鞘内注射钩吻素子对骨癌痛小鼠行为学及脊髓背角胶质细胞活化的影响  

Effects of intrathecal injection of koumine on behavior and activation of glial cells in spinal dorsal horn of mice with bone cancer pain

作　　者：张文杰 杨成伟 韩明明 康芳 李娟[2] 黄祥 ZHANG Wenjie;YANG Chengwei;HAN Mingming;KANG Fang;LI Juan;HUANG Xiang(Graduate School of Wannan Medical College,Wuhu 241002,China)

机构地区：[1]皖南医学院研究生院,芜湖市241002 [2]中国科学技术大学附属第一医院麻醉科

出　　处：《临床麻醉学杂志》2022年第8期851-856,共6页Journal of Clinical Anesthesiology

基　　金：国家自然科学基金青年项目(82101289);中央高校基本科研业务费专项资金(WK9110000169)。

摘　　要：目的 探究鞘内注射钩吻素子对骨癌痛(BCP)小鼠行为学和脊髓背角胶质细胞活化的影响。方法 实验一:选择雄性SPF级C57BL/6小鼠36只,4~6周龄,体重18~22 g。采用随机数表法将小鼠分为两组:假手术1组(S_(1)组)和BCP_(1)组,每组18只。S_(1)组向右侧股骨远端骨髓腔注入PBS溶液10μl,BCP_(1)组向右侧股骨远端骨髓腔接种含有2×10~5个Lewis肺癌细胞的PBS溶液10μl建立BCP模型。于建模前1 d、建模后4、7、10、14、21 d每组随机取6只小鼠测定含机械缩足阈值(MWT)和热缩足潜伏期(TWL)。于建模后7、14、21 d完成行为学测试后每组随机处死6只小鼠,取小鼠脊髓腰膨大处,采用Western blot法检测脊髓星形胶质细胞标记蛋白(GFAP)和小胶质细胞标记蛋白(Iba-1)含量。取小鼠右侧股骨行HE染色进行骨组织病理学检查。实验二:选择雄性SPF级C57BL/6小鼠54只,4~6周龄,体重18~22 g。采用随机数表法将小鼠分为三组:假手术2组(S_(2)组)、BCP_(2)组和BCP+钩吻素子组(K组),每组18只。S_(2)组和BCP_(2)组处理同S_(1)组和BCP_(1)组,K组向右侧股骨远端骨髓腔接种含有2×10~5个Lewis肺癌细胞的PBS溶液10μl建立BCP模型,建模后14 d向小鼠L_(4-5)或L_(5-6)间隙缓慢鞘内注射含钩吻素子30μg的20%DMSO溶液5μl。给药前1 h、给药后1、2、3 h每组随机取6只小鼠测定MWT和TWL。给药后1 h时每组随机处死12只小鼠,采用Western blot法检测脊髓GFAP和Iba-1蛋白含量,ELISA法检测脊髓IL-6、IL-1β和TNF-α浓度。结果 实验一:与S_(1)组比较,BCP_(1)组建模后7、10、14、21 d MWT明显降低、TWL明显缩短(P<0.05),建模后7、14、21 d脊髓GFAP和Iba-1蛋白含量均明显升高(P<0.05)。HE染色示S_(1)组无骨质破坏,骨髓腔内为正常骨髓细胞;BCP_(1)组骨质遭破坏,骨小梁、骨髓腔和周围软组织内大量Lewis肺癌细胞浸润。实验二:与S_(2)组比较,BCP_(2)组给药前1 h、给药后1、2、3 h MWT均明显降低、TWL均明显缩短Objective To investigate the effects of intrathecal injection of koumine on behavior and activation of glial cell in spinal dorsal horn of mice with bone cancer pain(BCP).Methods Experiment 1:Thirty-six male SPF C57 BL/6 mice,aged 4-6 weeks,weighing 18-22 g,were selected.The mice were randomly divided into two groups:sham operation group 1(group S_(1)) and group BCP_(1),18 mice in each group.In group S_(1),PBS solution 10 μl was injected into the bone marrow cavity of right distal femur.In group BCP_(1),PBS solution 10 μl containing 2 × 10~5 Lewis lung cancer cells was injected into the bone marrow cavity of right femur to establish BCP mouse model.Six mice in group S_(1) and group BCP_(1) were randomly selected to determine the mechanical withdrawal threshold(MWT) and thermal withdrawal latency(TWL) 1 day before modeling,4,7,10,14,and 21 days after modeling.Six mice were sacrificed randomly after the behavioral tests completed 7,14 and 21 days after modeling,and the expressions of astrocyte marker protein(GFAP) and microglial marker protein(Iba-1) in the spinal cord were detected by Western blot.The right femur of the mice was taken for HE staining.Experiment 2:Fifty-four male SPF C57 BL/6 mice,aged 4-6 weeks,weighing 18-22 g,were selected.The mice were randomly divided into three groups:sham operation group 2(group S_(2)),group BCP_(2) and BCP+koumine group(group K),18 mice in each group.PBS solution 10 μl was injected into the bone marrow cavity of group S_(2),and PBS solution 10 μl containing 2 × 10~5 Lewis lung cancer cells was injected into the bone marrow cavity of right distal femur to establish BCP mouse model in group BCP_(2).In group K,20% DMSO solution 5 μl containing koumine 30 μg was slowly injected into the L_(4-5) or L_(5-6) 14 days after establishment of BCP mouse model,and 6 mice were randomly selected to determine MWT and TWL 1 hour before administration,1,2,and 3 hours after administration.Twelve mice were randomly sacrificed 1 hour after administration.The expressions of GFAP and Iba-

关 键 词：骨癌痛 钩吻素子 星形胶质细胞 小胶质细胞 小鼠 

分 类 号：R285.5[医药卫生—中药学]