A new method to isolate and culture rat kupffer cells.
A new method to isolate and culture rat kupffer cells.
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分离培养大鼠库普弗细胞的新方法
DOI：
10.1371/journal.pone.0070832
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Liu ZJ
中科院分区：
文献类型：
--
作者：
Zeng WQ;Zhang JQ;Li Y;Yang K;Chen YP;Liu ZJ
以前的库普弗细胞(KCs)分离方法需要复杂的技能和繁琐的程序。很少有研究试图在体外探索KCs的自我更新能力。因此，本研究的目的是建立一种简便的大鼠KCs分离方法，并进一步研究KCs的体外有丝分裂潜能。通过一步灌流、酶组织处理、差速离心法和选择性贴壁法获得KCs。用四甲基偶氮唑盐比色法和碘化丙二钠流式细胞仪检测KCs的增殖能力。采用吞噬实验和ED-1、ED-2免疫荧光方法鉴定细胞表型。检测细胞表面抗原(MHCII、CD40、CD80、CD86)的表达和细胞因子(核因子-κ-B、肿瘤坏死因子-α、白介素6、白介素10)的产生，进行细胞功能鉴定。以一定的数量和合理的纯度分离出KCs。KCs至少能存活到第5代(P5)，在P3表现出与原代KCs同样强的吞噬活性(P0)。经脂多糖刺激后，P3细胞表面抗原表达和细胞因子产生的变化与P0细胞相似。我们的研究为KCs的分离提供了一种简单有效的方法，并发现自我更新的KCs具有与原代KCs相同的吞噬活性和功能。
Previous methods for Kupffer cells (KCs) isolation require sophisticated skills and tedious procedures. Few studies have attempted to explore the self-renewal capacity of KCs in vitro. Therefore, the aim of this study was to establish a simple method for rat KCs isolation and further investigate the mitotic potential of KCs in vitro. KCs were obtained by performing one-step perfusion, enzymatic tissue treatment, differential centrifugation and selective adherence. The proliferation ability of cultured KCs was determined by MTT assay and Propidium Iodide FACS analysis. Phagocytic assay and ED-1, ED-2 immunofluorescence were used to identify cell phenotype. After stimulation with LPS, the expression of surface antigens (MHCII, CD40, CD80, and CD86) and the production of cytokines (NF-κB, TNF-α, IL-6 and IL-10) were measured for cell function identification. KCs were isolated with certain numbers and reasonable purities. The KCs were able to survive until at least passage 5 (P5), and at P3 showed equally strong phagocytic activity as primary KCs (P0). After stimulation with LPS, the change in the expression of surface antigens and the production of cytokines for P3 cells was similar to that for P0 cells. Our study provides a simple and efficient method for KCs isolation, and reveals that self-renewing KCs have the same phagocytic activity and functions as primary KCs.
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