CAJ | 학술논문

目的探讨脊髓背角IL-17在神经病理性痛大鼠星形胶质细胞活化中的作用.方法在体实验成年健康SPF级雄性SD大鼠64只,6～8周龄,体重180～ 200 g,由江苏大学实验动物中心提供.采用随机数字表法,将其分为3组:对照组(C组,n=16)、假手术组(S组,n=24)和神经病理性痛组(NP组,n=24).采用切断L5,6脊神经的方法制备神经病理性痛模型.于模型制备前、模型制备后1、3、5、7、10、14d时测定机械痛阈.于模型制备后7和14 d,采用qRT-PCR法测定脊髓背角IL-17、IL-6、IL-1β、TNF-α的mRNA表达水平.于模型制备7d,测定脊髓背角星形胶质细胞的活化水平.离体实验采用随机数字表法将原代培养的乳鼠星形胶质细胞分为4组:空白对照组(C组,n=22)、10 ng/ml IL-17组(I10组,n=18)、50 ng/ml IL-17组(I50组,n=18)和100 ng/ml IL-17组(I100组,n=22).I10组、I50组和I100组分别用含上述3种浓度IL-17的培养基进行孵育,于孵育或培养24、48、72 h时,采用MTT法检测星形胶质细胞的增殖水平.采用qRT-PCR法检测IL-6、IL-1β、TNF-α的mRNA表达水平.结果在体实验与C组比较,NP组模型制备后3-14 d机械痛阈降低,模型制备后7d脊髓背角IL-17、IL-6、IL-1β的mRNA表达上调,星形胶质细胞活化水平升高(P＜0.05或0.01),S组模型制备后各时点机械痛阈差异无统计意义(P＞0.05).离体实验与C组比较,I10组和I50组星形胶质细胞孵育48 h时增殖水平升高,I100组孵育48和72 h时星形胶质细胞增殖水平升高,IL-6、IL-1β的mRNA表达上调(P＜0.05或0.01),S组各时点星形胶质细胞增殖水平差异无统计学意义(P＞0.05).结论脊髓背角IL-17表达上调可能参与了大鼠神经病理性痛的维持,其机制与促进星形胶质细胞活化,诱发中枢炎性反应有关.
목적 탐토척수배각IL-17재신경병이성통대서성형효질세포활화중적작용.방법 재체실험 성년건강SPF급웅성SD대서64지,6～8주령,체중180～ 200 g,유강소대학실험동물중심제공.채용수궤수자표법,장기분위3조:대조조(C조,n=16)、가수술조(S조,n=24)화신경병이성통조(NP조,n=24).채용절단L5,6척신경적방법제비신경병이성통모형.우모형제비전、모형제비후1、3、5、7、10、14d시측정궤계통역.우모형제비후7화14 d,채용qRT-PCR법측정척수배각IL-17、IL-6、IL-1β、TNF-α적mRNA표체수평.우모형제비7d,측정척수배각성형효질세포적활화수평.리체실험채용수궤수자표법장원대배양적유서성형효질세포분위4조:공백대조조(C조,n=22)、10 ng/ml IL-17조(I10조,n=18)、50 ng/ml IL-17조(I50조,n=18)화100 ng/ml IL-17조(I100조,n=22).I10조、I50조화I100조분별용함상술3충농도IL-17적배양기진행부육,우부육혹배양24、48、72 h시,채용MTT법검측성형효질세포적증식수평.채용qRT-PCR법검측IL-6、IL-1β、TNF-α적mRNA표체수평.결과 재체실험여C조비교,NP조모형제비후3-14 d궤계통역강저,모형제비후7d척수배각IL-17、IL-6、IL-1β적mRNA표체상조,성형효질세포활화수평승고(P＜0.05혹0.01),S조모형제비후각시점궤계통역차이무통계의의(P＞0.05).리체실험여C조비교,I10조화I50조성형효질세포부육48 h시증식수평승고,I100조부육48화72 h시성형효질세포증식수평승고,IL-6、IL-1β적mRNA표체상조(P＜0.05혹0.01),S조각시점성형효질세포증식수평차이무통계학의의(P＞0.05).결론 척수배각IL-17표체상조가능삼여료대서신경병이성통적유지,기궤제여촉진성형효질세포활화,유발중추염성반응유관.
Objective To investigate the role of interleukin-17 (IL-17) in spinal dorsal horns in neuropathic pain (NP) in rats and its effect on activation of astrocytes.Methods In vivo experiment Sixty-four male SPF Sprague-Dawley rats,aged 6-8 weeks,weighing 180-200 g,were randomly divided into 3 groups using a random number table:control group (group C,n =16),sham operation group (group S,n =24) and group NP (n =24).The animals were anesthetized with intraperitoneal pentobarbital sodium,the L5,6 spinal nerves of the left side of the rat were gently separated and exposed,tightly ligated with 5-0 silk suture and transected.In group S,the L5,6 spinal nerves of the left side of the rat were only exposed.In group C,no operation was performed.Mechanical pain threshold was measured at day 1 before operation and days 1,3,5,7,10 and 14 after operation.The expression of IL-17,IL-6,IL-1β and tumor necrosis factor-alpha (TNF-α) mRNA in the spinal dorsal horn was determined using quantitative real-time PCR at day 7 and day 14 after operation.At day 7 after operation,the activation of astrocytes in the spinal dorsal horn was detected.In vitro experiment Primarily cultured astrocytes of neonatal rats were randomly divided into 4 groups using a random number table:control group (group C,n=22),10 ng/ml IL-17 group (I10 group,n=18),50 ng/ml IL-17 group (I50 group,n-18) and 100 ng/ml IL-17 group (I100 group,n=22).In I10,I50 and I100 groups,the astrocytes were incubated with the culture medium containing 10,50 and 100 ng/ml IL-17,respectively.The proliferation of astrocytes was detected by MTT at 24,48 and 72 h of incutation or culture.The expression of IL-6,IL-1β and TNF-α mRNA was determined using quantitative real-time PCR.Results In vivo experiment Compared with group C,the mechanical pain threshold was significantly decreased at 3-14 days after operation,the expression of IL-17,IL-6 and IL-1β mRNA in the spinal dorsal horn was up-regualted at 7 days after operation,and the activation of astrocytes was increased in group NP,and no significant change was detected in the mechanical pain threshold at each time point after operation in group S.In vitro experiment Compared with group C,the proliferation of astrocytes was significantly increased at 48 h of incubation in I10 and I50 groups,the proliferation of astrocytes was significantly increased at 48 and 72 h of incubation,and the expression of IL-6 and IL-1β mRNA was up-regulated in I100 group,and no significant change was found in the proliferation of astrocytes in group S.Conclusion Up-regulated expression of IL-17 in spinal dorsal horns may be involved in the maintenance of NP,and the mechanism is related to promoted activation of astrocytes and induced inflammatory responses in rats.