农业生物技术学报2017，Vol.25Issue(8)：1307-1313,7.DOI:10.3969/j.issn.1674-7968.2017.08.011
豚鼠腹腔巨噬细胞的分离培养及免疫抗结核功能检测
Isolation and Cultivation of Guinea Pig (Cavia porcellus) Peritoneal Macrophage and Its Immunological Function in Response to Tuberculosis
摘要
Abstract
Macrophages are key cells associated with innate immunity,pathogen containment and modulation of the immune response and also the key regulators of tissue repair,regeneration and fibrosis.Commonly used model systems for studying macrophage biology have centered on macrophage-like leukemic cell lines,primary macrophages derived from model organisms and primary macrophages differentiated from blood monocytes.Although these cells have provided important insights into macrophage-associated biology,there are issues that need consideration.The primary drawback of understanding macrophage biology is that reliable and scalable macrophage models for cellular and genetic studies is scarce,limiting their utility in genetic studies.Based on these,in order to elucidate the role and mechanism of guinea pig macrophages in tuberculosis immunization,in the present study,guinea pig peritoneal macrophagcs were isolated and characterized by using the following methods:Morphological observation was by phase contrast microscope,cell viability analysis was performed using Trypan blue staining method,phagocytosis assay was carried out using the method of swallowing ink particles,the expression of intracellular enzyme was detected using α-naphthol acetate esterace (α-NAE) staining method,and the expression of cell surface specific antigen analysis was detected by flow cytometry.Furthermore,in order to study the effect of Bacillus Calmette-Guérin (BCG) on guinea pig macrophage apoptosis and its production of nitric oxide (NO),guinea pig macrophage was infected by BCG in vitro.The results showed that the guinea pig macrophage performed multiple cell morphology and maintained the characteristic of macrophage-specific morphology,which resembled round shape,oval shape,spindle shape and irregular shape under the microscope.Pseudopodium and bulge were observed in some of the cells.The cells had a large amount of cytoplasm and large nuclei.The result of Giemsa staining showed that the nuclei of macrophage was darker and horseshoe shaped,round or irregular shape,locating at one side of the cells.The macrophage had the ability of favourable phagocytosis,and the phagocytic rate was (94.3± 1.04)％.The results of nonspecific esterase staining showed that the rate of positive staining cells was (94.3 ± 1.44)％,which indicated that the purity of the isolated macrophages was higher.Meanwhile,flow-cytometric surface phenotyping of guinea pig macrophage revealed expression of classical macrophage markers such as CD 14,CD40 and CD68,and the percentages of positive staining of CD 14,CD40and CD68 were 97.6％,96.9％ and 92.1％ respectively.Moreover,the results showed that the production of NO in guinea pig macrophage which were infected by BCG was significantly increased (P＜0.01) compared with the control group.Meanwhile,the results of apoptosis analysis by FITC Annexin V Apoptosis Detection Kit assay combined with fluorescence activated cell sorter (FACS) showed that the ratio of apoptosis of the BCG infected group was (14.4±2.86)％ which was significantly higher than that of the control group (4.26±1.06)％ (P＜0.01).These results initially confirmed that pre-stimulation of starch broth solution could extract a large number of guinea pig peritoneal macrophages,and confirmed that the guinea pig macrophage had the function in tuberculosis immunity.And the present results may provide new experimental material and data support for the research on interaction between macrophage and Mycobacterium tuberculosis.关键词
豚鼠/巨噬细胞(M(φ))/卡介苗(BCG)/NO/凋亡Key words
Guinea pig/Macrophage (M(φ))/Bacillus Calmette-Guérin (BCG)/NO/Apoptosis分类
农业科技引用本文复制引用
李欧阳,洪丹萍,柯敏霞,麦丽丽,沈文强,何玉龙,刘立丽,吴月红..豚鼠腹腔巨噬细胞的分离培养及免疫抗结核功能检测[J].农业生物技术学报,2017,25(8):1307-1313,7.基金项目
国家自然科学基金项目(No.31260287)、浙江省自然科学基金项目(No.LY17C120001)和浙江理工大学科研启动基金项目(No.15042016-Y) （No.31260287）
