【摘要】 目的 采用Transwell小室构建的小鼠胰岛细胞株MIN6和巨噬细胞株RAW264.7共培养系统，探讨酰基化Ghrelin能否保护胰岛b细胞免遭巨噬细胞介导的炎症损伤。 方法 实验分为单独RAW264.7巨噬细胞组、单独MIN6胰岛细胞组、共培养组和Ghrelin干预组。RT-PCR和Western blot检测巨噬细胞上TLR4和脂肪酸结合蛋白(A-FABP)的表达;ELISA检测细胞上清白细胞介素1β(IL-1)、肿瘤坏死因子α(TNF-α)的浓度，以及葡萄糖刺激后MIN6细胞上清液的胰岛素浓度。结果(1)共培养系统中，巨噬细胞TLR4[mRNA及蛋白水平分别为(1.35±0.13) vs (0.93±0.03)，A-FABP为(1.99±0.10)vs(0.91±0.01)]的表达、细胞上清液IL-1β[(10.47±1.11)pg/ml]、TNF-α[(917.54±9.09)pg/ml]、蛋白水平[(0.91±0.09) vs (0.56±0.02)，A-FABP为(0.91±0.16) vs (0.51±0.05)]及单独胰岛细胞组分泌[IL-1β(4.77±0.49)pg/ml,TNF-α (672.78±3.07)pg/ml]较单独巨噬细胞或胰岛细胞组明显增高(P<0.05);(2)高糖刺激下，共培养系统中胰岛b细胞的胰岛素分泌水平较单独胰岛细胞组明显降低(P<0.05);(3)与共培养组比较，酰基化Ghrelin干预后共培养，巨噬细胞TLR4表达水平[按10、100、500 nmol/L的Ghrelin浓度，mRNA及蛋白水平分别为(0.77±0.07)、(0.61±0.03);(0.51±0.03)、(0.46±0.02);(0.31±0.03)、(0.35±0.03)。A-FABP分别为(1.06±0.04)、(0.61±0.02);(0.43±0.05)、(0.43±0.04);(0.27±0.01)、(0.27±0.05)及细胞上清液中IL-1β水平[(8.37±0.33)、(6.11±0.49)、(2.48±1.11)pg/ml)]和TNF-α[(656.82±38.87)、(595.98±19.29)、(486.50±17.93)pg/ml)]均降低(P<0.05)，且呈剂量依赖性。结论 巨噬细胞与胰岛b细胞共培养后，炎症通路活化，炎症因子释放，胰岛b细胞的胰岛素分泌功能受损;酰基化Ghrelin可剂量依赖性削弱巨噬细胞和胰岛b细胞共培养后炎症通路的活化和炎症因子的释放，但不能完全阻止胰岛b细胞胰岛素分泌功能的降低。

　　【关键词】Ghrelin;共同培养技术;胰岛细胞;巨噬细胞

　　【Abstract】 Objective To investigate if acylated ghrelin can protect pancreatic islet b cells from

　　inflammatory injury mediated by macrophages by using established Co-culture system for RAW264.7 macrophages and MIN6 insulinoma cells through Transwell chamber. Methods Six experimental groups were set up：macrophages control group, insulinoma cells control group, co-culture group，ghrelin treated group. After treatment, the mRNA and protein levels of TLR4 and A-FABP in RAW264.7 macrophages were detected by

　　RT-PCR and Western blotting, respectively. The concentrations of IL-1β and TNF-α in the supernatant ,as well as

　　insulin levels in the supernatant of MIN6 cells after glucose stimulation were determined by enzyme linked immunosorbant assay (ELISA). Results (1) Compared with macrophages control group and insulinoma cells control group, the mRNA and protein levels of TLR4 and A-FABP as well as the concentrations of TNF-α and IL-1β (P<0.05) in RAW264.7 macrophage; (2) Compared with insulinoma cells control group, co-culture under high-level glucose stimulation led to significantly decreased insulin serection (P<0.05); (3) Compared to co-culture control, acylated ghrelin treatment led to dose-dependent decrease of mRNA and protein expression levels of TLR4 and A-FABP, as well as the concentration of IL-1β and TNF-α(P<0.05) in the supernatant of RAW264.7 macrophage cells. Conclusions Co-culture of macrophages and pancreas islet b cell can activate inflammation pathway which leads to release of inflammatory factors and reduction of secretion ability of pancreas islet b cell; acylated ghrelin can decrease the activation of inflammatory pathway and the release of inflammatory factors in response to the co-culture in a dose dependent manner, but it can’t prevent completely the decline of secretion function in pancreas islet b cell.

　　【Key words】Ghrelin; Coculture techniques; Macrophage ;Islet b cells;Macrophages