脊髓背角γ氨基丁酸转运体-1在大鼠骨癌痛中的作用

Role of gamma-aminobutyric acid transporter-1 in the spinal cord in a rat model of bone cancer pain

目的评价脊髓背角γ-氨基丁酸转运体-1（GAT-1）在大鼠骨癌痛中的作用。方法清洁级健康雌性SD大鼠80只，体重150～180g，采用随机数字表法，将其随机分为5组（n=16）：假手术组（Ⅰ组）；骨癌痛组（Ⅱ组）采用胫骨上段骨髓腔接种Walker-256乳腺癌细胞的方法制备大鼠骨癌痛模型；假手术＋GAT-1选择性抑制剂NO-711组（Ⅲ组）和骨癌痛＋NO-711（Ⅳ组）于术后第14天鞘内注射NO-71120μg，1次／d，连续3d；骨癌痛＋生理盐水组（V组）于术后第14天鞘内注射10μl生理盐水，1次／d，连续3d。于术前1d、术后第3、5、7、10、14、16天时测定大鼠机械痛阈，术后第16天机械痛阚测定后处死大鼠，取腰段脊髓，采用Westernblot法检测脊髓GAT-1的表达，采用免疫荧光双标法观察Ⅰ组和Ⅱ组大鼠患侧脊髓GAT-1和星形胶质细胞标志物胶质纤维酸性蛋白（GFAP）免疫反应阳性产物的共表达。结果与Ⅰ组和Ⅲ组比较，Ⅱ组、Ⅳ组和Ⅴ组术后第7—16天机械痛阈降低，Ⅱ组和V组术后GAT-1表达上调（P〈0．05）；与Ⅱ组和V组比较，Ⅳ组术后第16天鞘内给药后机械痛阈升高，脊髓背角GAT-1表达下调（P〈0．05）；与Ⅰ组比较，Ⅱ组患侧脊髓GFAP和GAT-1共表达增加（P〈0．05）。结论脊髓背角GAT-1的表达上调参与了大鼠骨癌痛的形成与维持，该作用可能与脊髓星形胶质细胞的活化有关。

Objective To investigate the role of gamma-aminobutyric acid transporter-1 （GAT-1） in the spinal cord in a rat model of bone cancer pain. Methods Eighty female SD rats weighing 150-180 g were randomly divided into 5 groups （n = 16 each） ： sham operation group（ group Ⅰ）, bone cancer pain group（ group Ⅱ ）, sham operation ＋ NO-711 group（group Ⅱ）, Ⅳgroup BCP ＋ NO-711 group（group Ⅳ） and BCP ＋ vehicle group （ group Ⅴ）. Bone cancer pain was induced by inoculating Walker-256 mammary gland carcinoma cells into medul- lary cavity of tibia. NO-711 （20 μg, 10μl） was administered intrathecally once a day for 3 consecutive days from the 14th day after operation. Mechanical withdrawl threshold （MWT） of mechanical stimulus was determined the day before operation and at days 3, 5, 7, 10, 14 and 16 after operation. The animals were sacrificed on the 16th day after operation, and then the spinal cords were removed for determination of the expression of GAT- 1 and double immunostaining of GAT-1 and glial fibrillary acidic protein （GFAP, astrocyte marker）. Results MWT were significantly decreased in groups Ⅱ, Ⅳand Ⅴ as compared with groups Ⅰand Ⅲ. The expression of GAT-1 significantly up-regulated in groups Ⅱ, Ⅴas compared with groups Ⅰand Ⅲ, NO-711 significantly increased MWT, while decreased the expression of GAT-1 in group Ⅳcompared with groups Ⅱand Ⅴ. The expression of GAT-1 up-regulation appeared colocalizes with in astrocytes activation in spinal dorsal horn. Conclusion The up-regulation of expression of GAT-1 in spinal cord is involued in the development and maintenance of bone cancer pain, which may be related to the astrocytes activation.