骨癌痛模型大鼠患侧腰段脊髓p38α和p38β的细胞定位及其意义

目的 观察骨癌痛模型大鼠患侧腰段脊髓p38d和p38B的分布及细胞定位,探讨其在骨癌痛中的作用机制。方法 选择雌性SD大鼠20只,随机分为2组,每组10只：A组（对照组）：左侧胫骨上段骨髓腔注入3μl Hanks液;B组（模型组）：左侧胫骨上段骨髓腔注入3μl MADB-106大鼠乳腺癌细胞（4．8×10^3／μl）。术前及术后14d,各组大鼠隔日观察机械痛及辐射热痛阈值变化。第14d,取大鼠脊髓L4～6节段,采用免疫组织化学ABC法和荧光双标法观察骨癌痛模型大鼠患侧腰段脊髓p38α和p38β免疫反应阳性产物的分布变化和细胞定位。结果 A组大鼠对机械、辐射热痛刺激的缩爪阈值在术后各时间点组内相比,差异无统计学意义;B组大鼠对辐射热痛刺激缩爪阈值在术后的前6d与A组相比,差异有统计学意义（P〈0．05）;术后第14d,与A组大鼠对机械痛刺激缩爪阈值和辐射热痛阈值相比,B组差异有统计学意义（P〈0．05）。B组大鼠患侧腰段脊髓背角Ⅰ～Ⅳp38ct和p38B免疫反应吸光值明显高于A组,两组相比差异有统计学意义（均P〈0．05）。荧光双标显示患侧腰段脊髓背角p38d与神经元特异性核蛋白标志物NeuN有共表达,p38B与小胶质细胞标志物OX-42有共表达。结论 脊髓p38d和p38B参与了骨癌痛的发生和发展,p38d主要在脊髓神经元表达,而p38B则在脊髓小胶质细胞中表达。

The cellular location and significance of p38alpha/beta isoforms in the lumbar spinal cord of the bone cancer pain rats

OBJECTIVE: To examine the cellular distribution and location of p38alpha/beta isoforms in the lumbar spinal cord of the bone cancer pain rats. METHODS: Twenty SD female rats weighing 180 - 220 g were randomly divided into 2 groups (n = 10 each): group A (control group): intra-tibial injection of 3 microl Hank's solution; group B (model group): intra-tibial injection of 3 microl MADB-106 mammary gland carcinoma cells of rats (4.8 x 10(3)/microl). Mechanical withdrawal threshold and radiant heat threshold of rats' hind paws were measured every other day from one day before operation until 14 days later. The lumbar 4 ~ 5 spinal cord was removed on the 14th day. The cellular distribution and location of the spinal p38alpha/beta immunoreactivity were detected by immunohistochemistry SABC and double immunofluorescence methods. RESULTS: No significant differences in mechanical withdrawal threshold and radiant heat threshold were found at all time points in group A. During the first 6 days of post-operation there was obvious difference in radiant heat stimulus between group A and B (P < 0.05); on the 14th day after operation, mechanical pain threshold and radiant heat threshold in group B were significantly different from that of group A (P < 0.05). The p38alpha/beta immunoreactivity of group B in laminae I approximately IV of dorsal horn showed stronger staining than group A (P < 0.05). Double immunofluorescence confocal micrographs showed that spinal p38alpha and the neuronal marker neuronal N (NeuN) were colocalized in the dorsal horn, indicating that p38alpha was expressed in neurons. Double immunofluorescence micrographs demonstrated that antibodies againsted p38beta and the microglia marker OX42 labeled the same cell, indicating that p38beta was expressed in microglia. CONCLUSION: Our studies indicate that p38alpha and p38beta are involved in the generation and maintenance of bone cancer pain states. p38alpha is predominantly expressed in neurons, while p38beta is expressed in microglia.