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目的探讨脂多糖(lipopolysaccharide,LPS)对高糖环境下肝脏枯否细胞(Kupffer cells,KCs)增殖和分泌功能及超微结构的影响.方法将小鼠原代KCs培养扩增后随机分为4组:高糖组[(high glucose,HG),25.0 mmol/L D-葡萄糖],正常对照组[(normal control,CON),11.1 mmol/L D-葡萄糖],LPS+高糖组[(LPS+high glucose,LPS-HG),25.0 mmol/L D-葡萄糖],LPS+正常对照组[(LPS normal+control,LPS-CON),11.1 mmol/L D-葡萄糖].培养24 h后,LPS-HG和LPS-CON组分别加入等量LPS,继续培养6 h后,四甲基偶氮唑盐比色法测定细胞增殖,流式细胞仪检测细胞周期,Luminex xMAP技术检测细胞上清液中肿瘤坏死因子-α(tumor necrosis factor alpha,TNF-α)、白介素(interleukin,IL)-1β、IL-6水平,透射电镜观察KCs细胞超微结构.结果MTT比色法测定结果显示,KCs经LPS干预6 h后吸光度(absorbance,OD)测值显著升高,HG和LPS-HG组的OD测值明显低于相对照的CON组和LPS-CON组(P<0.05).流式细胞仪技术检测结果显示,KCs经LPS干预6 h后G0/G1期细胞分布明显下降,S期+G2/M期细胞分布明显升高(P<0.01).HG和LPS-HG组的G0/G1期细胞分布显著高于相对照的CON和LPS-CON组,HG和LPS-HG组的S期+G2/M期细胞分布显著低于相对照的CON和LPSCON组(P<0.01);Luminex xMAP技术结果显示,KCs经LPS干预6 h后TNF-α、IL-1β、IL-6水明显升高,TNF-α和IL-1β的变化更为显著.电镜观察KCs结果显示,HG组可见自噬体形成,CON组个别细胞胞质内仅见少量空泡;LPS-HG组可见大量空泡及自噬体形成,LPS-CON组可见空泡形成而未见自噬体.结论LPS能激活并强化高糖环境中肝脏KCs的增殖和分泌功能,高糖环境和LPS均可引发肝脏KCs自噬等超微结构改变.
Objective To investigate the effects of lipopolysaccharide (LPS) on proliferation, secretion and ultrastructure of hepatic Kupffer cells (KCs) under high glucose conditions.Methods Primary cultured KCs were randomly divided into 4 groups Group: high glucose (HG), 25.0 mmol / L D-glucose], normal control (CON), 11.1 mmol / L D-glucose and LPS + LPS-HG, 25.0 mmol / L D-glucose, LPS-HG and 11.1 mmol / L D-glucose] And LPS-CON group were added the same amount of LPS, continue to culture for 6 h, tetramethylzirconate colorimetric assay for cell proliferation, cell cycle detection by flow cytometry, Luminex xMAP technology to detect cell supernatant tumor necrosis The ultrastructures of KCs were observed by transmission electron microscopy.Results The results of MTT assay showed that KCs were intervened by LPS After 6 h, the absorbance (OD) was significantly increased, and the OD values in HG and LPS-HG groups were significantly lower than those in CON and LPS-CON groups (P <0.05). Flow cytometry The results showed that the distribution of cells in G0 / G1 phase was significantly decreased and the cell distribution was significantly increased in S phase and G2 / M phase (P <0.01) after treated with LPS for 6 h.G0 / G1 phase cells in HG and LPS-HG groups The distribution of S phase and G2 / M phase in HG and LPS-HG groups was significantly lower than that in CON and LPSCON groups (P <0.01). The results of Luminex xMAP The results showed that the changes of TNF-α and IL-1β in KCs were significantly increased after KCs intervention with LPS for 6 h, and the changes of TNF-α and IL-1β were more obvious.KEMCs results showed that the autophagosome , Only a few vacuoles were found in the cytoplasm of CON cells, a large number of vacuoles and autophagosomes were found in LPS-HG group, and no vacuoles formed in LPS-CON group.Conclusion LPS can activate and strengthen high The proliferation and secretion of hepatic KCs in the sugar environment, high glucose environment and LPS can induce the autophagy and other ultrastructural changes in the liver KCs.