恒河猴外周血单核巨噬细胞体外培养方法的建立

Establishment of a method for culture in vitro of peripheral blood monocytes from rhesus macaque

目的 建立一种简单、经济、高效的培养恒河猴外周血单核巨噬细胞（monocyte-derived macrophage,MDM）的方法.方法 用肝素钠抗凝管采集成年恒河猴（Macaca mulatta）全血,密度梯度离心法分离外周血单核细胞（peripheral blood mononuclear cells,PBMCs）.同时用无抗凝剂采血管采集同一只猴外周血,自凝后分离血清.将猴PBMCs置于CellBIND Surface的96孔（0.8 ×10^6个细胞/孔）或48孔培养板（3×10^6个细胞/孔）中,用含不同百分比的猴自体血清或胎牛血清（fetal calf serum,FCS）的RPMI 1640培养液培养24 h后洗弃未贴壁细胞,加入含有猴自体血清或FCS的新鲜培养基继续培养7d后观察细胞形态学.分化良好的猴单核巨噬细胞贴壁能力强,占据板底大部分区域.胞体形态多样,多数呈长梭形.用巨噬细胞标记受体（CD14）抗体染色判断细胞纯度.并用细菌内毒素（LPS）刺激分化的巨噬细胞,检测巨噬细胞炎性因子的表达.此外,用猴艾滋病毒（SIVmac17E-Br、SIV-mac251）和人-猴嵌合体艾滋病毒（SHIV KU-1）感染分化良好的猴巨噬细胞,检测病毒在猴巨噬细胞中的复制.结果 在含2％猴自体血清的RPMI 1640培养条件下,大多数（＞85％）猴单核细胞能在24 h内贴壁,体外分化5-7d后,猴巨噬细胞纯度大于96％.相比而言,含较高浓度（4％,8％或10％）猴自体血清或FCS的RPMI 1640培养基对猴单核细胞的贴壁和分化作用较差.分化良好的猴巨噬细胞对LPS刺激敏感,可产生多种巨噬细胞炎性因子.此外,这些细胞对SIV或SHIV均易感,产生感染性病毒.结论 含2％猴自体血清的RPMI 1640培养基适于原代猴单核细胞的贴壁和分化.该方法简单、花费少,无需生长因子,且分化效果好,是培养猴艾滋病毒及开展相关免疫学实验的重要手段.

Objectives To establish a simple, inexpensive and efficient technique for in vitro culture of monocyte- derived macrophages （MDM） from rhesus macaques of Chinese origin. Methods Peripheral blood of healthy rhesus ma- caques （Macaca mulatta） were obtained in heparinized vacutainer collection tubes. Peripheral blood mononuclear cells （PBMCs） were isolated from blood by Ficoll gradient centrifugation. Serum was isolated from peripheral blood of the autol- ogous animals. PBMCs were plated in 48-well-plate （3 x 106 cells/well） or 96-well-plate （0.8 x 106 cells/well） for 24 h. After removal of non-adherent cells from the culture, monocytes were cultured in RPMI 1640 supplemented with different proportions （2% , 4% , 8% , 10% ） of autologous serum or fetal calf serum （FCS） for 7 days. To examine the biological function of MDM, lipopolysaccharide （LPS） was added to MDM cuiture to determine inflammatory cytokine production.Also, MDM cultures were tested for the susceptibility to simian immunodeficiency virus （SIV） or simian-human immunode- ficiency virus （SHIV） infections. Results The cell cultures with RPMI1640 containing 2% autologous serum yielded the best results with regard to macrophage morphology, the response to LPS stimulation and susceptibility to SIV or SHIV infec- tion. The purity of adherent macrophages under condition of 2% autologous serum culture was higher than 96%. Conclu- sions RPMI 1640 with 2% autologous serum is suitable for culture in vitro of peripheral blood monocytes from rhesus ma- caques. This technique is simple, inexpensive, no need for growth factor and highly effective to obtain adherent and well differentiated macaque monocytes. Therefore, this method provides an important tool for culture of macaque AIDS viruses and for related immunological research.