CAJ | 학술논문

目的观察白细胞介素-1β(IL-1β)对人心脏成纤维细胞基质金属蛋白酶-2(MMP-2)的作用及相关机制.方法将3～6代人心脏成纤维细胞接种于6孔培养板内接受各种干预措施.待实验细胞接受刺激12 h后,用RT-PCR法观察MMP-2的mRNA表达量.细胞接受各种刺激24 h后,收集细胞总蛋白和培养上清,通过凝胶酶谱法进行上清MMP-2的活性检测,采用Western blot法测定细胞诱导型一氧化氮合酶(iNOS)蛋白的表达,采用Griess法衡量细胞上清中一氧化氮(NO)水平.结果 IL-1B作用人心脏成纤维细胞24 h后,促进了细胞MMP-2的活性增加,其中4 ng/ml的IL-1β刺激作用达到高峰,与对照组相比活性增加了(170.24±13.12)%(P＜0.01),并且该浓度IL-1β能时间依赖性地促进心脏成纤维细胞MMP-2的活性增加.IL-1β作用心脏成纤维细胞12 h后,与正常对照组相比4 ng/ml和10 ng/ml IL-β组MMP-2 mRNA表达明显增加(P＜0.01).IL-1β(4 ng/ml)可以明显促进细胞NO水平升高(P＜0.01),而NOS抑制剂NG-甲基-L-精氨酸(10-3 mol/L)显著抑制了IL-1β诱导的MMP-2 mRNA表达(P＜0.01)、MMP-2活性(P＜0.01)以及NO水平的升高(P＜0.01).通过Western blot发现在生理水平的心脏成纤维细胞上未能检测到iNOS蛋白的表达,而不同浓度的IL-1β明显促进了细胞iNOS的蛋白水平的升高.结论 IL-1β能促进人心脏成纤维细胞MMP-2的mRNA表达和活性,并提示其作用与细胞iNOS-NO水平有关.
목적 관찰백세포개소-1β(IL-1β)대인심장성섬유세포기질금속단백매-2(MMP-2)적작용급상관궤제.방법 장3～6대인심장성섬유세포접충우6공배양판내접수각충간예조시.대실험세포접수자격12 h후,용RT-PCR법관찰MMP-2적mRNA표체량.세포접수각충자격24 h후,수집세포총단백화배양상청,통과응효매보법진행상청MMP-2적활성검측,채용Western blot법측정세포유도형일양화담합매(iNOS)단백적표체,채용Griess법형량세포상청중일양화담(NO)수평.결과 IL-1B작용인심장성섬유세포24 h후,촉진료세포MMP-2적활성증가,기중4 ng/ml적IL-1β자격작용체도고봉,여대조조상비활성증가료(170.24±13.12)%(P＜0.01),병차해농도IL-1β능시간의뢰성지촉진심장성섬유세포MMP-2적활성증가.IL-1β작용심장성섬유세포12 h후,여정상대조조상비4 ng/ml화10 ng/ml IL-β조MMP-2 mRNA표체명현증가(P＜0.01).IL-1β(4 ng/ml)가이명현촉진세포NO수평승고(P＜0.01),이NOS억제제NG-갑기-L-정안산(10-3 mol/L)현저억제료IL-1β유도적MMP-2 mRNA표체(P＜0.01)、MMP-2활성(P＜0.01)이급NO수평적승고(P＜0.01).통과Western blot발현재생리수평적심장성섬유세포상미능검측도iNOS단백적표체,이불동농도적IL-1β명현촉진료세포iNOS적단백수평적승고.결론 IL-1β능촉진인심장성섬유세포MMP-2적mRNA표체화활성,병제시기작용여세포iNOS-NO수평유관.
Objective To investigate the effect of interleukin-1β(IL-1β)on expression and activity of matrix metallopmteinage-2(MMP-2)of cultured human cardiac fibroblasts and related signaling pathway.Methods Primary human cardiac fibmblasts seeded in 6-well tissue culture plates and cultured to 80%to 90%confluence were harvested at passage 3 to 6 and exposed to IL-1β at vaious concentrations for 24 h,culture supernatant and cell protein were obtained.MMP-2 mRNA was determined by RT-PCR.The activity of MMP-2 wag analyzed by zymography and the expression of inducible nitric oxide synthese(iNOS)Drotein level was detected by Western blot analysis.Assessment of NO production in the culture supernatant wag performed using the Griess method.Results IL-1β(4 ng/ml)significandy increased MMP-2 activity of cultured fibmblagts in a time-dependent manner.MMP-2 mRNA expression wag significantly upregulated by IL-1β(4 ng/ml and 10 ng/ml,all P＜0.01).Moreover,IL-1β also significantly increased NO production in supernatant(P＜0.01)and these effects could be significantly blocked by cotreatment with L-NMMA(10-3 mol/L,all P＜0.01).Western blot analysis showed that iNOS could not be detected in unstimulated human cardiac fibroblasts but could be detected in cardiac fibroblasts exposed to IL-1β.Conclusion IL-1β increased MMP-2 activity and transcription of human cardiac fibroblasts via iNOS-NO