脊髓背角小胶质细胞组织蛋白酶S在大鼠骨癌痛维持中的作用

Role of cathepsin S in spinal microglia in maintenance of bone cancer pain in rats

目的 探讨脊髓背角小胶质细胞组织蛋白酶S（CatS）在大鼠骨癌痛维持中的作用.方法 雌性未交配SD大鼠50只,4～6周龄,体重150～ 180 g,采用随机数字表法,将其分为5组（n=10）：假手术组（S组）、骨癌痛组（BCP组）、假手术＋CatS抑制剂吗啉亮氨酸高苯丙氨酸乙烯基苯基砜（LHVS）组（S＋L组）、骨癌痛＋二甲基亚砜组（BCP＋D组）和骨癌痛＋LHVS组（BCP＋L组）.左侧胫骨骨髓腔内接种浓度为2×107/ml Walker256细胞5μl制备大鼠骨癌痛模型.分别于造模后10、11、12d时S＋L组和BCP＋L组鞘内注射LHVS 50 nmol/10l,1次/d,BCP＋D组给予等容量二甲基亚砜.分别于造模前1d（基础状态）及造模后3、6、9、10、11、12 d（T0-6）测定机械缩爪反应阈（MWT）,分别于鞘内给药前、鞘内给药后0.5、1.0、3.0、6.0、9.0、12.0、24.0 h时测定（MWT）.处死大鼠,取L4-6脊髓组织,采用免疫组化法测定OX-42表达.结果 与S组比较,BCP组、BCP＋D组和BCP＋L组T2-6时MWT降低,脊髓背角OX-42表达上调（P＜0.01）,S＋L组MWT和脊髓背角OX-42表达差异无统计学意义（P＞0.05）；与BCP组比较,BCP＋L组T4-6时MWT升高,脊髓背角OX-42表达下调（P＜0.01）,BCP＋D组MWT和脊髓背角0X-42表达差异无统计学意义（P＞0.05）.S＋L组和BCP＋D组鞘内给药后3.0、6.0、9.0h时MWT低于BCP＋D组（P＜0.01）.结论 脊髓背角小胶质细胞CatS激活参与了大鼠骨癌痛的维持.

Objective To investigate the role of cathepsin S （CatS） in spinal microglia in the maintenance of bone cancer pain （BCP） in rats.Methods Fifty unmated female Sprague-Dawley rats,aged 4-6 months,weighing 150-180 g,were randomly divided into 5 groups with 10 rats in each group：sham operation group （group S） ; group BCP; sham operation ＋ CatS inhibitor morpholinurea-leucine-homophenylalanine-vinyl phenyl sulfone （LHVS） group （group S ＋ L）; BCP ＋ dimethyl sulfoxide （DMSO） group （group BCP ＋ D）; BCP ＋ LHVS group （group BCP ＋ L）.BCP was induced by inoculating 2 × 107/ml Walker256 mammary gland carcinoma cells 5 μl into the medullary cavity of the left tibia,while in S and S ＋ L groups,Hank＇ s solution 5μl was injected into left tibia instead of Walker256 cells.At 10,11 and 12 days after inoculation,the rats in S ＋ L and BCP ＋ L groups received an intrathecal injection of LHVS 50 nmol/10μl,and the rats in group BCP ＋ D received an intrathecal injection of 5 ％ DMSO 10 μl.Mechanical paw withdrawal threshold （MWT） was measured at 1 day before inoculation （baseline） and 3,6,9,10,11 and 12 days after inoculation （T0-6）.MWT was measured before intrathecal administration and at 0.5,1.0,3.0,6.0,9.0,12.0 and 24.0 h after intrathecal administration.The rats were sacrificed and the L4-6 segments of the spinal cord were removed for determination of the expression of OX-42 in the spinal dorsal horn by immunohistochemistry.Results Compared with group S,MWT was significantly decreased at T2-6 and the expression of OX-42 in spinal dorsal horn was up-regulated in BCP,BCP ＋ D and BCP ＋ L groups （P ＜ 0.01）,and no significant changes in MWT and expression of OX-42 in spinal dorsal horn were found in S ＋ L group （P ＞ 0.05）.Compared with group BCP,MWT was significantly increased at T4-6 and the expression of OX-42 in spinal dorsal horn was down-regulated in group BCP＋ L （P ＜ 0.01）,and no significant change in MWT and the expression of OX-42 in spinal dorsal horn was found in group BCP ＋ D （P ＞ 0.05）.MWT was significantly decreased at 3.0,6.0 and 9.0 h after intrathecal administration in S ＋ L and BCP ＋ D groups as compared with BCP ＋ D group （P ＜ 0.01）.Conclusion Activation of CatS in spinal microglia is involved in the maintenance of BCP in rats.