CAJ | 학술논문

目的本研究构建了共表达小鼠白细胞介素-18结合蛋白(IL-18BP)和IL-4的重组腺病毒载体(Ad mIL-18BP/mIL-4),将其用于胶原诱导型关节炎(CIA)小鼠局部关节基因治疗,以探讨对小鼠滑膜组织环氧化酶-2(COX-2)和诱导型一氧化氮合酶(iNOS)及其诱导产物前列腺素E2(PGE2)、一氧化氮(NO)表达水平的影响.方法采用雄性的DBA-1/BOM小鼠,建立CIA的小鼠模型,经共表达小鼠IL-18BP和IL-4的重组腺病毒载体局部关节基因治疗后,分别采用半定量反转录一聚合酶链反应(RT-PCR)法和Westem印迹法检测滑膜组织COX-2、iNOS mRNA的表达水平和蛋白表达水平,用竞争ELISA和硝酸酶还原法检测关节滑膜液PGE2和NO的含量,并设AdLacZ组和磷酸盐缓冲液(PBS)组分别为病毒对照组和组织对照组.结果 AdmIL-18BP/mIL-4治疗组滑膜组织COX-2、iNOS mRNA表达水平明显低于AdLacZ对照组(0.15 vs 0.42,P<0.01;0.05 vs 0.77,P<0.01)和PBS对照组(0.15 vs 0.65,P<0.01;0.05 vs 0.64,P<0.01);AdmIL-18BP/mIL-4治疗组滑膜组织COX-2、iNOS的蛋白表达水平明显低于AdLacZ对照组(0.08 VS 0.92,P<0.01;0.11 vs 1.00,P<0.01)及PBS对照组(0.08 vs 0.77,P<0.01;0.11 vs 0.84,P<0.01);AdmIL-18BP/mIL-4治疗组关节滑膜液中PGE2及NO水平明显低于AdLacZ对照组[(0.68±0.06)vs(2.58±0.21)ng/ml,P<0.01;(23.4±2.5)vs(60.0±11.3) μmol/L,P<0.01]和PBS对照组[(0.68±0.06)vs(2.57±0.20)ng/mL,P<0.01;(23.4±2.5)vs(60.3±13.4)μmol/L,P<0.01].结论共表达小鼠IL-18BP和IL-4的重组腺病毒基因治疗能明显下调COX-2和iNOS的表达水平,减少其诱导产物PGE2、NO的合成,这将为RA的基因治疗提供新的治疗策略.
목적 본연구구건료공표체소서백세포개소-18결합단백(IL-18BP)화IL-4적중조선병독재체(Ad mIL-18BP/mIL-4),장기용우효원유도형관절염(CIA)소서국부관절기인치료,이탐토대소서활막조직배양화매-2(COX-2)화유도형일양화담합매(iNOS)급기유도산물전렬선소E2(PGE2)、일양화담(NO)표체수평적영향.방법 채용웅성적DBA-1/BOM소서,건립CIA적소서모형,경공표체소서IL-18BP화IL-4적중조선병독재체국부관절기인치료후,분별채용반정량반전록일취합매련반응(RT-PCR)법화Westem인적법검측활막조직COX-2、iNOS mRNA적표체수평화단백표체수평,용경쟁ELISA화초산매환원법검측관절활막액PGE2화NO적함량,병설AdLacZ조화린산염완충액(PBS)조분별위병독대조조화조직대조조.결과 AdmIL-18BP/mIL-4치료조활막조직COX-2、iNOS mRNA표체수평명현저우AdLacZ대조조(0.15 vs 0.42,P<0.01;0.05 vs 0.77,P<0.01)화PBS대조조(0.15 vs 0.65,P<0.01;0.05 vs 0.64,P<0.01);AdmIL-18BP/mIL-4치료조활막조직COX-2、iNOS적단백표체수평명현저우AdLacZ대조조(0.08 VS 0.92,P<0.01;0.11 vs 1.00,P<0.01)급PBS대조조(0.08 vs 0.77,P<0.01;0.11 vs 0.84,P<0.01);AdmIL-18BP/mIL-4치료조관절활막액중PGE2급NO수평명현저우AdLacZ대조조[(0.68±0.06)vs(2.58±0.21)ng/ml,P<0.01;(23.4±2.5)vs(60.0±11.3) μmol/L,P<0.01]화PBS대조조[(0.68±0.06)vs(2.57±0.20)ng/mL,P<0.01;(23.4±2.5)vs(60.3±13.4)μmol/L,P<0.01].결론 공표체소서IL-18BP화IL-4적중조선병독기인치료능명현하조COX-2화iNOS적표체수평,감소기유도산물PGE2、NO적합성,저장위RA적기인치료제공신적치료책략.
Objective A recombinant adenoviral vector containing mIL-18BP and mIL-4 fusion gene(AdmIL-18BP/mIL-4) was constructed and used to investigate the role of mIL-18BP and mIL-4 in medula-ring the expressions of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) and their inducing products PGE2, NO in murine collagen-induced arthritis. Methods Male DBA-1/BOM mice were used in this study. Mice with CIA were intra-articularly injected with 107 pfu/6μl of AdmIL-18BP/mIL-4.Intra-articular injections of AdLacZ or PBS were used as controls. The mRNA expression of COX-2, iNOS in synovial tissue was analyzed by semi-quantitative RT-PCR. Expression of COX-2 and iNOS protein was estimated by Western blot method. The production of PGE2 and NO in synovia was detected by competitive ELISA and enzyme reduction of nitrate. Results The expression of COX-2, iNOS mRNA in routine synovial tissue of AdmIL-18BP/mIL-4 treatment group was significantly lower than that of AdLacZ group (0.15 vs 0.42,P<0.01 ; 0.05 vs 0.77, P<0.01) and PBS group (0.15 vs 0.65, P<0.01; 0.05 vs 0.64, P<0.01 ). And the protein expression of COX-2, iNOS from AdmIL-18BP/mIL-4 treatment group was also obviously lower than that of AdLacZ group (0.08 vs 0.92, P<0.01; 0.11 vs 1.00, P<0.01) and PBS group (0.08 vs 0.77, P<0.01; 0.11 vs 0.84, P<0.01 ). The PGE2 and NO production in synovia of AdmIL-18BP/mIL-4 treatment group was significantly lower than that of AdLacZ group [(0.68x0.06) vs (2.58±0.21)ng/mL, P<0.01; (23.4+2.5) vs (60.0±11.3)μmol/L, P<0.01 ] and PBS group [(0.68±0.06) vs (2.57±0.20)ng/mL, P<0.01; (23.4+2.5) vs (60.3±13.4)μmol/L, P<0.01]. Conclusion These data indicat that local over-expre-ssion of mIL-18BP and mIL-4 can down-regulate COX-2, iNOS and their induced product PGE2, NO in CIA mice. The combination treatment with mIL-18BP and mIL-4 is a promising therapeutic target for RA.