鞘内注射DREAM-shRNA对神经病理性痛大鼠的镇痛效应

Analgesic effect of intrathecal DREAM-shRNA in rats with neuropathic pain

目的评价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的镇痛效应。方法健康雄性SD大鼠，体重280—320g，采用坐骨神经慢性缩窄性损伤（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（Sham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天鞘内注射含DREAM．shRNA的慢病毒5μl、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、生理盐水5μl，连续注射7d。于CCI前1d（基础状态）、CCI后第7～14天（T1～8）测定热痛阈和机械痛阈，于CCI后第15天测定脊髓背角绿色荧光蛋白（GFP）的表达水平。结果与基础值比较，NP组和BV组热痛阈降低，Sham组T1-8时热痛阈降低，RNAi组T1-4.6时热痛阈降低，4组CCI后各时点机械痛阈降低（P〈0．05或0．01）；与T1时比较，NP组和BV组其余时点热痛阈和机械痛阈降低，RNAi组L1-5时热痛阈降低，R时热痛阈和机械痛阈升高（P〈0．05或0．01）；与Sham组比较，NP组和BV组热痛阈和机械痛阈降低，RNAi组T2时机械痛阈降低，T8时升高（P〈0．05），热痛阈差异无统计学意义（P〉0．05）；与NP组比较，RNAi组热痛阈和机械痛阈升高（P〈0．05）。仅RNAi组脊髓背角有GFP表达，其余3组脊髓背角未见GFP表达。结论鞘内连续注射DREAM-shRNA可在一定程度上缓解大鼠神经病理性痛。

Objective To investigate the analgesic effect of intrathecal （IT） DREAM-short hairpin RNA （shRNA） in rats with neuropathic pain. Methods Healthy male SD rats weighing 280-320 g were used in this study. Neuropathic pain was induced by chronic constrictive injury （CCI） to sciatic nerve. A microspinal catheter was inserted into subarachnoid space according to Yaksh method at day 3 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 （S）,group Ⅱ neuropathic pain （NP）,group Ⅲ RNA interference （RNAi） and group Ⅳ blank vector （BV）. Lentivirus with DREAM-shRNA 5μl was injected IT at day 8 after CCI in group RNAi （ Ⅲ ）. Blank vector 5 /A was injected IT once a day for 7 days, starting from the day 8 after CCI in group BV （ Ⅳ ）. The thermal pian threshold and mechanical pian threshold were measured at day 1 before CCI （To, baseline） and at day 7-14 after CCI （T1-8）. The animals were killed at day 15 after CCI. The lumbar segment of the spinal cord （L4-6） was removed for determination of expression of green fluorescent protein （GFP） in the dorsal horn of the spinal cord by immuno-fluorescent method. Results The thermal threshold was significantly decreased at T1-4 as compared with the baseline at To in all 4 groups and returned to the baseline levels at T5-8 in group S and RNAi （group Ⅰ and Ⅲ ） but remained low in group NP （ Ⅱ ） and BV （Ⅳ ）. The mechanical pian threshold was significantly decreased after CCI/sham operation and was significanty higher at Ts in group RNAi than in other 3 groups. The green fluorescence was observed in group RNAi but not in other 3 groups. Conclusion IT DREAN-shRNA can ameliorate hyperalgesia in rats with neuropathic pain.