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目的探讨树突细胞联合细胞因子诱导的杀伤细胞(DC-CIK)生物学特性和体外杀瘤机制。方法从白血病患儿外周血分离单个核细胞,经过γ干扰素(IFN-γ)、抗CD3单克隆抗体(CD3McAb)、IL-2诱导并与树突细胞(DC)共培养后,获得DC-CIK。采用MTT法检测DC-CIK对多种白血病细胞株的杀伤作用。在予以10mg.L-1、20mg.L-1小鼠抗人淋巴细胞功能相关抗原-1(LFA-1)单克隆抗体处理后,流式细胞仪检测CD4+CD25+Treg细胞比例,RT-PCR与Westernblot方法检测Foxp3基因表达水平。结果诱导后的DC-CIK细胞形态规则,其对肿瘤细胞B95、Jhhan、M07e均表现出杀伤活性。其中对B95细胞的杀伤作用较强,在效靶比为51、101时DC-CIK对B95细胞的杀伤作用均>60%,而对Jhhan、M07e的作用不明显。在予以10mg.L-1、20mg.L-1LFA-1单克隆抗体处理后,与阴性对照组比较,2个处理组均使CD4+CD25+Treg细胞表达增加(t=6.783、7.282,Pa<0.05)。RT-PCR与Westernblot结果表明与阴性对照组比较,2个处理组均使Foxp3基因mRNA表达水平增加(t=11.671、10.909,Pa<0.05),Foxp3基因蛋白表达水平增加(t=16.734、9.562,Pa<0.05)。结论 DC-CIK具有强大的体外杀瘤作用,Foxp3基因参与LFA-1介导的DC-CIK杀瘤途径,其杀瘤机制表现为CD4+CD25+Treg细胞途径受抑制。
Objective To investigate the biological characteristics of dendritic cells combined with cytokine-induced killer cells (DC-CIK) and the mechanism of killing tumor cells in vitro. Methods Mononuclear cells were isolated from peripheral blood of leukemia children and induced by IFN-γ, anti-CD3 monoclonal antibody (CD3McAb) and IL-2 and co-cultured with dendritic cells (DCs) CIK. The killing effect of DC-CIK on a variety of leukemia cell lines was detected by MTT assay. After treated with 10μg.L-1, 20mg.L-1 mouse anti-human lymphocyte function-associated antigen-1 (LFA-1) monoclonal antibody, the proportion of CD4 + CD25 + Treg cells was detected by flow cytometry, PCR and Western blotting were used to detect Foxp3 gene expression. Results The morphology of DC-CIK cells after induction showed regular cytotoxic activity against tumor cells B95, Jhhan and M07e. The cytotoxicity against B95 cells was stronger. When the target ratio was 51,101, the killing effects of DC-CIK on B95 cells were both> 60%, while the effect on Jhhan and M07e was not obvious. After treatment with 10 mg.L-1 and 20 mg.L-1 LFA-1 monoclonal antibodies, the expression of CD4 + CD25 + Treg cells was increased in both treatment groups compared with the negative control group (t = 6.783, 7.282, Pa < 0.05). The results of RT-PCR and Western blot showed that Foxp3 mRNA expression increased (t = 11.671,10.909, Pa <0.05) and Foxp3 gene protein increased (t = 16.734,9.562, Pa <0.05). Conclusions DC-CIK has a strong killing effect in vitro. The Foxp3 gene is involved in the DC-CIK killing pathway mediated by LFA-1, and its killing mechanism is inhibited by the pathway of CD4 + CD25 + Treg cells.