synaptic plasticity is due tomodifications of existing synapses or the formation of new synaptic connections is stillunknown. Here we have shown that sarcoma implantation induced excitabilitysynaptogenesis which drives the development of bone cancer pain. But the mechanisms ofsynapse formation induced by sarcoma inoculation are still unknown.
New synapse establishment requires an interaction between axons and dendrites,accompanied by the appositional organization of pre-and postsynaptic specializations.Thus, the axon guidance cues are required to help establish specific connections between aneuron and its multiple cellular targets. Slit is a largeextracellular matrix protein that wasidentified and implicated in axonal guidance and branching during the development ofnervous system. Roundabout (Robo) was found to encode a protein which is atransmembrane receptor of slit and a member of the immunoglobulin superfamily. Slits actthrough receptors of the Robo in axon chemorepulsion and guidance cues during thedevelopment of the central nervous system. The small GTPase RhoA, a signaling proteinregulating neuronal morphogenesis, plays a critical role in regulationof axonalre-generation.In our chip array experiments, we have identified tSlit2upregulation andRobo1and RhoA downregulation in rats with bone cancer pain. Thus, it seems reasonable to hypothesize that Slit2/Robo1via RhoA mediate the synaptogenesis and contribute to thespinal sensitization of bone cancer pain. To test this notion, siRNA was used to knock downSlit2and Robo1in vivo and in vitro, a carcinoma tibia implantation rat model was used totest the bone cancer-related pain behaviors, and synaptogenesis was examined in vivo andvitro. We have shown thatcarcinoma inoculation induces excitatory synaptogenesis andbone cancer pain behaviors which are reversed by Slit2knockdown but aggravated byRobo1knockdown. In vitro experiments, neurite outgrowth and synaptogenesis of culturedneurons are inhibited by knockdown Slit2, but enhanced by Robo1knockdown. And thatcarcinoma implantation induces an increase of Slit2and decrease Robo1andRhoA.whileSlit2knockdown results in increase of Robo1and RhoA via N-terminal of Slit2directly bounding to Robo1and Robo1knockdown decreased RhoA with Slit2uneffected.These results indicate thatSlit2inhibiting Robo1and then RhoA promotessynaptogenesisand contributes to bone cancer pain. Our findingssuggest a new spinal sensitizationmechanism underlying the development of bone cancer pain.
Methods and results
1. Unique gene expression profiles in spinal cord of rats with bone cancer painMethods To identify altered genes that might contribute to spinal sensitization of chronicpain, we conducted a time-course mRNA profiling experiment on carcinoma tibiaimplantation, L5spinal nerve ligation (SNL), and intraplantar complete Freund’s adjuvant(CFA) injection rat models. The mRNA expression in lumbar spinal cord of respectiveanimals was examined using Affymetrix Rat Genome230.2microarrays that include30,000probe sets.The gene expression profiles in the chronic pain rats were compared withthe corresponding data of sham.
Results We identified2245,1989and1679probe sets that were upregulated respectivelyon days3,7,14post-CFA injection,1570,1560and1494probe sets that were upregulated respectively on days7,14,21post-SNL, and1381,1740and1908probe sets that wereupregulated respectively on days7,14,21post-carcinoma implantation.These genes mayinvolve the onset or maintenance of chronic pain. By comparing the microarray resultsamong the three rat models, we were able to identify a large set of genes whose expressionswere upregulated at each time point as a unique result of CFA injection, SNL andcarcinoma implantation (352,196and216probe sets identified respectively). These genesmay be involved in the maintenance of inflammatory, neuropathic and bone cancer pain,respectively. When the genes whose expressions were upregulated were pooled together,1093,884and344genes overlapped among three time points and344gene sets wereoverlap among three models,.This suggests that these three types of pain on the one handshared a similar mechanisms, but on the other hand have a special gene expression profilethemselves.
2. Slit2/Robo1promotes synaptogenesis and contributes to bone cancer painMethods A Local injection of carcinoma cells directly into rat tibia were used to mimicclinical bone cancer pain and the siRNA lentivirus were injected into the ipsilateral dorsalhorn to specially knock down Slit2or Robo1expression. Coimmunoprecipitation assayusing L3-5spinal cord of carcinoma implantation rats and cultured neurons was used to testwhether functions of Slit2in bone cancer pain was mediated through direct interaction withRobo1. Immunoprecipitation was performed using antibody to Slit2and Robo1, Slit2weredetectable by western blotting using antibodies to Robo1, Slit2. Paw withdrawal thresholdwas measurement of mechanical allodynia.Tthe subcellular distribution and expression ofSlit2, Robo1and RhoA were tested by mmunofluorescent stain, western blot and RT-PCR.Synapse was examined with Transmission Electron Microscopy and excitatory synapse wasexamined using double immunofluorescent labeling with the anti-Synaptophysin (Syn), apresynaptic vesicle protein and anti-PSD95antibody, a major scaffolding protein in theexcitatory postsynaptic density (PSD).
