脂多糖对高糖环境下肝脏Kupffer细胞增殖和分泌及超微结构的影响  

Effect of lipopolysaccharide on proliferation,secretion and ultrastructure of murine liver Kupffer cells under high glucose conditions

作　　者：闫洪锋 孙宏伟[2] 王平[2] 张诗琳 杨建武[2] 徐冰心[3] 周金莲[4] 李成林[2] 崔彦[1,2] 

机构地区：[1]解放军医学院306医院普通外科,北京市100101 [2]解放军第306医院普通外科,北京市100101 [3]解放军第306医院特种医学实验研究中心,北京市100101 [4]解放军第306医院病理科,北京市100101

出　　处：《世界华人消化杂志》2017年第5期412-419,共8页World Chinese Journal of Digestology

基　　金：全军医学科研“十二五”重点课题基金资助项目,No.BWS11J051;试验技术研究计划重点课题基金资助项目,No.SYFD1500128;解放军第306医院医药卫生重点科研课题基金资助项目,No.13ZD/12~~

摘　　要：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.AIM To investigate the effect of lipopolysaccharide（LPS） on the proliferation,secretion and ultrastructure of liver Kupffer cells（KCs）under high glucose conditions.METHODS Murine liver KCs were cultured,amplified,and then randomly divided into a high glucose group[（HG）,25.0 mmol/L D-glucose],a normal glucose group[（CON）,11.1 mmol/L D-glucose],a LPS＋high glucose group[（LPS-HG）,25.0 mmol/L D-glucose],and a LPS＋normal glucose group[（LPS-CON）,11.1 mmol/L D-glucose）].The KCs in each group were cultured for 24 h,and then LPS was added for the LPS-HG and LPS-CON groups.After 6 h of continuous cultivation,cell proliferation and cell cycle were detected by MTT colorimetric assay and flow cytometry,respectively.Cell supernatants were collected to determine the levels of tumor necrosis factor alpha（TNF-α）,interleukin（IL）-1β and IL-6 by Luminex xMAP technique.The ultrastructure of KCs was observed by transmission electron microscopy（TEM）.RESULTS MTT colorimetric assay showed that the optical density（OD） of murine liver KCs treated with LPS for 6 h under high glucose conditions increased significantly,but the OD values decreased significantly in the HG and LPS-HG groups compared with those of the CON and LPS-CON groups（P〈0.05）.Flow cytometry revealed that high glucose arrested the cell cycle in G0/G1 phase;the percentage of G0/G1 phase cells decreased and that of S＋G2/M phase cells increased significantly（P〈0.01） after KCs were treated with LPS for 6.The Luminex xMAP assay showed that the levels of TNF-α,IL-1β and IL-6 increased significantly after murine liver KCs were treated with LPS for 6 h under high glucose conditions,and the changes in TNF-a and IL-ip were more obvious.TEM revealed obvious ultrastructural alterations of KCs treated with LPS for 6 h under high glucose conditions.A large number of autophagosomes were observed in the LPSHG group,and only few were noted in the HG group.Only vacuolar degenerations were visible in the CON and LPS-CON groups.CONC

关 键 词：高糖环境 脂多糖 枯否细胞 细胞增殖 分泌功能 超微结构 

分 类 号：R587.1[医药卫生—内分泌]