肝细胞来源的肝前体样细胞对巨噬细胞的调控作用研究  

Study on the regulatory effect of hepatocellular derived liver precursor-like cells on macrophages

作　　者：彭媛 周徐 景宏舒 朱雪晶 史瑶平 王涛[1] 崔丹[1] 施东华[1] 丁敏[1] 鄢和新 翟博[1] PENG Yuan;ZHOU Xun;JING Hong-Shu;ZHU Xue-Jing;SHI Yao-Ping;WANG Tao;CUI Dan;SI Dong-Hua;YAN He-Xin;ZHAI Bo(Department of Interventional Oncology,Renji Hospital,Jiaotong University School of Medicine,Shanghai 200127,China;Celliver Biotechnology Inc.,Shanghai 200127,China)

机构地区：[1]上海交通大学医学院附属仁济医院肿瘤介入科,200127 [2]上海赛立维生物科技有限公司

出　　处：《肝脏》2021年第6期684-687,695,共5页Chinese Hepatology

基　　金：国家自然科学基金(82070619)。

摘　　要：目的探究人类肝细胞来源的肝前体样细胞(HepLPC)对巨噬细胞亚群M1及M2的影响。方法采用肝细胞扩增培养体系(TEM)将原代肝细胞体外扩增转分化为HepLPC,培养过程收集HepLPC条件培养上清,离心后冻存备用。巨噬细胞为C57小鼠骨髓来源(BMDM),通过红细胞裂解处理后,加入小鼠GM-CSF 40 ng/mL培养,刺激诱导7 d,贴壁所得为实验所需巨噬细胞。处于静息状态的巨噬细胞,通过LPS 100 ng/mL刺激产生炎性状态的M1型巨噬细胞以及IL-440 ng/mL刺激产生具有抗炎及具有组织修复功能的M2型巨噬细胞。在定向诱导巨噬细胞极化的基础上,将HepLPC条件培养上清与不同极化状态的巨噬细胞共同培养,收集不同时间点的培养上清,细胞RNA和细胞蛋白,通过流式细胞术、定量PCR、ELISA等方法综合评价HepLPC条件培养上清对巨噬细胞不同亚群的影响。结果小鼠GM-CSF刺激诱导BMDM贴壁,得到M0型巨噬细胞。F4/80+(巨噬细胞总标记)表达占比92.8%。流式细胞检测结果显示F4/80+,CD11c+表达占比88.1%;F4/80+,CD206+表达占比97.0%。LPS刺激6 h,M1相关炎性基因表达上调:IL6表达上调(823.200±174.500)倍,IL1β表达上调(8.389±0.029)倍,iNOS表达上调(24.650±1.196)倍;IL-4刺激6h,M2相关基因表达上升:CD206表达上调(114.000±3.579)倍,IL10表达上调(2.634±0.028)倍,ARG1表达上调(53.260±8.083)倍;M1型、M2型巨噬细胞诱导成功。在此基础上,HepLPCs-CM共培养后,M1型巨噬细胞极化相关基因的表达下调:IL6表达为(346.300±20.810),IL1β表达为(11.290±0.10),iNOS表达为(169.800±9.711);相关炎性因子下降:IL6分泌为(138.700±32.130)pg/(mL×10^(5) cell),IL1β分泌为(0.710±0.019)pg/(mL×10^(5) cell),iNOS分泌为(0.095±0.001)pg/(mL×10^(5) cell)。M2型巨噬细胞极化相关基因表达上调:CD206表达为(40.88±0.1768),IL10表达为(22.2±0.1414),ARG1表达为(32.25±0.2121);IL10分泌增加,为(108.052±0.472)pg/(mL×10^(5) cell)。结论HepLPC条件培养�Objective To investigate the effects of human hepatocellular derived liver precursor like cells(HEPLPCS)supernatant on macrophage subsets M1 and M2,respectively.Methods The primary liver cell line was purchased from Ziride liver,and the hepatocyte amplification and culture system(TEM)developed by the previous team was used to amplify and transdifferentiate the primary liver cells into liver precursor like cells(HEPLPCS)in vitro.During the culture process,the supernatant of the HEPLPCS was collected,and after centrifugation,the cells were frozen and stored for later use.Macrophages were derived from bone marrow of C57 mice(BMDM).After erythrocyte lysis treatment,mice M-CSF(40 ng/mL)was added for culture,and stimulated and induced for 7 days.Adherent macrophages were obtained for the experiment.Macrophages in the resting state were stimulated by LPS(100 ng/mL)to produce m1-type macrophages in the inflammatory state and IL-4(40 ng/mL)to produce M2-type macrophages with anti-inflammatory and tissue repair functions.On the basis of the directional induced macrophage polarization,the culture supernatant HepLPCs conditions with different polarization states of macrophages to jointly develop,collect the culture supernatant of different time points,cell RNA and protein,by flow cytometry quantitative PCR ELISA methods such as comprehensive evaluation HepLPCs culture supernatant on macrophages,the influence of different subgroup.Results M0 macrophages were obtained by inducing BMDM adherent by GM-CSF stimulation in mice.The purity of macrophages was identified by flow cytometry,and F4/80+(total macrophage marker)expression accounted for 92.8%.Directionally induced macrophage polarization:LPS and IL-4 were used to stimulate the cells for six h,respectively,and the cells were harvested for flow test and qPCR test.F4/80+,CD11c+expression accounted for 88.1%.F4/80+,CD206+expression accounted for 97.0%.After 6 hours of LPS stimulation,the expression of M1-related inflammatory genes were upregulated:IL6,IL1β,and iNOS were upregu

关 键 词：HepLPCs BMDM M1型巨噬细胞 M2型巨噬细胞 极化 

分 类 号：R575[医药卫生—消化系统]