CAJ | 학술논문

目的探讨大麻素CB2受体在小鼠骨癌痛形成中的作用.方法将C3H/HeJ小鼠84只以数字随机法分为肿瘤组(T组,n =30)、给药组(J组,n=12)、溶媒组(D组,n=12)和假手术组(S组,n =30).将含2×105个NCTC2472纤维肉瘤细胞的混悬液(含最低有效培养基α-MEM)注射到C3H/HeJ小鼠右侧股骨远端骨髓腔制作骨癌痛模型,S组小鼠注入不含肿瘤细胞的α-MEM.所有小鼠均于接种前及接种后第5、7、10、14天检测疼痛行为学指标,包括机械缩足阈值(PWMT)和热缩足潜伏期(PWTL).J组和D组分别于鞘内注射2μg大麻素CB2受体激动剂JWH015和4％二甲基亚砜(DMSO),注药体积均为5.0μl.两组于鞘内注射后1、6、12、24、48和72 h测定痛行为学.选取接种后5、7、10、14 d和给药后12 h的各组小鼠提取脊髓腰膨大进行免疫印迹检测CB2表达水平,取材均于行为学测定后进行.结果 (1)行为学测定:小鼠于接种后7d,PWMT为(1.27±0.28)g(P＜0.05),此后逐渐下降,于接种后14 d达到(0.53±0.20)g,鞘内注射JWH015后6 h PWMT升高至(1.00±0.20)g(P＜0.05),12 h达到高峰,PWMT为(1.40±0.39)g,48 h后逐渐衰减,72 h回复至给药前水平；小鼠于接种后10 d出现热痛敏,PWTL为(16.9±0.4)s(P＜0.05),接种后14 d为(11.5±0.7)s,鞘内注射JWH015后6 hPWTL回升至(15.7±1.9) g(P＜0.05),12 h达到(18.6±2.3)g,72 h回复至给药前水平(12.8±0.6)g(P＞0.05).(2) Western印迹结果:从接种后5d开始,CB2与β-肌动蛋白比值逐渐增高.与接种后5 d CB2/β-肌动蛋白(0.190±0.010)相比,接种后14 d比值增加到0.660±0.010 (P ＜0.05)；与D组0.903±0.006比较,鞘内注射JWH015后12hJ组CB2/β-肌动蛋白降低至0.510±0.010 (P ＜0.05).结论大麻素CB2受体在骨癌痛形成中具有重要作用.
목적 탐토대마소CB2수체재소서골암통형성중적작용.방법 장C3H/HeJ소서84지이수자수궤법분위종류조(T조,n =30)、급약조(J조,n=12)、용매조(D조,n=12)화가수술조(S조,n =30).장함2×105개NCTC2472섬유육류세포적혼현액(함최저유효배양기α-MEM)주사도C3H/HeJ소서우측고골원단골수강제작골암통모형,S조소서주입불함종류세포적α-MEM.소유소서균우접충전급접충후제5、7、10、14천검측동통행위학지표,포괄궤계축족역치(PWMT)화열축족잠복기(PWTL).J조화D조분별우초내주사2μg대마소CB2수체격동제JWH015화4％이갑기아풍(DMSO),주약체적균위5.0μl.량조우초내주사후1、6、12、24、48화72 h측정통행위학.선취접충후5、7、10、14 d화급약후12 h적각조소서제취척수요팽대진행면역인적검측CB2표체수평,취재균우행위학측정후진행.결과 (1)행위학측정:소서우접충후7d,PWMT위(1.27±0.28)g(P＜0.05),차후축점하강,우접충후14 d체도(0.53±0.20)g,초내주사JWH015후6 h PWMT승고지(1.00±0.20)g(P＜0.05),12 h체도고봉,PWMT위(1.40±0.39)g,48 h후축점쇠감,72 h회복지급약전수평；소서우접충후10 d출현열통민,PWTL위(16.9±0.4)s(P＜0.05),접충후14 d위(11.5±0.7)s,초내주사JWH015후6 hPWTL회승지(15.7±1.9) g(P＜0.05),12 h체도(18.6±2.3)g,72 h회복지급약전수평(12.8±0.6)g(P＞0.05).(2) Western인적결과:종접충후5d개시,CB2여β-기동단백비치축점증고.여접충후5 d CB2/β-기동단백(0.190±0.010)상비,접충후14 d비치증가도0.660±0.010 (P ＜0.05)；여D조0.903±0.006비교,초내주사JWH015후12hJ조CB2/β-기동단백강저지0.510±0.010 (P ＜0.05).결론 대마소CB2수체재골암통형성중구유중요작용.
Objective To explore the effects of cannabinoid 2 receptor (CB2) in the development of bone cancer pain in mice.Methods A total of 84 mice (C3H/HeJ) were randomly divided into 4 groups:tumor group ( Group T,n =30),medication administration group ( Group J,n =12),vehicle group ( Group D,n =12) and sham group ( Group S,n =30).And 2 × 105 osteolytic NCTC2472 cells in α-MEM were injected into medullary cavity of right distal femur to induce bone cancer pain in a murine model while sham mice received an injection of only α-MEM.All mice were tested for pain-related behaviors at preinoculation and at Days 5,7,10 and 14 post-inoculation.The tests included paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency ( PWTL).Group J and Group D were injected intrathecally with 2 μg JWH015 dissolved in 4％ DMSO and only 4％ DMSO respectively in a 5 μ1.volume.Pain behavior tests were performed before and at 1,6,24,48 and 72 h after an intrathecal injection.Lumbar intumescentia of mice in each group were harvested to examine the expression level of CB2by Western blot after pain behavior tests at Days 5,7,10 and 14 post-inoculation and 12 h after an intrathecal injection.Results (1) Pain behavior tests:Mechanical allodynia appeared at Day 7 postinoculation.The value of PWMT was ( 1.27 ± 0.28 ) g ( P ＜ 0.05 ) and it declined gradually to ( 0.53 ± 0.20) g at Day 14.The threshold of mechanical hyperalgesia increased to ( 1.00 ±0.20) g at 6 h after an intrathecal injection of JWH015,peaked at (1.40 ± 0.39) g at 12 h,became alleviated after 48 h and recovered to the pre-dosing levels at 72 h.Thermal hyperalgesia appeared at Day 10 post-inoculation.The value of PWTL was (16.9±0.4) s (P＜0.05) at Day 10 and declined to (11.5 ±0.7) s at Day 14 postinoculation.The threshold of thermal hyperalgesia increased to ( 15.7 ± 1.9 ) g at 6 h after an intrathecal injection of JWH015,peaked at ( 18.6 ± 2.3 ) g at 12 h,became alleviated after 48 h and recovered to the pre-dosing levels at 72 h.(2) Western blot:From Day 5 post-inoculation,the ratio of CB2/β-actin increased gradually.Compared with the ratio of 0.190 ± 0.010 at Day 5 post-inoculation,the ratio of CB2/β-actin increased to 0.660 ± 0.010 at Day 14 post-inoculation ( P ＜ 0.05 ) ; compared with the ratio of 0.903 ±0.006 in group D at 12 h after an intrathecal injection of JWH015,the ratio of CB2/β-actin 0.510 ±0.010significantly decreased (P ＜ 0.05).Conclusion The cannabinoid 2 receptor plays an important role in the formation of bone cancer pain.