CAJ | 학술논문

目的探讨脂肪细胞型脂肪酸结合蛋白(A-FABP)抑制剂对胰岛β细胞遭受巨噬细胞介导的炎症损伤的保护作用.方法采用Transwell小室构建小鼠胰岛细胞株MIN6和巨噬细胞株RAW264.7的共培养系统.在共培养前2h,在RAW264.7巨噬细胞中分别加入A-FABP的抑制剂(5μmol/L).共培养48 h后,用实时定量PCR和Western印迹法检测RAW264.7巨噬细胞上Toll样受体(TLR)4和脂肪酸结合蛋白(A-FABP)的表达,用ELISA方法检测细胞上清液中白细胞介素(IL-1)β和肿瘤坏死因子(TNF)-α的浓度,此外,用含葡萄糖3、30 mmol/L的碳酸氢钠缓冲液(KRBB液)孵育MIN6胰岛细胞1h,用ELISA方法测定细胞上清液中胰岛素的浓度.结果 (1)共培养后,巨噬细胞上TLR4和A-FABP的表达水平以及细胞上清液中IL-1β和TNF-α的浓度均明显高于巨噬细胞对照组(均P ＜0.05)；(2)共培养后,高糖刺激的胰岛素分泌水平明显低于胰岛细胞对照组[(16.0±2.2)比(41.1±6.6)ng/ml,P＜0.05]；A-FABP的抑制剂干预后再共培养,巨噬细胞上TLR4和A-FABP的表达水平以及细胞上清液中的IL-1β和TNF-α的浓度均显著低于共培养对照组(均P＜0.05),高糖刺激胰岛β细胞的胰岛素分泌水平显著高于共培养对照组[(31.4±3.3)比(16.0±2.2)ng/ml,P＜0.05].结论 (1)巨噬细胞与胰岛β细胞共培养后,可活化炎症通路,释放炎症因子,损伤胰岛β细胞的胰岛素分泌能力；(2) A-FA BP的抑制剂可抑制巨噬细胞和胰岛β细胞共培养后炎症通路的活化和炎症因子的释放,从而阻止胰岛β细胞胰岛素分泌能力的下降.
목적 탐토지방세포형지방산결합단백(A-FABP)억제제대이도β세포조수거서세포개도적염증손상적보호작용.방법 채용Transwell소실구건소서이도세포주MIN6화거서세포주RAW264.7적공배양계통.재공배양전2h,재RAW264.7거서세포중분별가입A-FABP적억제제(5μmol/L).공배양48 h후,용실시정량PCR화Western인적법검측RAW264.7거서세포상Toll양수체(TLR)4화지방산결합단백(A-FABP)적표체,용ELISA방법검측세포상청액중백세포개소(IL-1)β화종류배사인자(TNF)-α적농도,차외,용함포도당3、30 mmol/L적탄산경납완충액(KRBB액)부육MIN6이도세포1h,용ELISA방법측정세포상청액중이도소적농도.결과 (1)공배양후,거서세포상TLR4화A-FABP적표체수평이급세포상청액중IL-1β화TNF-α적농도균명현고우거서세포대조조(균P ＜0.05)；(2)공배양후,고당자격적이도소분비수평명현저우이도세포대조조[(16.0±2.2)비(41.1±6.6)ng/ml,P＜0.05]；A-FABP적억제제간예후재공배양,거서세포상TLR4화A-FABP적표체수평이급세포상청액중적IL-1β화TNF-α적농도균현저저우공배양대조조(균P＜0.05),고당자격이도β세포적이도소분비수평현저고우공배양대조조[(31.4±3.3)비(16.0±2.2)ng/ml,P＜0.05].결론 (1)거서세포여이도β세포공배양후,가활화염증통로,석방염증인자,손상이도β세포적이도소분비능력；(2) A-FA BP적억제제가억제거서세포화이도β세포공배양후염증통로적활화화염증인자적석방,종이조지이도β세포이도소분비능력적하강.
Objective To investigate the potential role of adipocytes fatty acid binding protein (A-FABP) inhibitor to prevent pancreatic islet cells from cytotoxic injury by inflammatory cytokines released from macrophage.Methods Co-culture system for RAW264.7 macrophage and MIN6 insulinoma cells was established through transwell combined with A-FABP inhibitor BMS309403 treatment for 48 h.Meanwhile,cultured RAW264.7 and MIN6 respectively were set up as controls.In the inhibitor group,BMS309403 preprocessing (5 μmol/L) was performed 2 h before co-culture.The expression of toll-like receptors(TLR) 4 and A-FABP in RAW264.7 macrophages was detected by RT-PCR and Western blotting,interleukin(IL)-1β and tumor necrosis factor(TNF)-α levels in the supernatant were detected by ELISA,Glucose-stimulated insulin level was detected by insulin radioimmuno-assay kits for the function of islets.Results (1) The mRNA and protein levels of both TLR4 and A-FABP in RAW264.7 macrophages as well as the concentrations of IL-1β and TNF-α in the supernatant were significantly higher in co-culture group than in macrophages control group(P ＜ 0.05).(2) Insulin secretion stimulated by high glucose was obviously decreased in co-culture group when compared with insulinoma cells control group [(16.0 ± 2.2) vs (41.1 ±6.6) ng/ml,P ＜0.05].After the treatment with A-FABP inhibitor,the mRNA and protein levels of both TLR4 and A-FABP as well as the concentrations of IL-1β and TNF-α in the supernatant were significantly lower than in co-culture control (P ＜ 0.05).However,insulin secretion stimulated by high glucose was significantly enhanced when compared with insulinoma cells control group [(31.4 ± 3.3) vs (16.0 ± 2.2) ng/ml,P ＜ 0.05].Conclusions This study demonstrated that co-culture of macrophage and islet cells can activate inflammation pathway,stimulate inflammatory cytokine release and decrease insulin secretion from islet cells.A-FABP inhibitor can protect islet cells from macrophage-mediated cytotoxicity and preserve its insulin secretory function.