山羊肺泡巨噬细胞的分离、培养与鉴定

目的建立一种高纯度的肺泡巨噬细胞分离培养方法,为研究巨噬细胞生物学特性,开展细胞内寄生菌存活机理研究提供材料。方法在无菌环境下以PBS多次灌洗山羊肺脏,收集细胞后,用外周血单核细胞分离液结合密度梯度离心法从混合细胞中分离出羊肺泡巨噬细胞;培养于含10%胎牛血清的细胞培养基中,倒置显微镜下观察细胞形态,并利用其对鸡红细胞的吞噬功能检测细胞活性;采用流式细胞术检测单核巨噬细胞表面特征性标志CD14。结果获得的贴壁细胞具典型的巨噬细胞形态学特征,有伪足和突起伸出,呈现圆形及不规则形状,胞浆丰富,胞体较大;培养的巨噬细胞54.5%吞噬了鸡红细胞,具有较强的吞噬活性;在体外连续培养近一个月,仍有93.7%的细胞能特异性表达CD14抗原,具有巨噬细胞特异性免疫表型。结论本研究获得的山羊肺泡巨噬细胞纯度高、生物活性好,为开展细胞内寄生菌的疾病机理研究提供了细胞模型。

Isolation, culture and identification of goat alveolar macrophages

Objective In order to study the biological characteristics of macrophages and provide the materials to study the survival mechanism of intracellular parasites, we conducted this study to establish a high-purity alveolar macrophage isolation and culture method. Methods Goat lungs were lavaged with normal saline in sterile environment several times, and cells were collected and then goat alveolar macrophages were purified by density gradient centrifugation using peripheral blood mononuclear cells (PBMC) solution. The isolated goat alveolar macrophages were cultured in cell culture medium containing 10% fetal bovine serum and cell morphology was observed under an inverted microscope every day,and the phagocytic activity of the cells was detected by chicken red blood cell phagocytosis test. Flow cytometry was used to detect CD14, a characteristic monocyte-macrophage surface marker. Results The adherent cells were characterized by typical macrophage morphology, pseudopodia and protrusions, showing round and irregular shape, rich cytoplasm, and large cell body. Of the cultured macrophages, 54.5% could phagocytize chicken erythrocytes and showed good phagocytic activity. After one month of in vitro culture, 93.7% of the cells were able to express CD14 antigen, which had a macrophage-specific immunophenotype. Conclusions The alveolar macrophages obtained in this study have high purity and good bioactivity, thus provide a cell model for studying the immune mechanism of intracellular parasites.