NMDA受体和NOS参与骨癌痛小鼠吗啡耐受的形成

目的：制备骨癌痛模型，并在骨癌痛模型上模拟慢性吗啡耐受的产生，以探讨癌痛吗啡耐受的表现及机制。方法：选用40只成年雄性C57BL/6小鼠，其中20只接种Lewis肺癌细胞2×106个建立骨癌痛模型，于接种后的第15天将骨癌痛组再随机分为模型吗啡组与模型盐水组。另外20只正常小鼠完全随机分为正常吗啡组与正常盐水组。模型吗啡组和正常吗啡组小鼠皮下注射吗啡10 mg/kg，每天2次, 连续7 d, 建立慢性吗啡耐受模型，模型盐水组和正常盐水组按体质量给予等体积生理盐水。从给药的第1天开始，隔天上午给药后1 h采用机械触痛法测量小鼠的50%缩足反应阈值，以此机械触痛阈值作为疼痛指标。第7天处死小鼠分别作脊髓NMDA受体亚单位1型(NMDAR1)免疫组织化学检测和脊髓一氧化氮合酶（NOS）的定量测定。 结果：第1,3,5,7天给药后1 h行为学测试结果发现，模型盐水组和正常盐水组小鼠整个过程中50%缩足反应阈值没有明显变化。模型吗啡组第1，3，5天50%缩足反应阈值与模型盐水组相比，差异有统计学意义(P＜0.05)；模型吗啡组第5天的50%缩足反应阈值与第1, 3天比较明显变小(P＜0.05)；第7天模型吗啡组与模型盐水组相比，差异无统计学意义（P＞0.05）。正常吗啡组第1，3，5天50%缩足反应阈值与正常盐水组相比，差异有统计学意义(P＜0.05)；第7天正常吗啡组与正常盐水组相比，差异无统计学意义（P＞0.05）。模型吗啡组的NMDAR1染色灰度值与正常吗啡组、正常盐水组及模型盐水组相比，差异有统计学意义(P＜0.05)；模型盐水组的NMDAR1染色灰度值与正常盐水组相比，差异有统计学意义(P＜0.05)。模型吗啡组、模型盐水组以及正常吗啡组的积分光密度(IOD)值分别与正常盐水组相比，差异均有统计学意义(P＜0.05)；模型吗啡组的IOD值与模型盐水组相比，差异有统计学意义(P＜0.05)。模型吗啡组、模型盐水组以及正常吗啡组脊髓NOS含量与正常盐水组相比，差异均有统计学意义(P＜0.05)，模型吗啡组脊髓NOS含量与模型盐水组相比，差异有统计学意义(P＜0.05)。结论： 脊髓NMDA受体和NOS可能在癌痛模型吗啡耐受中起重要作用。

NMDA receptor and NOS in morphine tolerance in mice with bone cancer

ObjectiveTo set up a mouse model with bone cancer to simulate the morphine tolerance and explore its mechanism. MethodsForty C57BL/6 male mice were divided into 4 groups: Group 1 and Group 2 were firstly set up as bone cancer pain models. Morphine (10 mg/kg) was sequentially administered subcutaneously twice daily in Group 1 and normal saline was administered in Group 2 as the control group. Similar to Group 1 and Group 2, morphine (10 mg/kg) was administered subcutaneously twice daily in Group 3 and normal saline was administered in Group 4 as the control group. To set up morphine tolerance model, we injected morphine continuously for 7 days. From Day 1 to Day 7 after the morphine injection, we measured the mice hind paw withdrawal threshold in the von Frey hair test every other day. NMDA receptor 1 (NMDA1) and nitric oxide synthase (NOS) were measured on Day 7 after the morphine injection. ResultsThe mice hind paw withdrawal threshold in the von Frey hair test in Group 1 increased on Day 1,3, and 5 after the morphine injection compared with the paw withdrawal threshold in Group 2 and had the same threshold as Group 2 on Day 7. The mice hind paw withdrawal threshold in the von Frey hair test in Group 3 increased on Day 1,3, and 5 after the morphine injection compared with the paw withdrawal threshold in Group 4 and had the same threshold as Group 4 on Day 7. The grey scales and integral optical density (IOD) of NMDAR1 and the level of NOS in the spinal dorsal horn were higher in Group 1, Group 2, and Group 3 compared with those in Group 4（P＜0.05 or P＜0.01）, and the grey scales and IOD of NMDAR1 in Group 2 was higher than that in Group 1(P＜0.05). ConclusionNMDA receptors and NOS may play important roles in morphine tolerance in mice with bone cancer pain.