脊髓CXCL13在大鼠骨癌痛形成中的作用

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.

Role of spinal cord CXCL13 in formation of bone cancer pain in rats

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.