摘要
本研究的目的是建立对外周血来源的树突状细胞(dendritic cell,DC)的无血清培养方案。以含胎牛血清(FCS)、人AB血清的培养液作为对照。分离健康志愿者外周血单个核细胞(PBMNC),在不同培养液中分别加入GM-CSF(100ng/ml)、IL-4(500U/ml)培养6天,再分别加入钙离子载体A23187(100ng/ml)继续培养24小时,于倒置显微镜下观察细胞形态,流式细胞术分析细胞表型,MTT比色法检测各组DC刺激同种异体外周血T细胞增殖的能力和DC刺激的T细胞对K562细胞的杀伤作用。结果表明:无血清培养组培养出典型形态的DC,其表面CD14分子的表达明显减少,CD83、HLA-DR、CDw123分子的表达明显增高,具有明显的刺激同种异体T细胞增殖的能力和DC刺激的T细胞对K562细胞的杀伤作用。无血清组与两个含血清的对照组比较,组间无显著性差异(P>0.05)。结论:无血清培养液培养的DC与含血清的培养液培养的DC相比,无显著性差异。DC无血清培养具有临床应用前景。
This study was aimed to investigate the protocol in vitro to incubate the dendritic cell （DC） derived from peripheral blood monocytes using serum-free medium X-VIVO 20. Peripheral blood monocytes from healthy donors were treated with 100 ng/ml GM-CSF and 500 U/ml IL-4, respectively. After cultivation for 6 days, they were treated with 100 ng/ml calcium ionophore A23187. After cultivation for 24 hours the cellular morphology was observed under invert microscope, the surface markers were analyzed by flow cytometry, the proliferation of allogenetic T cells was detected by MTT colorimetry, the specific cytotoxicity of T cells primed with DC was examined by MTU assay. The results showed that in all three groups with serum-free, fetal calf serum （FCS） and human AB serum mediums, cells displayed characteristic morphological features of DC. Simultaneously CD14 expression was decreased, and CD83, HLA-DR and CDw123 expression were increased on these cells. In addition, DCs cultured with these methods could evidently stimulate the proliferation of allogenetic T cell. As compared with the two controls of serum containing groups, the cultured cells in the serum-free groups showed almost the same allo-stimulatory capability and cellular morphology and surface markers, and T lymphocytes primed with the culture-derived DC exhibited the similar killing activity to K562 （ P 〉 0. 05 ）. It is concluded that there is no significance in DC numbers, morphology, epitope and ability to stimulate the proliferation of allogenetic T cells between DC induced by serum-free X-VIVO 20 medium and DC induced by serum-contained medium. DC cultured and induced by serum-free medium is worth using in practice widely.
出处
《中国实验血液学杂志》
CAS
CSCD
2006年第5期985-989,共5页
Journal of Experimental Hematology
