脊髓CXCL13在大鼠骨癌痛形成中的作用

摘要

Objective To investigate the role of spinal cord chemokine CXC ligand13(CXCL13) in the formation of rat bone cancer pain(BCP).Methods Twenty healthy female SD rats weighing 160-200 g were divided into four groups(n=5):sham operation group(S),BCP group(BP),small interference RNA(siRNA) negative control(NC-siRNA) group (NC) and CXCL13-siRNA group(CS).Normal saline was given by tibial medullary cavity injection in the S group.The tibial BCP model was established by tibial medullary cavity injection of equivalent Walker-256 breast cancer cells in the group BP,NC and CS.NC-siRNA lentivirus and CXCL13-siRNA lentivirus were injected intrathecally in the group NC and CS respectively.The mechanical pain threshold was measured on 1 d before model construction and on postoperative 7,9,14,21 d.The rats were killed after pain threshold measurement.The spinal cord and tibial tissue were taken.The co-expression of spinal CXCL13,microglia specific marker Iba-1 and neuron specific neucleoprotein NeuN was determined by using the immunofluorescence double standard staining,and expressions of CXCL13 and ionized calcium binding adaptor molecule-1 (Iba-1) protein and mRNA in spinal cord were detected by Western blot and RT-PCR;the HE staining microscopy was adopted to observe the tibial bone structure destroy situation.Results Compared with group S,the mechanical pain threshold in theBP group and NC group was decreased on 7-21 d after inoculation,CXCL13 expression in neuron was significantly increased and microglia was obviously activated,the expression of CXCL13 and Iba-1 protein and mRNA was significantly elevated (P＜0.05);compared with the NC group,the mechanical pain threshold on 9-21 d after model construction in the CS group was significantly increased,CXCL13 expression in neurons was significantly decreased,microglia activation was decreased and expression of CXCL13 and Iba-1 protein and mRNA was significantly decreased(P＜0.05);HE staining showed that the model groups appeared the tumor growth in bone marrow cavity,moreover which was eroded outwards and destroyed bone cortex,but no abnormality was found in the S group.Conclusion Spinal cord CXCL13 is involved in the BCP formation in rats by activating microglia.%目的 探讨脊髓趋化因子CXC配体13(CXCL13)在大鼠骨癌痛形成中的作用.方法 健康雌性SD大鼠20只,体质量160～200 g,分为4组(n=5):假手术组(S组)、骨癌痛组(BP组)、小干扰RNA(siRNA)阴性对照(NC-siRNA)组(NC组)和CXCL13-siRNA组(CS组).S组胫骨骨髓腔内注射生理盐水,BP组、NC组和CS组均采用胫骨髓腔内注射等量Walker-256乳腺癌细胞的方法建立大鼠胫骨癌痛模型,NC组和CS组分别鞘内注射NC-siRNA慢病毒和CXCL13-siRNA慢病毒10μL.分别于造模前1d及术后第7、9、14、21天时测定机械痛阈值,痛阈测定结束后处死大鼠,取脊髓和胫骨组织,采用免疫荧光双标染色检测脊髓CXCL13、小胶质细胞特异性标记物(Iba-1)和神经元特异核蛋白(NeuN)的共表达情况;采用Western-blot、RT-PCR法测定脊髓CXCL13、Iba-1的蛋白及mRNA表达;采用HE染色光镜下观察胫骨骨结构破坏情况.结果 与S组比较,BP和NC组接种后7～21 d机械痛阈下降(P＜0.05),CXCL13在神经元中表达显著上调,小胶质细胞明显活化(P＜0.05),CXCL13和Iba-1蛋白及mRNA水平明显升高(P＜0.05);与NC组比较,CS组造模后9～21 d机械痛阈升高(P＜0.05),CXCL13在神经元中表达明显下调,小胶质细胞活化减少(P＜0.05),CXCL13和Iba-1蛋白及mRNA水平明显下降(P＜0.05);HE染色显示BP、NC组、CA组均出现骨髓腔内肿瘤生长且向外侵蚀破坏骨皮质,S组未见异常.结论 脊髓CXCL13通过激活小胶质细胞参与大鼠骨癌痛的形成.