致敏的脐血源树突状细胞诱导CTL特异杀伤白血病细胞的实验研究

In Vitro Investigation on Specific Anti-leukemia Cell Effect of CTL Induced by Sensitized Dendritic Cells from Umbilical Cord Blood

探讨脐血单个核细胞(MNC)诱导的树突状细胞(DC)通过负载冻融的HL-60、K562细胞抗原体外诱导产生细胞毒性T淋巴细胞(CTL)对HL-60、K562的杀伤作用。取脐血12份,分离MNC。在MNC中加入细胞因子GM-CSF(granulocyte monocyte colony-stimulating factor)、IL-3(interleukin3)、SCF(stem cell factor)和EPO培养4周。使用CD83、CD1a、CD11c和CDw123单克隆抗体、流式细胞仪测定培养前后脐血DC抗原变化及扩增情况。DC通过负载HL-60、K562白血病细胞抗原致敏T淋巴细胞产生CTL。3H-TdR掺入试验测定DC免疫刺激活性,MTT法观察CTL对HL-60、K562细胞的特异性杀伤活性。结果表明:新鲜脐血CD1a+、CD11c+、CD83+、CDw123+细胞数分别为0.27×105/ml、5.87×105/ml、1.94×105/ml、2.73×105/ml。加入上述细胞因子培养的脐血MNC分化为CD1a+、CD11c+、CD83+、CDw123+DC,经培养2-4周,DC数明显增多,分别达11.02×105/ml、28.24×105/ml、10.57×105/ml、18.7×105/ml,此后逐渐减少。细胞因子诱导脐血DC具有免疫刺激活性,且DC与CBMNC细胞比例为1∶40时的刺激活性最佳。冻融法得到的HL-60、K562白血病细胞抗原致敏DC诱导的CTL对HL-60、K562细胞的杀伤率分别为(42.04±8.46)%和(31.25±11.07)%,与实验组比较有显著性差异(p<0.01)。结论:加入细胞因子GM-CSF、IL-3、SCF和EPO培养2-4周的脐血MNC可分化为CD1a+、CD11c+、CD83+、CDw123+DC。冻融法得到的HL-60、K562白血病细胞抗原致敏DC,其诱导的CTL对HL-60、K562细胞具有特异的杀伤作用。脐血DC作为抗原呈递细胞在肿瘤免疫治疗上将起到重要作用。

This study was aimed to investigate the specific anti-leukemia cell effect of cytotoxic T lymphocytes （CTLs） induced by HL-60 or K562 cell-sensitized dendritic cells （DCs） from umbilical cord blood. 12 units of human umbilical cord blood （UCB） were collected and the mononuclear cells （MNCs） were isolated from UCB, then cultured with granulocyte monocyte colony- stimulating factor （ GM-CSF）, interleukin 3 （ IL-3 ）, recombinant human stem cell factor（SCF） and EPO for 3 - 4 weeks. Flow cytometry was used to determine the number of DCs and cell surface antigens before and after culture with monoclonal antibodies including CD83, CD1 a, CD11 c and CDw123. HL-60 and K562 were frozen-thawed, and released their tumor antigen peptides（TAP）. The CTLs were produced by sensitizing T lymphocytes with DC-loaded HL-60 and K562 cell antigens. The test of 3H-TdR incorporation was used to detect the immunostimulation activity of DCs. MTT assay was applied to evaluate specific cytotoxicity of CTL on leukaemia cells. The results indicated that the MNCs of UCBs cultured with GM-CSF, IL-3, EPO and SCF were shown to differentiate into CD1a^＋CD11c^＋CD83^＋CDw123^＋DCs. Numbers of DCs from UCBs remarkably increased in 2 -4 weeks and then decreased. After culture with cytokines DCs increased （ 10.6 -28.2）×10^5/ml in actual numbers. The CTL induced by DC pulsed with HL-60, K562 frozen-thawed lysates were effective to kill HL-60 and K562. Cytotoxicity of CTL to HL60 and K562 were （42.04 ± 8.46 ） % and （ 31.25 ± 11.07 ） % respectively. It is concluded that the MNCs of UCBs cultured with cytokines of GM-CSF, SCF, EPO and IL-3 can differentiate into CD1a^＋ CD83^＋ CD11c^＋and CDw123^＋DCs. The CTL induced by DCs pulsed with HL-60, K562 frozen-thawed lysates can effectively kill HL-60 and K562. These DCs as antigen presenting cells play an importamt role in cancer immunotherapy.