鞘内注射DREAM—shRNA对骨癌痛-吗啡耐受大鼠痛觉过敏的影响  

Effects of intrathecal DREAM-shRNA on hyperaigesia in morphine-tolerant rats with bone cancer pain

作　　者：于鹏[1] 王晶[2] 程智刚[3] 王云姣[3] 周清河[1] 肖旺频[1] 姚明[4] 吴城[1] 

机构地区：[1]嘉兴学院附属第二医院麻醉科,314000 [2]嘉兴学院附属第二医院康复医学中心,314000 [3]中南大学湘雅医院麻醉科 [4]嘉兴学院附属第一医院疼痛科

出　　处：《中华麻醉学杂志》2013年第11期1353-1358,共6页Chinese Journal of Anesthesiology

基　　金：浙江省自然科学基金（Y2091107）

摘　　要：目的评价鞘内注射转录因子下游调控元件拮抗分子．短发夹RNA（DREAM—shRNA）对骨癌痛．吗啡耐受大鼠痛觉过敏的影响。方法健康雌性sD大鼠，体重200-230g，进行鞘内置管术。取鞘内置管成功的大鼠60只，采用随机数字表法，将其分为5组（n=12）：假手术组（s组）、骨癌痛组（P组）、骨癌痛＋吗啡耐受组（PM组）、骨癌痛＋吗啡耐受＋空白载体组（PMB组）、骨癌痛＋吗啡耐受＋RNAi组（PMR组）。左胫骨上段骨髓腔注射Walker256乳腺癌细胞5μl（1×10^5个／μl）建立大鼠骨癌痛模型。PM组、PMB组和PMR组大鼠于造模后第14～18天时皮下注射吗啡，每日单次注射剂量依次为10、20、30、40和60mg／kg，3次／d，第19天时皮下注射吗啡3mg／kg。造模后第20天时PM组鞘内注射生理盐水10μl，PMB组鞘内注射慢病毒空白载体5μl＋生理盐水5μl，PMR组鞘内注射DREAM-shRNA5μl＋生理盐水5μl，1次／d，连续5d。分别于造模前1d（T0）、造模后7d（T1）、14d（T2）、18d（T3）、19d（T4）、21d（T5）、25d（T6）和28d（T7）时，记录自发性缩足反射次数和后肢跛行程度评分，测定热刺激缩足反应潜伏期（PWTL）和机械刺激缩足反射阈（PWMT）。造模后第28天痛行为学测定结束后，处死大鼠，取L4脊髓组织，观察绿色荧光蛋白（GFP）表达，测定DREAMmRNA及其蛋白的表达。结果与s组比较，P组T2-3,5-7时，PM组、PMV组和PMR组L4-6时自发性缩足反射次数增加，后肢跛行程度评分降低，P组T1-3,5-7时，PM组、PMV组和PMR组T1,3-7时PWMT降低，P组、PM组、PMV组和PMR组脊髓DREAMmRNA及其蛋白表达上调（P〈0．01）；与P组比较，PM组和PMV组自发性缩足反射次数T2-3时减少，T4时增加，后肢跛行程度评分T2-3时升高，T4时降低，PMR组自发性缩足反射次数T2-3,6-7时减少，T4时增加，后肢跛行程度评分T2-3,6-7时升高，T4时降低，PM组和PMV组PWObjective To evaluate the effects of intrathecal DREAM-short hairpin RNA （shRNA） on hy- peralgesia in morphine-tolerant rats with bone cancer pain （BCP）. Methods Sixty healthy female Sprague-Dawley rats, weighing 200-230 g, in which intrathecal catheters were successfully implanted without complications, were randomly divided into 5 groups （n = 12 each） using a random number table： sham operation group （group S）, BCP group （group P）, BCP ＋ morphine tolerance group （group PM）, BCP ＋ morphine tolerance ＋ vehicle group （group PMV） and BCP ＋ morphine tolerance ＋ RNAi group （group PMR）. Bone cancer pain was induced by injection of breast cancer cells （Walker 256） 5μl （1× 10^5μl） into upper segment of bone marrow of the left tibia. Morphine tolerance was induced by subcutaneous injection of morphine three times a day for 5 consecutive days starting from 14th day after intra-tibia injection in PM, PMV and PMR groups. The single bolus of morphine was 10, 20, 30, 40 and 60 mg/kg from 14th day to 18th day, respectively. Morphine 3 mg/kg was injected subcu- taneously on 19th day after intra-tibia injection in PM, PMV and PMR groups. On 20th day after intra-tibia injec- tion, normal saline 10 μl, vehicle 5 μl ＋ normal saline 5μl and DREAM-shRNA 5μl ＋ normal saline 5 μl were injected intrathecally once a day for 5 consecutive days in PM, PMB and PMR groups, respectively. The frequency of spontaneous paw withdrawal was recorded and the extent of hind hmb claudication when the rats walked was scored and recorded, and thermal paw withdrawal latency （TWL） and mechanical paw withdrawal threshold （MWT） were measured 1 day before intra-tibia injection （To ） and at 7, 14, 18 , 19 , 21, 25 and 28 days after intra-tibia injection （T1-7） . The animals were sacrificed after the last measurement of pain threshold at T7 and the L4 segments of the spinal cord were removed for detection of the expression of green fluorescent protein （ GFP）, and DREAM mRNA and

关 键 词：RNA干扰 转录因子 注射 脊髓 骨肿瘤 疼痛 吗啡 药物耐受性 痛觉过敏 

分 类 号：R738[医药卫生—肿瘤]