背景:分枝杆菌细胞壁部分(MCWF)源自非致病性草分枝杆菌,在临床实践中用作免疫调节化合物,然而,它的作用方式需要进一步研究。
目的:通过酶联免疫吸附试验(ELISA)和定量逆转录(qRT)-PCR评估犬外周血单个核细胞(PBMC)中宿主对MCWF的反应。
方法:八只健康的拉布拉多猎犬。
方法:使用密度离心从全血中分离PBMC。用不同浓度的MCWF或细胞因子产生的有效刺激剂培养细胞。佛波醇12-肉豆蔻酸酯13-乙酸酯/离子霉素,或在细胞培养基中放置24、48和72小时。通过ELISA测量白细胞介素(IL)-4,IL-10和干扰素-γ(IFN-γ)的细胞因子,并通过qRT-PCR检测IL-4、IL-10、IL-13、IFN-γ、肿瘤坏死因子α(TNF-α)和转化生长因子-β。
结果:对于所有浓度的MCWF,在所有时间点均检测到IL-10信使核糖核酸(mRNA)的显着增加(p<0.05)。蛋白质分析反映了这一发现,IL-10的最大浓度为300.6±38.3μg/mL。与阴性对照相比,在所有浓度的MCWF(p<0.01)的24小时,刺激后IFN-γmRNA升高。对于0.5μg/dLMCWF,TNF-αmRNA仅在72h增加(p<0.05)。
结论:MCWF刺激PBMC导致促炎和调节细胞因子mRNA的升高。应考虑进一步研究MCWF作为全身给药的调节性免疫调节剂或过敏原特异性免疫疗法的佐剂的作用。
BACKGROUND: Mycobacterium cell wall fraction (MCWF) is derived from nonpathogenic Mycobacterium phlei and is used as an immunomodulatory compound in clinical practice, yet its mode-of-action requires further research.
OBJECTIVE: To evaluate the host response to MCWF in canine peripheral blood mononuclear cells (PBMCs) by using enzyme-linked immunosorbent assays (ELISA) and quantitative reverse transcription (qRT)-PCR for assessment of cytokines.
METHODS: Eight healthy Labrador retrievers.
METHODS: PBMCs were isolated from whole blood using density centrifugation. The cells were cultured with different concentrations of MCWF or a potent stimulator of cytokine production, phorbol 12-myristate 13-acetate/ionomycin, or left in cell culture medium for 24, 48 and 72 h. Cytokines were measured by ELISA for interleukin (IL)-4, IL-10 and interferon-gamma (IFN-γ), and by qRT-PCR for IL-4, IL-10, IL-13, IFN-γ, tumour necrosis factor alpha (TNF-α) and transforming growth factor-beta.
RESULTS: A significant increase of IL-10 messenger ribonucleic acid (mRNA) was detected at all time points for all concentrations of MCWF (p < 0.05). Protein analysis reflected this finding, with a maximum IL-10 concentration of 300.6 ± 38.3 μg/mL. Compared to the negative control, post-stimulation elevation of IFN-γ mRNA was noted at 24 h with all concentrations of MCWF (p < 0.01), and TNF-α mRNA was increased for 0.5 μg/dL MCWF only at 72 h (p < 0.05).
CONCLUSIONS: MCWF stimulation of PBMCs results in the elevation of both proinflammatory and regulatory cytokine mRNA. Further research into the role of MCWF as a systemically administered regulatory immunomodulator or adjuvant to allergen-specific immunotherapy should be considered.