山羊肺泡巨噬细胞永生化建立与表型鉴定

Establishment and phenotypic characterization of an immortalized goat alveolar macrophage line

摘要:

【背景】尽管已有多种人和鼠细胞系可以作为布鲁氏菌(Brucella)的体外侵染模型，但目前仍缺乏能够准确模拟其在体内感染过程的大动物体外细胞模型。【目的】获得具有单一克隆特性的、能稳定传代的山羊肺泡巨噬细胞系，优化布鲁氏菌体外侵染山羊细胞系模型。【方法】在无菌环境下用PBS灌洗山羊肺脏，分离得到原代山羊肺泡巨噬细胞；采用转染SV40大T抗原(SV40 large T antigen, SV40LT)、嘌呤霉素抗性筛选的方法建立了永生化山羊肺泡巨噬细胞系；使用细胞免疫荧光和流式细胞术检测单核巨噬细胞表面特征性标志物分化抗原簇14 (cluster of differentiation antigen 14, CD₁₄)，利用鸡红细胞吞噬实验检测其吞噬能力；随后将布鲁氏菌(Brucella) M5疫苗株侵染该细胞系，在不同时期统计胞内活菌数。【结果】成功分离并构建了山羊肺泡巨噬细胞系，并且具有典型的巨噬细胞形态学特征，呈现不规则形状，有伪足伸出，胞体较大，吞噬能力强；体外培养10 d，有95.40%原代细胞特异性表达CD₁₄，永生化后仍有92.4%的细胞特异性表达CD₁₄；相较于另外3种细胞，永生化后的肺泡巨噬细胞对布鲁氏菌敏感性与RAW264.7细胞近似，高于绵羊间质睾丸细胞(sheep testicular interstitial cell, STIC)。【结论】成功建立永生化山羊肺泡巨噬细胞系，该细胞系为研究布鲁氏菌的致病机制及免疫逃逸机制提供了体外试验对象。

关键词:SV40LT;慢病毒;布鲁氏菌;攻毒模型;流式细胞术

Abstract:

[Background] Although a variety of human and murine cell lines have been utilized as models for Brucella infection in vitro, there remains a lack of large animal cell models that can accurately simulate the infection process. [Objective] To obtain a stable passable goat alveolar macrophage line with single cloning characteristics and optimize the goat cell infection model of Brucella infection. [Methods] Goat alveolar macrophages were isolated by perfusing goat lungs with PBS under sterile conditions. An immortalized goat alveolar macrophage cell line was established by transfection with SV40 large T antigen (SV40LT) and puromycin resistance screening. The characteristic surface marker of mononuclear macrophages, CD₁₄, was detected by immunofluorescence and flow cytometry. The phagocytic ability of the established cell line was assessed with chicken red blood cells. Subsequently, the cell line was infected with Brucella vaccine strain M5, and the intracellular bacteria were counted at different time points. [Results] The alveolar macrophage cell line of goat was successfully isolated and constructed, and it had typical macrophage morphological characteristics, showing an irregular shape, pseudopodium protruding, large cells, and strong phagocytosis. After 10 d of cultivation, 95.40% of the primary cells specifically expressed CD₁₄, and 92.4% of the cells specifically expressed CD₁₄ after immortalization. Compared with the other three cell lines, the immortalized alveolar macrophage cells were sensitive to Brucella, with the sensitivity similar to that of RAW264.7 cells and higher than that of sheep testicular interstitial cell (STIC). [Conclusion] An immortalized goat alveolar macrophage cell line was successfully established, providing an in vitro experimental object for studying the pathogenicity and immune escape mechanism of Brucella.

Key words:SV40LT;lentivirus;Brucella;cell challenge model;flow cytometry