摘要/Abstract
摘要： 目的·研究脊髓趋化因子受体2（chemokine C-C motif receptor 2，CCR2）介导大鼠骨癌痛（bone cancer pain，BCP）维持的机制。方法·选取54只SD大鼠为研究对象，将其分为BCP组、假手术组、BCP+INCB3344（CCR2特异性阻断剂）组和BCP+溶剂对照组。向BCP组大鼠胫骨骨髓腔内注入Walker256乳腺癌细胞，建立骨癌痛模型；假手术组则注射等量的生理盐水。BCP+INCB3344组是在BCP模型建立第14日后，向大鼠鞘内注射INCB3344；BCP+溶剂对照组则注射等量的溶剂。通过检测BCP组大鼠和假手术组大鼠的机械痛阈值来判断鉴定BCP模型是否成功。采用蛋白质印迹检测假手术组和BCP组大鼠脊髓后角CCR2表达情况。通过机械痛行为的测试，观察鞘内给予INCB3344对BCP模型大鼠机械痛阈的影响。通过全细胞膜片钳记录观察BCP组、BCP+INCB3344组和BCP+溶剂对照组大鼠脊髓胶状质层（substantia gelatinosa，SG）神经元自发兴奋性突触后电流（spontaneous excitatory postsynaptic currents，sEPSCs）、α-氨基-3-羟基-5-甲基-4-异唑丙酸（α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid，AMPA）电流和N-甲基-D-天冬氨酸（N-methyl-D-aspartic acid，NMDA）电流间的差异。结果·与假手术组相比，术后14 d时BCP组大鼠的机械痛阈下降且术侧脊髓后角CCR2的表达增加（P0.000，P0.009），而鞘内注射INCB3344 4 h时可显著升高BCP大鼠的机械痛阈（P0.002）。BCP组大鼠SG神经元sEPSCs的频率和幅度、AMPA电流和NMDA电流幅度均高于假手术组（均P0.000），而鞘内给予INCB3344可显著抑制BCP大鼠的上述指标（均PP0.001）和幅度（P0.020）。结论·脊髓后角的CCR2可通过增强AMPA和NMDA受体功能来介导BCP大鼠脊髓后角兴奋性突触传递效能的增强，该作用可能是BCP维持的重要机制。
关键词: 趋化因子受体2, 骨癌痛, 脊髓, 自发兴奋性突触后电流, 胶状质层
Abstract:
Objective · To investigate the mechanism of spinal chemokine C-C motif receptor 2 (CCR2)-mediated maintenance of bone cancer pain (BCP) in rats. Methods · Fifty-four SD rats were divided into BCP group, sham operation group, BCP+INCB3344 (CCR2 specific antagonist) group, and BCP+vehicle control group. Walker256 breast cancer cells were injected into the tibia medullary cavity of rats in the BCP group to establish the BCP model, while the rats in the sham operation group were injected with the same amount of saline. The rats in the BCP+INCB3344 group received intrathecal injection of INCB3344 on the 14th day after the establishment of BCP model, while the BCP+vehicle control group rats were injected with the same amount of vehicle. The mechanical pain thresholds of BCP group rats and sham operation group rats were measured to judge the success of BCP model. The s of CCR2 in the dorsal horn of spinal cord in the sham operation group rats and the BCP group rats were detectedWestern blotting. The effects of intrathecal administration of INCB3344 on the mechanical pain threshold of BCP rats were observedmechanical pain behavior test. Whole-cell patch-clamp recordings were used to investigate the differences of spontaneous excitatory postsynaptic currents (sEPSCs), α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid (AMPA) and N-methyl-D-aspartic acid (NMDA)-induced currents of spinal substantia gelatinosa (SG) neurons of rats in the BCP group, the BCP+INCB3344 group and the BCP+vehicle control group. Results · Compared with the sham operation group, the mechanical pain threshold of BCP group rats reduced significantly on the 14th day after operation (P0.000), and the of CCR2 in ipsilateral spinal cord of BCP group rats increased significantly (P0.009). After intrathecal injection of INCB3344 for 4 h, the mechanical pain threshold of BCP+INCB3344 group rats was significantly increased (P0.002). The frequency and amplitude of sEPSCs and the amplitude of AMPA and NMDA-induced currents in SG neurons of BCP group rats were significantly higher than those of the sham operation group rats (all P0.000), while intrathecal administration of INCB3344 could significantly inhibit the above-mentioned indices in the BCP+INCB3344 group (all PP0.001) and amplitude (P0.020) of sEPSCs in SG neurons in BCP rats. Conclusion · CCR2 expressing in the spinal cord mediates the enhancement of excitatory synaptic transmission efficacy in the spinal dorsal horn of BCP ratsenhancing the functions of AMPA and NMDA receptors, which may be an important mechanism for the maintenance of BCP.
Key words: chemokine C-C motif receptor 2 (CCR2), bone cancer pain (BCP), spinal cord, spontaneous excitatory postsynaptic currents (sEPSCs), substantia gelatinosa (SG)
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