摘要
目的 研究下游调控元件拮抗分子DREAM对骨癌痛-吗啡耐受大鼠脊髓谷氨酸转运体-1（GLT-1）表达的影响.方法 健康雌性SD大鼠60只,体质量200 ～ 230 g,按随机数字表法分为癌痛组（CP组,n=48）和假手术组（S组,n=12）.于接种前1 d（T0）,接种后第7天（T1）、14天（T2）、18天（T3）、19天（T4）、21天（T5）、25天（T6）和28天（T7）检测机械痛阈.术后T2～ T3,随机取CP组中36只大鼠行皮下吗啡注射,每日单次剂量依次为10、20、30、40和60 mg/kg,3次/d.T4时,皮下注射吗啡3 mg/kg.术后20 ～ 25 d,依鞘内注射物质不同,将经吗啡处理过的大鼠分为吗啡耐受组（MT组,n=12）、RNAi组（R组,n=12）和载体组（V组,n=12）.术后28 d,取其L4脊髓,检测术侧脊髓DREAM和谷氨酸转运体-1（GLT-1）的表达变化.结果 （1）机械痛阈：与S组比较,CP组、MT组、V组和R组在T1、T3、T5～T7时降低,CP组在T2时降低,MT组、V组和R组在T4时降低;与CP组比较,MT组、V组及R组在T2 ～T3时升高（P＜0.01）,T4时降低（P＜0.01）,R组在T5时降低（P＜0.01）,MT组和V组在T5～T7时降低（P＜0.01）.与MT组和V组比较,R组在T6～T7时升高（P＜0.01）.（2）脊髓DREAM基因mRNA和DREAM蛋白含量：与S组比较,其他各组脊髓DREAM基因mRNA和DREAM蛋白含量均显著增高（P＜0.01）;与CP组比较,MT组和V组表达均增加（P＜0.01）,R组表达减少（P＜0.01）;与MT组和V组比较,R组表达减少（P＜0.01）.（3）GLT-1表达：与S组比较,其他各组GLT-1表达均减少（P＜0.01）;与CP组比较,MV组、V组和R组表达G LT-1表达均减少（P＜0.01）.结论 DREAM参与骨癌痛-吗啡耐受机械痛敏的维持,对脊髓GLT-1的表达未见明显影响.
Objective To investigate the effects of downstream regulatory element antagonist modulator （DREAM） on the expression of glutamate transporter-1 （GLT-1） in spinal cord in rats with bone cancer pain and morphine tolerance.Methods Sixty female healthy Sprague-Dawley （SD） rats weighing 200 ～230 g were randomly divided into tow groups,group Ⅰ cancer pain （CP,n =48） and group Ⅱ Sham （S,n =12）.Cancer pain in each group was produced by inoculation of syngenetic Walker 256 rat mammary gland carcinoma cells （5 × 105） to left tibia.Pain threshold to mechanical stimulus was measured before （baseline） and after the surgical procedure.From 14 d to 18 d after the inoculation of carcinoma cells,36 rats from group CP received subcutaneous injection of morphine at 3 times per day with doses increasingly from 10 mg/kg initially to 20 mg/kg,30 mg/kg,40 mg/kg,and 60 mg/kg.Equal volume of normal saline was applied to the 12 rats left in group CP.On 19th day after the carcinoma cells inoculation once subcutaneous injection of morphine at 3mg/kg was performed in all rats in group CP.From the next day,the rats in group CP ever receiving injections of morphine for 5 days were randomized into thre subgroups,including subgroups morphine tolerance （MT,n =12）,vehicle （V,n =12）,and RNAi （R,n =12）.They were injected intrathecally with 20 μl of normal saline （NS）,10 μl vehicle plus 10 μ1 NS,and 10 μ1 of DREAM-shRNA plus l0 μ1 NS,respectively,once a day for 5 days.Focusing on the affected limb,mechanical pain threshold was measured one day before surgery （T0）,and at day7 （T1）,day 14 （T2）,day 18 （T3）,day 19 （T4）,day21 d （T5）,day 25 （T6）,and day 28 （T7） after surgery.The animals were sacrificed at day 28 after the procedure.The lumbar 4 segments in rats were removed for detection of DREAM and GLT-1.Results The mechanical threshold was significantly decreased at T1 compared to the baseline in all groups,returned to the baseline at T2 ～ T7 in group S,at T4 in group CP,and at T2 in group MT,V,and R,but remained low at T5 ～T7 in group CP,and at T3 ～T7 in group MT,V,and R.Compared to that at T1,it was decreased at T2 ～T3 and T5 ～ T7 in group CP,at T4 ～ T7 in group MT and V,and at T4 ～ T5 in group R,going back to the baseline at T4 in group CP and at T2 in group MT,V and R,and increased at T6 ～ T7 in group R.Compared to that in group S,the mechanical threshold in group CP,MT,V and R was decreased,and lower at T2 in group CP and at T4 in group MT,V and R.Compared to that in group CP,the mechanical threshold was significantly higher at T2 ～ T3 but lower at T4 in group MT,V,and R,decreased at T5 in group R and at T5 ～ T7 in group MT and V.The mechanical threshold was increased at T6 ～ T7 in group R and higher than that in groups MT and V.The expression of DREAM,compared to that in group S,was down-regulated in other groups.Compared to group CP,increment was shown in groups MT and V,and decrease was exhibited in group R.It was cut down in group R compared to that in groups MT and V.Compared to group S,GLT-1 was decreased in other groups.It was down-regulated in groups MT,V and R compared to group CP.Conclusions DREAM is involved in the development of allodynia after morphine tolerance in rats with bone cancer pain.No evidence in this study supports a link between DREAM and GLT-l in spinal cord.
出处
《中国医师杂志》
CAS
2014年第12期1593-1597,共5页
Journal of Chinese Physician
基金
浙江省自然科学基金（Y2091107）
关键词
基因表达调控
肿瘤
骨肿瘤/并发症
疼痛/病因学
吗啡/药理学
药物耐受性
谷氨酸/代谢
Gene expression regulation, neoplastic
Bone neoplasms/complications
Pain/etiology
Morphine/pharmacology
Drug tolerance
Glutamic acid/metabolism
