人外周血树突状细胞的分离与鉴定

Isolation and identification of dendritic cells from human peripheral blood

目的:体外分离培养并鉴定人外周血树突状细胞,并观察其抗原呈递功能。方法:实验于2005-05/2006-11在南方医科大学南方医院肿瘤中心生物治疗实验室完成。从人类白细胞抗原A2表达阳性的健康人外周血中分离获得单个核细胞。培养5h后洗涤贴壁细胞,加入含有10%人AB血清的RPMI1640培养基,及重组人粒细胞-巨噬细胞集落刺激因子和重组人白细胞介素4,于培养的第1,3,6天对树突状细胞的形态、表型进行分析,并定期检测树突状细胞的纯度与得率。抽取与以上树突状细胞不同来源的其他健康人外周血。经淋巴细胞分离液分离后,获取非贴壁细胞,用含10%人AB血清的1640培养基重悬,加入白细胞介素2继续孵育6d,作为同种异体T淋巴细胞。将树突状细胞分为两组,一组按常规方法培养6d,另一组在培养至第5天时加入黑色素瘤抗原基因A3编码的多肽继续培养24h。在经紫外线处理后的96孔板中,分别加入树突状细胞悬液1×104,5×103,2×103,1×103细胞/每孔,以自身T淋巴细胞作为对照,每孔设3个复孔,分别加入1×105淋巴细胞/每孔。评价树突状细胞刺激T淋巴细胞增殖的能力。结果:①单个核细胞体外培养至第6天,可获得大量、90.81%高纯度的树突状细胞,能够较高地表达21.8%CD1a、99.0%HLA-DR、63.4%CD80、18.9%CD83和80.6%CD86。②将诱导培养6d获得的两组树突状细胞作为刺激细胞,以不同的浓度与同种异体淋巴细胞混合,均可产生增殖反应;经过黑色素瘤抗原基因A3编码的多肽处理的各种比例的树突状细胞,较相应未经黑色素瘤抗原基因A3编码的多肽处理的树突状细胞激发淋巴细胞增殖的能力明显增强,浓度相对较高的树突状细胞刺激效果最明显,能够强烈地激发同种混合淋巴细胞增殖。结论:得到了一群较高程度表达CD83、CD86和HLA-DR分子、体外可强烈激发同种异体T淋巴细胞增殖的树突状细胞群。

AIM： To isolate, culture and identify in vitro of dendritic cells （DCs） from human peripheral blood, and observe the function of antigen presentation. METHODS： The experiment was done from Mayo2005 to November 2006 in Biotherapy Laboratory Tumor Center, Nanfang Hospital, Southern Medical University. Peripheral blood monocytes were isolated from the healthy volunteers （who had known the facts）, and then cultured for 5 hours. The adherent cells were washed and then cultured with RPMI1640 medium containing 10% AB serum, granulocyte-macrophage colony stimulating factor （GM-CSF） and intedeukin-4 （IL-4）. The cellular morphous and phenotypes were examined, respectively at days 1, 3 and 6, and the cellular purity and yield were also detected. Peripheral blood was obtained from different healthy volunteers （who had known the facts）. The blood was isolated with lymphocyte separating-medium, and non-adherent cell were cultured with 1640 medium containing 10% AB serum and intedeukin-2 （IL-2）. At the sixth day, the allogeneic T lymphocytes were harvested. The DCs were divided into two groups： one group was cultured with regular process for 6 days; another group was cultured with Melanoma Associated Antigen Gene A3 （MAGE-A3） peptide on the 5= day. After another 24 hours, 1×10^4,5×10^3,2×10^3,1×10^3/per well of DCs were added in ultraviolet-treated 96-well plate. The auto-lymphocytes were used as control, and there were 3 sub-wells in eaqh well, and 1×10^5 lymphocytes were added in each well to evaluate the ability of DCs stimulating lymphocytes proliferation. RESULTS：①A large number of DCs with high purity （90.81%） were generated culturing for six days in vitro. DCs expressed highly 21.8%CD1a, 99.0%HLA-DR, 63.4%CD80, 18.9%CD83 and 80.6%CD86. ②Lymphocyte proliferation was induced by mixing allogeneic lymphocytes with variant proportional DCs cultured for 6 days. The ability of stimulating lymphocyte proliferation was significantly strengthened by various proportional DCs with MAGE-A3 pepUde than corresponding proportional DCs without MAGE-A3 peptide. The most powerful effectiveness was developed with higher density of DCs, which could intensively stimulating allogeneic lymphocyte proliferation in. mixed lymphocyte reaction. CONCLUSION： A group of dendritic cells, which highly express CD83, CD86 and HLA-DR molecules, and stimulate strongly allogeneic T lymphocyte proliferation in vitro, are obtained.