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目的利用低速离心和差速贴壁的特性,探索一种高效分离纯化BALB/c小鼠Kupffer细胞(KC)的方法。方法采用0.5 g/L 4型胶原蛋白酶原位灌注和消化肝脏组织,低速分离肝脏的细胞后差速贴壁分离纯化KC,与Percoll密度梯度离心分离方法比较KC的产量和活性,应用F4/80免疫荧光染色鉴定细胞形态、流式细胞术检测F4/80的表达、墨汁吞噬实验进行吞噬功能鉴定。构建KC缺氧/复氧模型,流式细胞术检测主要组织相容性复合体(MHC)Ⅱ、CD40、CD68和CD86的表达,ELISA检测肿瘤坏死因子α(TNF-α)的表达。结果每只小鼠肝脏可获得(5.83±0.54)×106个KC,细胞存活率达92%,Percoll密度梯度分离仅能获得(2.19±0.43)×106个KC,F4/80免疫荧光染色显示贴壁后KC呈梭形或棘状,流式细胞术鉴定F4/80阳性细胞占比接近90%,吞墨实验可见胞质内大量被吞噬的碳颗粒。KC在缺氧复氧模型下MHC-Ⅱ、CD40、CD86分子表达增多,并分泌TNF-α参与炎症反应。结论成功建立了BALB/c小鼠KC分离纯化的方法。
Objective To explore a method for efficient isolation and purification of Kupffer cells (KC) in BALB / c mice by using the characteristics of low speed centrifugation and differential adherence. Methods The hepatic tissue was perfused and digested with 0.5 g / L collagenase 4 in situ, and the KCs were separated and purified by differential adherent method. The yield and activity of KC were compared with Percoll density gradient centrifugation method. F4 / 80 Cell morphology was identified by immunofluorescence staining, F4 / 80 expression was detected by flow cytometry, and phagocytosis was assessed by ink phagocytosis assay. The model of KC hypoxia / reoxygenation was established. The expressions of major histocompatibility complex (MHC) Ⅱ, CD40, CD68 and CD86 were detected by flow cytometry. The expression of tumor necrosis factor α (TNF-α) was detected by ELISA. Results The liver cell survival rate of each mouse was (5.83 ± 0.54) × 106 KC, the cell survival rate was 92%. Percoll density gradient was only (2.19 ± 0.43) × 106 KC, F4 / 80 immunofluorescence staining showed The posterior wall KC was spindled or spiculate. F4 / 80 positive cells accounted for nearly 90% by flow cytometry. A large amount of carbon particles were swallowed in the cytoplasm after the ink swabbing experiments. The expression of MHC-Ⅱ, CD40 and CD86 in KC increased under hypoxia and reoxygenation, and TNF-α was involved in inflammatory reaction. Conclusion The method of isolation and purification of KC in BALB / c mice was successfully established.