CAJ | 학술논문

目的：探索用 RNA 干扰（RNAi）法沉默脊髓脂质运载蛋白-2（lipocalin-2，LCN2）的表达及其对正常大鼠吗啡耐受形成的影响。方法鞘内置管成功的♂SD 大鼠48只，体质量180～220 g，随机均分为4组，每组12只：Ⅰ组对照组、Ⅱ组吗啡耐受组、Ⅲ组错义小干扰 RNA（mismatch siRNA，MM siRNA）组、Ⅳ组 LCN2小干扰 RNA（LCN2 siRNA）组。所有大鼠鞘内置管术后 d 6确定导管位置，记为 d 0。d 2～8连续7 d，每天2次进行皮下注射，每次注射剂量10μg·g －1，Ⅰ组大鼠皮下注射生理盐水（normal saline，NS），Ⅱ－Ⅳ组大鼠皮下注射吗啡建立吗啡耐受模型。各组大鼠每天皮下给药前分别鞘内注射10μL DEPC 溶液、10μL DEPC 溶液、10μL 含4μg MM siRNA 的 DEPC 溶液和10μL 含4μg LCN2 siRNA 的 DEPC 溶液。d 1、d 9，测定所有大鼠的基础热缩足反应潜伏期（paw withdrawal thermal latency，PWTL）及皮下注射吗啡后45 min 的 PWTL，并计算吗啡的最大可能镇痛效应百分比值（％ maximal possible effect，％MPE）。d 9行为学测试结束后，取脊髓腰膨大，用 Western blot 法检测磷酸化 p38丝裂原活化蛋白激酶（p-p38 MAPK）及 LCN2蛋白表达水平，用免疫组织荧光染色法检测小胶质细胞标记物Iba1的表达。结果d 9，与Ⅰ组比，Ⅱ、Ⅲ组大鼠％MPE 值明显下降（P ＜0.05），腰段脊髓 LCN2、p-p38 MAPK、Iba1表达明显增加（P ＜0.05）。与Ⅱ组比，Ⅳ组大鼠的％MPE 值明显增加（P ＜0.05），脊髓 LCN2、p-p38MAPK、Iba1表达明显下降（P ＜0.05）。结论鞘内注射 LCN2 siRNA 沉默脊髓LCN2的表达能够部分抑制吗啡耐受的形成，该现象可能与其抑制脊髓背角小胶质细胞的活化及脊髓 p-p38 MAPK 的表达有关。
목적：탐색용 RNA 간우（RNAi）법침묵척수지질운재단백-2（lipocalin-2，LCN2）적표체급기대정상대서마배내수형성적영향。방법초내치관성공적♂SD 대서48지，체질량180～220 g，수궤균분위4조，매조12지：Ⅰ조대조조、Ⅱ조마배내수조、Ⅲ조착의소간우 RNA（mismatch siRNA，MM siRNA）조、Ⅳ조 LCN2소간우 RNA（LCN2 siRNA）조。소유대서초내치관술후 d 6학정도관위치，기위 d 0。d 2～8련속7 d，매천2차진행피하주사，매차주사제량10μg·g －1，Ⅰ조대서피하주사생리염수（normal saline，NS），Ⅱ－Ⅳ조대서피하주사마배건립마배내수모형。각조대서매천피하급약전분별초내주사10μL DEPC 용액、10μL DEPC 용액、10μL 함4μg MM siRNA 적 DEPC 용액화10μL 함4μg LCN2 siRNA 적 DEPC 용액。d 1、d 9，측정소유대서적기출열축족반응잠복기（paw withdrawal thermal latency，PWTL）급피하주사마배후45 min 적 PWTL，병계산마배적최대가능진통효응백분비치（％ maximal possible effect，％MPE）。d 9행위학측시결속후，취척수요팽대，용 Western blot 법검측린산화 p38사렬원활화단백격매（p-p38 MAPK）급 LCN2단백표체수평，용면역조직형광염색법검측소효질세포표기물Iba1적표체。결과d 9，여Ⅰ조비，Ⅱ、Ⅲ조대서％MPE 치명현하강（P ＜0.05），요단척수 LCN2、p-p38 MAPK、Iba1표체명현증가（P ＜0.05）。여Ⅱ조비，Ⅳ조대서적％MPE 치명현증가（P ＜0.05），척수 LCN2、p-p38MAPK、Iba1표체명현하강（P ＜0.05）。결론초내주사 LCN2 siRNA 침묵척수LCN2적표체능구부분억제마배내수적형성，해현상가능여기억제척수배각소효질세포적활화급척수 p-p38 MAPK 적표체유관。
Aim To explore the effect of knockdown spinal cord LCN2 by RNAi on the development of mor-phine tolerance in normal rats.Methods After suc-cessful intrathecal implantation, fourty-eight male Sprague-Dawley rats weighing 1 80 -220 grams were randomly divided into 4 groups (n =1 2):group I:control group,group II:morphine tolerance group, group Ⅲ:mismatch siRNA group,group IV:LCN2 siRNA group.The sixth day after intrathecal implanta-tion,rats were tested to ensure the position of cathe-ters,and it was recorded as d 0.On d 2 -8,rats were subcutaneously (s.c)injected of normal saline (NS) (group I)or morphine (group Ⅱ,Ⅲ,Ⅳ)1 0 μg· g -1 twice a day at 8:00 and 1 6:00.Before everyday s. c injection,rats were intrathecally injected of 1 0 μL DEPC solution (group Ⅰ,Ⅱ),1 0 μL DEPC solution containing 4 μg mismatch siRNA (group III)and 4 μg LCN2 siRNA solution (group IV).Paw withdrawal la-tencies to thermal stimuli (PWTL)were tested before morphine injection and 45 minutes after morphine in-jection on d 1 and d 9.The percentage of maximal pos-sible effect (% MPE)was calculated later.Animals were sacrificed on d 9 after the behavioral test and the lumbar enlargement segments of the spinal cord were removed for detecting the expression of phosphorylated-p38 mitogen-activated protein kinase (p-p38 MAPK) and LCN2 by Western blot and microglia marker Iba1 by immunofluorecence.Results On d 1 ,there was no significant difference in %MPE among four groups. On d 9,compared to group Ⅰ,%MPE was signifi-cantly reduced (P <0.05)while p-p38MAPK,LCN2 and Iba1 were markedly up-regulated in group Ⅱ andⅢ (P <0.05 ).On d 9,compared to group Ⅱ,%MPE was significantly increased while p-p38MAPK, LCN2 and Iba1 were markedly reduced in group IV (P<0.05).Conclusion Using LCN2 siRNA to knock-down spinal LCN2 relieves the development of mor-phine tolerance in normal rats possibly through inhibi-ting the activation of microglia and p38 MAPK in the spinal cord.