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目的研究K562冻融抗原负载的健康供者来源的树突状细胞(DC)诱导细胞毒性T淋巴细胞(CTL)体外杀伤慢性粒细胞白血病(CML)细胞的毒性效应。方法利用健康供者外周血单个核细胞诱导分化为DC,采用反复冻融法从K562中提取的可溶性相关抗原负载DC;流式细胞学检测负载抗原前后DC表面分子表达的变化;ELISA法检测DC上清中IL-12和IFNγ的含量;混合淋巴细胞反应(MLR)测定DC体外刺激T细胞增殖的能力;乳酸脱氢酶法检测K562冻融抗原负载DC诱导的抗原特异性CTL对CML细胞的杀伤作用。结果与未经抗原负载的DC相比,经K562抗原负载的DC表面分子表达明显上调,CD1a(27·40±5·00)%、(15·40±2·34)%,CD80(61·35±5·35)%、(42·00±2·77)%,CD83(93·30±3·48)%、(25·15±4·02)%,CD86(85·25±4·39)%、(37·25±3·20)%,CD40(89·80±7·18)%、(35·95±4·06)%,HLA-DR(49·50±5·45)%、(17·15±3·61)%,DC分泌IL-12和诱导T细胞分泌IFNγ的能力增加(P<0·05),具有很强的刺激T细胞增殖能力,且刺激强度在24h最强,48h降低,冻融抗原负载DC后激活的CTL在体外对K562的杀伤率为77·35%,显著高于未经抗原负载的DC(P=0·001)。结论经K562细胞冻融抗原负载DC激活的CTL在体外具有更强的增殖能力和杀伤CML细胞的作用。
Objective To study the toxic effects of dendritic cells (DCs) derived from K562 cells on the cytotoxic T lymphocytes (CTLs) in vitro killing chronic myeloid leukemia (CML) cells. Methods DCs were differentiated into DCs from peripheral blood mononuclear cells of healthy donors. DCs were extracted from K562 cells by repeated freeze-thaw cycles. DCs were analyzed by flow cytometry before and after antigen-loaded DCs. DCs were detected by ELISA The content of IL-12 and IFNγ in the supernatant; the ability of DC to stimulate the proliferation of T cells in vitro by mixed lymphocyte reaction (MLR); the effect of DC-induced antigen-specific CTL loaded by K562 freeze-thaw antigen on lactate dehydrogenase Killing effect. Results Compared with non-antigen-loaded DCs, the expression of DCs molecules on K562 antigen-loaded DCs were significantly upregulated, with CD1a (27.4 ± 5.00)%, (15 · 40 ± 2.34)% and CD80 35 ± 5 · 35%, 42 · 00 ± 2 · 77%, CD83 (93 · 30 ± 3.48)%, (25 · 15 ± 4 · 02)%, CD86 (37 · 25 ± 3.20)%, CD40 (89 · 80 ± 7 · 18)%, (35.95 ± 4.60)%, HLA-DR %, (17.15 ± 3.61)% respectively. The ability of DCs to secrete IL-12 and induce the secretion of IFNγ by T cells increased (P <0.05), and had a strong stimulation of T cell proliferation. Strongest, and decreased at 48h. CTLs activated by freeze-thaw antigen loaded DCs exhibited a K-kill rate of 77.35% in vitro, which was significantly higher than that of non-antigen-loaded DCs (P = 0.001). Conclusion CTL activated by K562 cells freeze-thawed antigen-loaded DC has a stronger ability to proliferate and kill CML cells in vitro.