趋化因子CXCL13在大鼠骨癌痛发生和吗啡镇痛调节中的双重作用及机制研究

In recent studies, chemokines play important roles in the development and maintanence of cancer-induced bone pain (CIBP). In our previous studies, upregulation of chemokine CXCL10 in spinal cord was a critical initiation for cancer-induced bone pain. Furthermore, CXCL10 could also regulate morphine analgesia in mice. In the results of gene microarray, the expression of CXCL13 in spinal cord of CIBP rats were significantly increased on day 7, 14 and 21 after modeling. Based on these backgrouds, we hypothesis that upregulation of spinal CXCL13 may participate in the development of CIBP and regulate morphine analgesia. In this project, we will detect the expression of CXCL13 and its receptor CXCR5 in the spinal cord of CIBP rats and the coexpression of CXCR5 and opiate receptor (MOR) by several molecular biological ways, including immunofluorescence, real-time PCR, western blots and co-immunoprecipitation, et al. Furthermore, the role of spinal CXCL13 in the development of cancer pain in rats will be studied by behavior evaluation. Finally, the activation of downstream signal pathways of CXCR5 and MOR involved in the development of CIBP and regulation of morphine analgesia will be detected. This project will provide a novel mechanism for the development of cancer pain and a new target for cancer pain manegement in clinich.

趋化因子在骨癌痛的发生和维持中发挥着重要作用。本课题组前期证实脊髓内趋化因子CXCL10的表达上调是大鼠骨癌痛发生的重要促使因素，还可以减弱吗啡的镇痛作用。本课题组的基因芯片结果显示，骨癌痛大鼠脊髓内趋化因子CXCL13的表达在模型建立后7、14和21天的表达均明显增高。结合相关研究背景，我们推测脊髓内CXCL13的表达上调可能参与骨癌痛发生和吗啡镇痛调节。本课题拟通过免疫荧光、western、IP等分子生物学方法检测CXCL13及其受体CXCR5在骨癌痛大鼠脊髓内的表达情况、以及趋化因子受体CXCR5与阿片受体MOR的共表达和相互作用；通过行为学检测观察CXCL13在骨癌痛大鼠疼痛形成中的作用，并检测其对吗啡镇痛效果的影响；最后探讨CXCL13参与骨癌痛发生和吗啡镇痛调节所涉及的下游信号通路的活化及其相互关系。本课题将为探讨骨癌痛的发生机制提供新的方向，并为骨癌痛的治疗提供新的靶点。

目的：研究表明，中枢神经系统内趋化因子的表达上调与骨癌痛的发生有关。本课题分别研究了趋化因子CXCL13在骨癌痛发生中的作用、CXCL13对吗啡镇痛作用的影响以及其下游机制。.方法：通过von frey丝测定大鼠机械痛阈的变化，通过western blot, real-time PCR和免疫荧光检测了趋化因子CXCL13和受体CXCR5以及信号通路蛋白（p-p38，p-ERK，p-AKT）的表达变化，并确定CXCL13和CXCR5在神经细胞的定位。.结果：在建模后7,14,21天后，骨癌痛大鼠脊髓内CXCL13蛋白和mRNA的表达均明显上调，CXCL13表达于神经元内，而CXCR5表达于星形胶质细胞，CXCL13通过受体CXCR5参与骨癌痛的发生过程，是癌痛条件下的一个致痛因素，此外，脊髓内CXCL13的表达上调可对抗阿片类药物-吗啡的镇痛作用。此作用通过调节p38, AKT和ERK信号蛋白磷酸化水平来实现，CXCL13可使p38, AKT和ERK磷酸化水平明显增加，而吗啡可使这些信号蛋白的磷酸化水平明显降低，CXCL13可对抗吗啡诱导的p38, AKT和ERK活化抑制。.结论：CXCL13通过激活p38, AKT和ERK信号通路参与骨癌痛的发生过程，CXCL13与吗啡对p38, AKT和ERK信号通路作用相反，并能对抗吗啡的镇痛作用，其可能机制是通过阿片受体的异源性脱敏，本研究为骨癌痛的治疗提供了新的研究思路和靶点。