目的 探讨肝脏M1型巨噬细胞流式细胞计数方案及M1型巨噬细胞极化与非酒精性脂肪性肝病（nonalcoholic fatty liver disease，NAFLD）相关性。 方法 高脂饮食喂养建立NAFLD小鼠模型，12只C57BL/6小鼠按照随机数字表法分为普通饮食（normal diet，ND）组、高脂饮食（high fat diet，HFD）组喂养24周，检测小鼠血糖、血脂等代谢指标，实时荧光定量PCR法检测小鼠M1型巨噬细胞相关因子，HE染色观察肝脏病理表现。利用Percoll分离液梯度离心法收集肝脏Kupffer细胞，流式细胞仪检测小鼠肝脏M1型巨噬细胞（分选方案：应用FSC-A、SSC-A分群，去除肝脏中的红细胞及杂质；FITC CD45（+）/PE-cy7 CD11c^low，将白细胞分群；APC CD115（+）/Percp cy5.5 CD11b^high筛选单核细胞，应用Apc-cy7 F4/80^low/PE Ly-6C^high分选出M1巨噬细胞）。 结果 与普通饮食组相比，24周HFD小鼠各项代谢指标明显升高，体质量[（28.35±1.71） g vs. （38.43±4.41） g，P<0.001]、肝脏重量 [（1.03±0.18） g vs. （1.85±0.41） g，P=0.003]、空腹血糖[（7.07±0.58） mmol/L vs. （10.23±1.58） mmol/L，P<0.001]、胰岛素[（18.62±3.84） pg/mL vs. （28.84±8.3） pg/mL，P<0.001]、甘油三酯[（2.97±0.67） mmol/L vs. （4.05±0.99） mmol/L，P=0.01]、总胆固醇[（0.23±0.06） mmol/L vs. （0.55±0.17） mmol/L，P<0.001]、谷丙转氨酶（alanine aminotransferase，ALT）5.67（3.16，9.23） U/L vs. 35.86（19.68，58.33） U/L，P=0.003]和谷草转氨酶（aspartate aminotransferase，AST）[53.14（38.18，64.40） U/L vs. 155.10（113.60，192.20） U/L，P<0.001]，NAFLD小鼠M1型巨噬细胞极化明显增加[9.95%（3.10，12.00） vs. 42.00%（26.50，45.50），P=0.003]。HFD诱导的小鼠肝脏中重组人白细胞介素-1β（human interleukin-1 beta protein，IL-1β）、白细胞介素-6（interleukin-6，IL-6）、F4/80和肿瘤坏死因子（tumor necrosis factor-α，TNF-α）mRNA水平明显升高。 结论 流式细胞检测方案可应用于肝脏M1型巨噬细胞检测，NAFLD炎症反应明显加重，M1型巨噬细胞极化与NAFLD发生呈正相关。

Objective To investigate the flow cytometry cell sorting regimen for M1 macrophages and the association between M1 macrophage polarization and nonalcoholic fatty liver disease（NAFLD）. Methods High-fat diet（HFD） was used to establish a mouse model of NAFLD. Twelve C57BL/6 mice were randomly divided into control group（normal diet） and HFD group using a random number table and were fed for 24 weeks. Metabolic markers including blood glucose and blood lipids were measured；quantitative real-time PCR was used to measure the factors associated with M1 macrophages in mice；HE staining was used to observe liver pathology. The Percoll gradient centrifugation method was used to collect liver Kupffer cells，and flow cytometry was used to measure M1 macrophages in mouse liver（sorting regimen：FSC-A/SSC-A for grouping and removing red blood cells and impurities in the liver；FITC CD45（+）/PE-cy7 CD11c^low for grouping leukocytes；APC CD115（+）/Percp cy5.5 CD11b^high for the screening of monocytes；Apc-cy7 F4/80^low/PE Ly-6C^high for separating M1 macrophages）. Results Compared with the control group at week 24，the HFD group had significant increases in the indicators of body weight [（28.35±1.71） g vs. （38.43±4.41） g，P<0.001），liver weight [（1.03±0.18） g vs. （1.85±0.41） g，P=0.003），fasting blood glucose [（10.23±1.58） mmol/L vs. （7.07±0.58） mmol/L，P˂0.001）]，insulin [（18.62±3.84） pg/mL vs. （28.84±8.3） pg/mL，P˂0.001）]，triglyceride [（2.97±0.67） mmol/L vs. （4.05±0.99） mmol/L，P=0.01）]，cholesterol[（0.23±0.06） mmol/L vs. （0.55±0.17） mmol/L，P<0.001）]，alanine aminotransferase [5.67（3.16，9.23） U/L vs. 35.86（19.68，58.33） U/L，P=0.003]，and aspartate aminotransferase [53.14（38.18，64.40） U/L vs. 155.10 （113.60，192.20） U/L，P<0.001]，and there was a significant increase in M1 macrophage polarization in NAFLD mice [42.00%（26.50，45.50） vs. 9.95%（3.1，12），P=0.003]. There were significant increases in the mRNA levels of IL-β，IL-6，F4/80，and TNF-α in the liver of mice induced by HFD. Conclusion The flow cytometry sorting regimen can be used to measure M1 macrophages in the liver. Significant aggravation of inflammatory response is observed in NAFLD，and M1 macrophage polarization is positively correlated with the onset of NAFLD.