猪PRRS抗性筛选单核细胞来源巨噬细胞模型的建立

Establishment of porcine monocytes-derived macrophages(MDMs) cell model for evaluation of PRRS resistance

采用密度梯度离心法分离猪外周血单核细胞(PBMCs),通过添加GM-CSF刺激使PBMCs转化为外周血单核细胞来源巨噬细胞(MDMs);比较了猪繁殖与呼吸综合征病毒(PRRSV)感染MDMs和肺泡巨噬细胞(PAMs)不同时间的病毒载量及病毒TCID50,研究了PRRSV体外感染猪MDMs后复制和滴度的变化规律。结果表明:PBMCs转化的MDMs具有很好的墨汁吞噬能力,MAC387标记阳性;GM-CSF刺激PBMCs转化为MDMs的最适质量浓度为20 ng.mL-1,刺激时间为72 h时MDMs转化率最高;PRRSV体外感染12 h时MDMs中病毒载量达到最高值,随后迅速下降;细胞上清液的病毒滴度在12～24 h进入平台期,然后转入衰亡期,与PRRSV在PAMs中增殖趋势相似。本试验建立的PRRS抗性筛选MDM模型,具有受检猪只损伤小、简便易行的特点,可代替PAM细胞模型用于评价猪对PRRS的抗性。

Monocytes-derived macrophages（MDMs） are important target cells of porcine reproductive and respiratory syndrome virus （PRRSV）. Peripheral blood mononuclear cell （PBMCs）, the precursor cells of MDMs, can be obtained conveniently and less expensively than porcine alveolar macrophages（PAMs）. The establishment of MDM eell model for evaluation of PRRS resistance is helpful for the pig breeding of resistance. PBMCs were isolated by density gradient eentrifugation. The transformation of PBMCs into MDMs was induced by GM-CSF. The virus load and viral titer of MDMs and PAMs were compared in different time post PRRSV in- fection. Results showed that MDMs had an active ink phagocytosis capacity and MAC387 positive staining. Moreover, the optimal con- centration of GM-CSF was 20 ng. mL-1 , and the proper time was 72 h. Furthermore, MDMs viral load reached a peak at 12 h post in- fection, then decreased rapidly, the virus titers of cell supernatant reached plateau phase during 12-24 h. PRRSV proliferation trend in MDMs was similar with that in PAMs. This experiment successfully established a non-destructive and simple MDMs model to evaluate PRRS resistance to replace traditional PAMs.