SD大鼠肝枯否细胞分离培养的改良方法与鉴定

目的枯否细胞(Kupffer cells,KC)是肝内巨噬细胞群,活化的KC参与多种生物、病理学反应过程。然而,KC分离、培养技术进展缓慢。为研究KC致炎、抗炎活化机制,旨在建立经济、简便、有效、稳定分离培养SD大鼠肝KC的方法。方法选用健康雄性SD大鼠,采用0.05%IV型胶原酶灌注肝,经25%和50%percoll密度梯度离心,提取肝KC。荧光显微镜下观察细胞自发荧光情况,锥虫蓝拒染实验鉴定细胞活力,倒置显微镜下观察KC培养过程中细胞形态变化,吞噬墨水、latex珠实验以及免疫细胞化学染色法鉴定细胞纯度。结果大鼠肝KC得率为(3.22±0.31)×107(n=24),细胞活力为(92.35±0.13)%,细胞纯度均在(94.62±0.24)%。结论该方法简单、可行,为进一步研究KC功能和作用机制奠定基础。

A modified method for primary isolation and culture of Kupffer cells in the liver of SD rats

Objective Kupffer cells(KC) is a macrophage population settled in the liver,and activated KCs participate in a variety of biological and pathological reactions.Hitherto there is no efficient method to isolate and culture KCs.This article is to study the inflammatory and anti-inflammatory activation mechanisms of KCs,and to build an economical,convenient,effective and stable method for the isolation and culture of KCs in the rat liver.Methods We perfused the livers of healthy male SD rats with 0.05% type IV collagenase,and obtained KCs following 25% and 50% Percoll gradient centrifugation.We observed the cell spontaneous fluorescence by fluorescent microscopy,determined the cell activity by the trypan blue excluding test,examined the morphological changes during cell culture process under the inverted microscope,and identified the cell purity using immunohistochemistry and phagocytosis of ink and latex beads.Results The yield of KCs in the liver was(3.22±0.31)×107per rat,with a survival rate of(92.35±0.13)% and purity of(94.62±0.24)%.Conclusion This modified method is simple and feasible for primary isolation and culture of KCs in the liver of SD rats,and may help further studies on the function and mechanism of KCs.