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目的比较无血清培养液AIM-V和RPMI1640+胎牛血清(FBS)培养液培养大肠癌患者外周血来源树突状细胞(DCs)的形态和免疫表型,检测DCs对T细胞的活化作用。方法对2003年3月至2004年10月首都医科大学附属北京朝阳医院消化内科收治的10例大肠癌患者,采集其外周血分离单核细胞,分别使用2种细胞培养液进行体外培养。观察细胞形态,检测细胞表面CD1a,CD80,CD86,CD83,CD14及HLA-DR的表达。再用丝裂霉素作用后的DCs作为刺激细胞,按照刺激细胞反应细胞(S∶R)1∶1,1∶10,1∶20,1∶40,1∶80的比例进行同种异体混合淋巴细胞反应(MLR),检测DCs刺激同种异体T细胞增殖的能力。结果大肠癌患者外周血单核细胞经2种细胞培养液培养后都可以诱导出DCs。细胞外型均呈毛刺状。细胞均表达CD1a、CD80、CD86和人类白细胞抗原(HLA-DR),不表达CD14、CD83,为不成熟DCs的特点。两组DCs细胞表型表达差异无显著性(P>0.05)。按S∶R不同比例进行同种异体MRL所得刺激指数分别为2.7,4.1,3.1,2.5,1.6,表明树突状细胞(DCs)在不同比例下都刺激T细胞增殖。结论使用AIM-V无血清培养液和传统的含FBS细胞培养液均可成功诱导培养大肠癌患者外周血来源的DCs,但使用AIM-V可以降低含血清培养液应用人体后引起的过敏反应等风险,更适合DCs的临床使用,可以替代含血清培养液。MLR结果显示,DCs在各种S∶R比例下均有刺激T细胞增殖的能力,但S∶R比为1∶10时最佳。
Objective To compare the morphology and immunophenotype of peripheral blood-derived dendritic cells (DCs) cultured with AIM-V and RPMI1640 + fetal bovine serum (FBS) in serum-free medium and detect the activation of T cells by DCs. Methods From March 2003 to October 2004, 10 patients with colorectal cancer who were admitted to Department of Gastroenterology, Beijing Chaoyang Hospital, Capital Medical University were enrolled. Peripheral blood mononuclear cells (PBMCs) were collected and cultured in vitro using two kinds of cell culture medium. The cell morphology was observed and the expression of CD1a, CD80, CD86, CD83, CD14 and HLA-DR on the cell surface was detected. DCs treated with mitomycin were used as stimulator cells and allogeneic cells were mixed at a ratio of 1: 1, 1:10, 1:20, 1:40 and 1:80 for stimulating cellular reaction cells (S: R) Lymphocyte reaction (MLR), the ability of DCs to stimulate allogeneic T cell proliferation. Results DCs were induced in peripheral blood mononuclear cells of patients with colorectal cancer after culture in two kinds of cell culture medium. Cells were burr-shaped appearance. Cells express CD1a, CD80, CD86 and human leukocyte antigen (HLA-DR), do not express CD14, CD83, is characterized by immature DCs. There was no significant difference in the phenotypes of DCs between the two groups (P> 0.05). Stimulation indices of allogeneic MRLs at different ratios of S: R were 2.7, 4.1, 3.1, 2.5, and 1.6, respectively, suggesting that dendritic cells (DCs) stimulate T cell proliferation at various ratios. Conclusion Both AIM-V serum-free medium and traditional FBS-containing culture medium can successfully induce DCs derived from peripheral blood of patients with colorectal cancer. However, the use of AIM-V can reduce the allergic reactions caused by the application of human serum with serum-containing medium Risk, more suitable for the clinical use of DCs, can replace serum containing medium. MLR results showed that DCs had the ability of stimulating T cell proliferation at various S: R ratios, but the S: R ratio of 1:10 was the best.