鞘内注射DREAM—shRNA对神经病理性痛大鼠脊髓背角p-CREB表达的影响  

Effects of intrathecal DREAM-shRNA on expression of p-CREB in spinal dorsal horn in a rat model of neuropathic pain

作　　者：于鹏[1] 肖旺频[1] 姚明[1] 程智刚[2] 王云姣[2] 李靖怡[2] 郭曲练[2] 

机构地区：[1]嘉兴学院附属第二医院麻醉科,浙江省嘉兴市314000 [2]中南大学湘雅医院麻醉科,长沙市410008

出　　处：《中华麻醉学杂志》2009年第8期708-711,共4页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金资助项目（30872427）;湖南省自然科学基金资助项目（05JJ40136）

摘　　要：目的探讨鞘内注射转录因子下游调控元件拮抗因子．短发夹RNA（DREAM-shRNA）对神经病理性痛大鼠脊髓背角磷酸化环磷酸腺苷反应元件结合蛋白（p-CREB）表达的影响。方法成年健康雄性SD大鼠，体重280～320g，采用坐骨神经慢性压迫（CCI）法建立大鼠神经病理性痛模型。于CCI后第3天鞘内置管。取鞘内置管成功的大鼠24只，随机分为4组，每组6只，假手术组（S组）：仅暴露坐骨神经，不结扎；神经病理性痛组（NP组）：于CCI后第8天鞘内注射生理盐水10出；RNA干扰组（RNAi组）：于CCI后第8天鞘内注射DREAM—shRNA5μl和生理盐水5μl；空白载体组（BV组）：于CCI后第8天鞘内注射慢病毒空白载体5μl和生理盐水5μl，各组连续注射7d。于CCI前1d（T0，基础状态）、CCI后第7～14天（T1-8）时测定机械痛阈。于CCI后第15天时测定脊髓背角绿色荧光蛋白（GFP）和p-CREB的表达水平。结果与基础值比较，各组各时点机械痛阈降低（P〈0．05或0．01）；与T1时比较，NP组和BV组T5-8时机械痛阈降低，RNAi组B时机械痛阈升高（P〈0．05或0．01）；与S组比较，NP组和BV组机械痛阈降低，RNAi组R时机械痛阈降低，B时升高，NP组、RNAi组和BV组脊髓背角p-CREB达上调（P〈0．05）；与NP组比较，RNAi组T6-8时机械痛阈升高，脊髓背角p-CREB表达下调（P〈0．05）。RNAi组脊髓背角可见大量绿色荧光，即GFP表达阳性，其余3组脊髓背角未见绿色荧光，即GFP无表达。结论鞘内注射DREAM—shRNA缓解大鼠神经病理性痛的机制可能与抑制脊髓背角p-CREB的表达有关。Objective To investigate the effects of intrathecal （IT） DREAM-short hairpin RNA （DREAM-shRNA） on expression of phosphorylated cyclic AMP response element binding protein （p-CREB） in the spinal dorsal horn in a rat model of neuropathic pain. Methods Adult male SD rats weighing 280-320 g were anesthetized with intraperitoneal 10% chloral hydrate. Neuropathic pain was induced by chronic constrictive injury （CCI） to sciatic nerve. IT catheters were placed according to the method described by Yaksh on 3rd day after CCI. Twenty-four rats in which IT catheter was successfully implanted were randomly divided into 4 groups （ n = 6 each） ： group Ⅰ sham operation （ group S） ; group Ⅱ neuropathic pain （group NP） ; group Ⅲ RNA interference （group RNAi） and group Ⅳ blank vector （group BV）. Lentivirus with DREAM-shRNA 5 μl was injected IT in group RNAi,and blank vector 5 μl in group BV,and once a day for 7 days, starting from the day 8 after CCI. The mechanical pain threshold was measured at day 1 before CCI （T0 ,baseline） and day 7-14 after CCI （T1-8）. The animals were killed on 15th day after CCI. The L4-6 lumbar segment of the spinal cord was removed for determination of the expression of green fluorescent protein （GFP） and p-CREB by immuno-fluorescent method. Results The mechanical pain threshold was significantly decreased as compared with the baseline at T0 in all 4 groups and returned to the baseline levels at T5-8 in group S and RNAi, but remained low in group NP and BV. The mechanical pain threshold was significantly lower after CCI/sham operation and significanty higher at T8 in group RNAi than in the other 3 groups. The expression of p-CREB in the spinal dorsal horn was up-regulated in group NP, RNAi and BV as compared with group S, and in group NP and BV as compared with group RNAi. The green fluorescence was observed in group RNAi but not in the other 3 groups. Conclusion IT DREAM-shRNA can ameliorate neuropathic pain in rats through inhibiting the expre

关 键 词：RNA干扰 转录因子 注射 脊髓 神经痛 DNA结合蛋白 环AMP反应性 脊髓 

分 类 号：R741[医药卫生—神经病学与精神病学]