脊髓CX3CR1在骨癌痛大鼠吗啡耐受形成中的作用

摘要

Objective To observe changes of CX3 CR1 protein expression in spinal dorsal horn of morphine tolerant rats with bone cancer pain,so as to explore the role of spinal CX3CR1 in the process of morphine tolerance in the rats,as well as interaction between CX3CR1 and microglia. Methods Forty-eight female adult Sprague-Dawley rats were randomly divided into four groups(n = 12) : control( C) group , sham operation( S) group, bone cancer pain-morphine tolerance( BM) group and BM+ minocycline(BM+m)group. The rats in S group received sham operation of tibia. In BM group tibial cancer pain models were established by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells , and 9 days later the rats were intrathecally administered morphine 20 μg/kg twice daily for 7 days. The rats in BM+m group were intrathecally administered minocycline 250 μg/kg for three days following the same treatment in BM group. The mechanical withdrawal threshold(MWT)and mechanical withdrawal duration( MWD) of all the rats were respectively measured pre-operation and at postoperative day( POD) 3 , 6 , 9 ,12,15,18. The rats were killed at POD 18 and the expression of CX3CR1 protein and OX-42 in spinal lumber 4-6 tissues was respectively detected by using Western blot and immunohistochemistry methods. Results Stable morphine tolerance models were established in BM group at 7th day after intrathecal administration of morphine. The MWT was lower,and MWD was higher in BM group than those in C group and S group(all P＜0. 01). There was statistically significant difference in MWT and MWD between BM group and BM+m group at POD 18(all P＜0. 05). The expression of CX3CR1 protein and OX-42 in BM group was obviously higher than that in C group and S group(all P＜0. 01)after morphine tolerance formed. The expression of CX3CR1 protein and OX-42 in BM+m group was significantly decreased after intrathecal administration of minocycline(all P＜0. 05). Conclusion Spinal CX3CR1 may play an important role in the development of morphine tolerance in rats with bone cancer pain,and its expression is elevated by activating microglia.%目的 观察骨癌痛-吗啡耐受大鼠脊髓背角CX3C趋化因子受体1(CX3CR1)蛋白表达的变化,探讨CX3CR1在骨癌痛大鼠吗啡耐受形成中的作用及其与小胶质细胞之间的关系.方法 成年雌性SD大鼠48只,随机分为4组(n=12):对照组(C组)、假手术组(S组)、骨癌痛-吗啡耐受组(BM组)、BM+米诺环素组(BM+m组).C组不作任何处理;S组大鼠仅手术暴露右侧胫骨上段;BM组大鼠右侧胫骨上段骨髓腔注入Walker 256乳腺癌细胞10 μL(4×105个细胞/mL)制作骨癌痛模型,第9天开始鞘内给予20 μg/kg吗啡,2次/d,连续7 d;BM+m组在BM组处理方法的基础上,手术后第16天再连续3 d鞘内注射米诺环素250 μg /kg.分别于术前,术后第3、6、9、12、15、18天测定大鼠机械缩足阈值(MWT)和机械缩足持续时间(MWD),各组于术后18 d处死大鼠,取脊髓L4～6节段组织,采用Western blot法测定CX3CR1蛋白表达,免疫组化法检测小胶质细胞标记物OX-42水平.结果 BM组大鼠在鞘内给予吗啡第7天(即术后第15天)形成较稳定的吗啡耐受,表现为MWT值下降,明显低于C组和S组(均P＜0.01),而MWD上升,明显高于C组和S组(均P＜0.01),术后第18天两指标与BM+m组比较差异也有统计学意义(均P＜0.05).BM组大鼠吗啡耐受形成后脊髓背角CX3CR1蛋白和OX-42表达明显高于C组和S组(均P＜0.01),而BM+m组注射米诺环素后脊髓CX3CR1蛋白和OX-42表达低于BM组(均P＜0.05).结论 脊髓CX3CR1可能在骨癌痛大鼠慢性吗啡耐受的形成中发挥重要作用,小胶质细胞活化使脊髓CX3CR1蛋白表达上调.