巴马猪NK细胞表型鉴定及猪CIK细胞诱导方法建立

Isolation,phenotype identification and activation of natural killer(NK) cells in Bama miniature pigs

目的获得巴马猪外周血淋巴细胞中NK细胞的表型及比例的数据,建立一种高效率猪CIK(cytokine-induced killer)细胞体外诱导培养的方法。方法分离巴马小型猪外周血淋巴细胞,通过检测CD2^+/CD8^+/CD3^-细胞以表征猪NK细胞的表型;通过优化诱导培养条件,提高诱导淋巴细胞中CIK的比例,建立高效率CIK细胞体外诱导方法。结果利用优化的诱导培养条件,在诱导培养第5天时CD3^-/CD2^+/CD8^+的NK细胞的比例可高达43.63%,较最初分离时NK细胞比例提升了5.59倍;细胞增殖活性实验表明诱导培养第5天时可见明显的3个荧光峰,表明诱导后细胞发生了3次分裂,理论上较最初分离增加了8倍;诱导细胞的qPCR表型分析表明该细胞群体在诱导培养第5天时相关的表面标志有明显的上升也与流式分析结果一致。结论建立了高效的猪CIK细胞体外诱导培养的方法。

Objective To describe the phenotype of NK cells in Bama miniature pigs,and establish an efficient activation and culture method for porcine cytokine-induced killer（ CIK） cells in vitro. Methods The porcine peripheral blood mononuclear cells（ PBMCs） were isolated by Percoll gradient centrifugation,and the phenotype of NK cells was tested by detecting the CD2^＋/ CD8^＋/ CD^3- cell compartment. To establish an efficient activation and culture method for porcine CIK cells,we optimized the culture conditions to improve the CIK activation efficiency. Results Using the optimized induction culture conditions,the ratio of CIK（ CD2^＋/ CD8^＋/ CD3^-） cells was up to 43. 63% at the fifth day,approximately 5. 59 times increased compared with the initially separated PBMCs. Cell proliferation experiments showed that three obvious fluorescence peaks were observed on the fifth day. The results indicated that the induced CIK cells underwent three times cell division,in theory,about increased 8-fold compared with the initial separation of PBMCs. Furthermore,the qRT-PCR result of the surface markers of porcine NK cells also showed a similar variation tendency as the flow cytometry results. Conclusions Our findings demonstrate the successful establishment of an efficient activation and culture method for porcine CIK cells in vitro.