豚鼠腹腔巨噬细胞的分离培养及免疫抗结核功能检测

发布时间：2018-11-01 13:27

【摘要】：巨噬细胞(macrophages,Mφ)在机体正常发育、体内平衡、组织修复和对病原体的免疫反应中起着重要作用。为了研究豚鼠(Cavia porcellus)Mφ在结核免疫中的作用及机制,本研究通过淀粉肉汤溶液预刺激的方法分离培养了豚鼠腹腔Mφ,并进行了细胞形态学观察、细胞活力、吞噬功能、非特异性酯酶染色和细胞表面特异性抗原表达检测等一系列鉴定。在此基础上,利用结核分枝杆菌(Mycobacterium tuberculosis,MTB)卡介苗(Bacillus Calmette-Guérin,BCG)感染分离培养的Mφ,并分析BCG对MφNO的产生和细胞凋亡的影响。结果表明,分离培养的腹腔Mφ具有Mφ所特有的形态特征、较强细胞活力和吞噬能力,并可检测到特异性的白细胞分化抗原14(cluster of differentiation 14,CD14)、CD40和CD68等的表达。与未感染的对照组相比,BCG感染24 h后Mφ产生的NO极显著增加(P0.01),细胞的凋亡率也极显著升高(P0.01)。表明淀粉肉汤溶液刺激的方法可成功获取豚鼠腹腔Mφ,同时证实BCG感染后能诱导其产生的NO增多,并且细胞凋亡率增加,提示其可能在抗结核免疫中起重要作用。研究结果为MTB和Mφ相互作用研究提供细胞模型和数据支撑。
[Abstract]:Macrophages (macrophages,M 蠁) play an important role in normal development, balance in vivo, tissue repair and immune response to pathogens. In order to study the role and mechanism of guinea pig (Cavia porcellus) M 蠁 in tuberculosis immunity, we isolated and cultured guinea pig peritoneal M 蠁 by starch broth solution prestimulation, and observed the cell morphology, cell viability and phagocytosis. A series of non-specific esterase staining and cell surface specific antigen expression detection and a series of identification. On this basis, M 蠁 was infected by Bacillus Calmette-Gu 茅 rin,BCG (Bacillus Calmette-Gu 茅 rin,BCG), and the effect of BCG on the production and apoptosis of M 蠁 NO was analyzed. The results showed that the isolated and cultured peritoneal M 蠁 had the characteristic morphological characteristics of M 蠁, strong cell viability and phagocytosis, and the expression of specific leukocyte differentiation antigen 14 (cluster of differentiation 14 CD14), CD40 and CD68 could be detected. Compared with the uninfected control group, the NO produced by M 蠁 was significantly increased (P0.01) and the apoptosis rate was significantly increased (P0.01) after 24 h of BCG infection. The results showed that the starch broth solution stimulation method could successfully obtain M 蠁 in guinea pig abdominal cavity. It was also confirmed that BCG infection could induce the increase of NO production and the increase of apoptosis rate, which suggested that it might play an important role in anti-tuberculosis immunity. The results provide cell model and data support for the study of interaction between MTB and M 蠁.
【作者单位】：浙江理工大学生命科学学院;
【基金】：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) 浙江理工大学科研启动基金项目(No.15042016-Y)
【分类号】：R392

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