Results Immunofluorescence stain indicated that Slit2, Robo1and RhoA were colocalizedin neuron of dorsal spinal cord. Carcinoma implantation resulted in an increaseof Slit2, but decrease of Robo1and RhoA in ipsilateral dorsal horn. Transfection of dorsalhorn neurons with siRNAto slit2reduced the level of endogenous slit2protein, butincreased Robo1and RhoA, while knockdown of Robo1decreased RhoA and had no effecton Slit2. These Immunohistochemical studies were confirmed by RT-PCR and immunoblotanalysis. The results of immunoprecipitation suggested that Slit2could bind to Robo1directly in vivo and vitro.Taken together, carcinoma implantation upregulated Slit2whichinhibited the expression of Robo1and subsequent RhoA via Slit2binding to Robo1. Wehad showed that carcinoma inoculation resulted in significant bone cancer-related painbehaviors on the ipsilateral paw, but not on the contralateral side. Bone cancer pain wereattenuated by Slit2knockdown but aggravated by Robo1knockdown. These resultsindicated that upregulation of Slit2and thereby downregulation of Robo1and then RhoAwere necessary, but not sufficient for the development of bone cancer pain. Quantitativeanalysis synapse by Transmission Electron Microscopyshowed that synapse numberbroadly increased in cancer bearing rats and knockdown of slit2resulted in a significantreduction in the synapse number.Moreover, the opposite effect was observed upon Robo1knockdown, which resulted in a significant increase in the synapse number. Together withthe quantitative experiments of slit2, Robo1and RhoA expression, the results suggestedthat upregulation of slit2induced by carcinoma implantation resulted in an increase ofsynapse in dorsal horn by inhibition of Robo1and RhoA. Using confocal microscopy, weshowed that both the PSD95and Syn expression increased in ipsilateral dorsal hornbut notin contralateral of cancer bearing rats. Quantified analysis of the magnified images showedthat synapse number was significantly higher in cancer bearing rats compared with sham.Moreover, injection of siSlit2-LV into dorsal horn resulted in decreasing synaptic formation,but siRobo1-LV led to an opposite effects.RT-PCR and immunoblot analysis revealed thatcarcinoma implantation induced a prominent increase both in the levels of PSD95andSynwhich were reversed by knockdown of Slit2, but further increased by Robo1 knockdown. These results indicated that upregulation of Slit2inhibited Robo1andpromoted the excitatory synaptogenesis in dorsal horn post-carcinoma implantation.
3. Slit2/Robo1promotesneurite outgrowth and contributes to synaptogenesisMethods To test whether upregulation of Slit2promote excitability synapse formationthrough inhibiting Robo1and subsequent removing the RhoA-inhibiting effect on theprocesses of axon enlongation and arborization, we first investigate the subcellulardistribution and interaction among Slit2, Robo1and RhoA. Cortial neurons from embryos(E17–E19) of pregnant rats were cultured, transfectedat5day in vitro (DIV)with a plasmidencoding green fluorescent protein(GFP) together with anRNAi to Slit2or Robo1or acontrol RNAi, and examined at10DIV. To unambiguously visualize multiple branchesfrom a single neuronal cell body, neurons were cultured at low density. We usedimmunocytochemistry to investigate the subcellular distribution of Slit2, Robo1and RhoA.Neuronal branches were examined using immunohistochemistrical staining ofmicrotubule-associated protein2(MAP2), a neuronal marker. We next examinedtheexcitability synapse formation of cortial neurons cocultured with astroglia from embryosof pregnant rats. The siRNA lentivires transfected cells at5DIV and and fixed10days laterfor staining with antibodies that recognize Synand PSD95and Syn.To quantify the numberof synapses formed on the normal or transfected neuron, we counted the number of apposedSyn/PSD95puncta along dendrites of normal or GFP-expressing neurons.
Results We used immunocytochemistry to investigate the subcellular distribution of Slit2,Robo1and RhoA and found that they were broadly co-expressed onneuronal soma anddendritesas well as axons. Knockdown Slit2inhibited Robo1and RhoA, while knockdownRobo1resulted in decrease of RhoA but not Slit2. The results were further confirmed byRT-PCR and western blot. Together with immunoprecipitation study descripted above usingcultured-neurons, these results indicated that Slit2bound to and inhibited Robo1, which inturn inhibited RhoA in vitro. These results were agreed with those of animal experimentupon. Using immunohistochemistrical staining of microtubule-associated protein2(MAP2), we showed that Slit2knockdown strongly reduced neurite number and length, whileknockdown of Robo1enhanced neurite outgrowth. Together with quantitative analysis ofSlit2, Robo1and RhoA expression in neuron, the results indicated that Slit2promoted axonelongationand branching probably due to inhibiting Robo1and sequent blockingRhoA-inhibition on neurite extension. Thus we next examined the synapse formation ofneurons cocultured with astroglia from embryos of pregnant rats. Quantify the number ofsynapses showed that knockdown of Slit2resulted in a significant decrease inexcitatorysynaptic numbe. Conversely, knockdown of Robo1induced an increase of excitatorysynapse.
4. Statistical analysis
All data are presented as means±SD. The statistical significance of difference betweenvalues was determined by analysis of variance (ANOVA). For all analyses, significancewas set at P <0.05.
Conclusion
1. We have identified hundreds of altered genes that may mediate the onset ormaintenance of inflammatory, neuropathy and bone cancer pain respectively. When thegenes whose expressions were upregulated were pooled together, lots of gene sets wereoverlap among three models.Thissuggests that these three types of pain on the one handshared a similarmechanisms, but on the other hand have a special gene expression profilethemselves.
2. Slit2mediates axon guidance and branch and promotes excitatory synaptogenesis viainhibition of Robo1and subsequent RhoA–inhibition of axon remodeling and contributesbone cancer pain.
Significance
In the present study, we have identified the unique gene expression profiles in the spinalcord from animals with bone cancer pain may provide insight into the widespread factorsthat drive spinal cord sensitivity of pain. This, in turn, will contribute to the development ofmore effective treatments for bone cancer pain.Synaptic plasticity is fundamental to spinal sensitivity of bone cancer pain. Here we haveshown that excitatory synaptogenesis contributes to bone cancer pain and described thatSlit2, Robo1and RhoA act as such cues that promote neurite outgrowth and guides theaxon for synapse formation. These results havedemonstrated a molecular mechanism ofsynaptogenesis in bone cancer pain.This maybe lead to a new treatment target for bonecancer pain.
引文
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