CAJ | 학술논문

目的探讨细菌脂蛋白(BLP)信号转导分子Toll样受体2(TLR2)和白细胞介素-1受体相关激酶1(IRAK-1)与BLP耐受发生的关系.方法在人胚肾293(HEK293)细胞中过表达TLR2以及IRAK-1蛋白,利用蛋白质免疫印迹法和双荧光素酶报告基因检测实验观察其对BLP耐受的影响.结果以BLP刺激稳定转染并表达TLR2的HEK293细胞可以剂量依赖性地诱导NF-κB活化,而且BLP预处理HEK-TLR2细胞可诱导BLP耐受的发生.在HEK-TLR2细胞中过表达IRAK-1可以剂量依赖性地增加NF-κB的活化并逆转BLP耐受:转染前BLP活化组与耐受组的NF-κB活化程度分别为0.329±0.010和0.168±0.010;转染0.02μg IRAK-1质粒后活化组与耐受组的NF-κB活化程度分别为0.493±0.010和0.427±0.035,均比转染前明显升高,差异均有统计学意义(均P＜0.01).结论 HEK293细胞中过表达TLR2不能阻止BLP耐受的诱导,但是在HEK-TLR2细胞中过表达IRAK-1可部分逆转BLP耐受,提示IRAK-1蛋白表达水平的变化在BLP耐受发生中起关键作用;IRAK-1可作为细菌感染和脓毒症治疗的重要靶点.
목적 탐토세균지단백(BLP)신호전도분자Toll양수체2(TLR2)화백세포개소-1수체상관격매1(IRAK-1)여BLP내수발생적관계.방법 재인배신293(HEK293)세포중과표체TLR2이급IRAK-1단백,이용단백질면역인적법화쌍형광소매보고기인검측실험관찰기대BLP내수적영향.결과 이BLP자격은정전염병표체TLR2적HEK293세포가이제량의뢰성지유도NF-κB활화,이차BLP예처리HEK-TLR2세포가유도BLP내수적발생.재HEK-TLR2세포중과표체IRAK-1가이제량의뢰성지증가NF-κB적활화병역전BLP내수:전염전BLP활화조여내수조적NF-κB활화정도분별위0.329±0.010화0.168±0.010;전염0.02μg IRAK-1질립후활화조여내수조적NF-κB활화정도분별위0.493±0.010화0.427±0.035,균비전염전명현승고,차이균유통계학의의(균P＜0.01).결론 HEK293세포중과표체TLR2불능조지BLP내수적유도,단시재HEK-TLR2세포중과표체IRAK-1가부분역전BLP내수,제시IRAK-1단백표체수평적변화재BLP내수발생중기관건작용;IRAK-1가작위세균감염화농독증치료적중요파점.
Objective To investigate Toll-like receptor 2(TLR2)and interleukin-1 receptorassociated kinase 1(IRAK-1)in bacterial lipoprotein(BLP)tolerance.Methods Western blotting was used to confirm the over expression of TLR2 and IRAK-1 in human embryo kidney 293(HEK293)cells.Plasmids for dual luciferase reporter gene with nuclear factor-κB promoter(pNF-κB-Luc)or CMV promoter (phRL-CMV internal control vector)were used to detect the NF-κB activation and the induction of BLP tolerance in HEK-TLR2 cells.Results BLP stimulation resulted in dose-dependent NF-κB activation in HEK293 cells stably expressing TLR2.And BLP pretreatment could reduce NF-κB activation and induce BLP tolerance in HEK-TLR2 cells.The NF-κB activation was 0.329±0.010 and 0.168±0.010 in BLP-activated and BLP-tolerant HEK-TLR2 cells,respectively.After transfection with 0.02μg IRAK-1 plasmid,NF-κB activation in the two groups was 0.493±0.010 and 0.427±0.035,respectively(both P＜0.01).So over expression of IRAK-1 could increase NF-κB activation in a dose-dependent manner.Conclusion These results demonstrated that over expression of IRAK-1 could reverse BLP tolerance,whereas over expression of TLR2 failed to prevent the induction of BLP tolerance.Therefore reduced IRAK-1 protein expression is an important mechanism in the development of BLP-induced tolerance,suggesting that it could be a potentially important target for future therapeutic strategies in bacterial infection and sepsis.