术中失血来源的树突状细胞用于肝癌治疗的体外实验研究

Induction of dendritic cells from intra-operative lost blood and their effects on CIK against liver cancer cells in vitro

目的探讨从肝癌患者术中失血来源的单个核细胞中培养树突状细胞（DC）的可行性，为个体化的DC介导免疫治疗提供新的细胞来源。方法采集9例原发性肝细胞癌患者术中失血及8例脐血，分离其单个核细胞，其中贴壁的单个核细胞经重组人粒细胞巨噬细胞集落刺激因子（RHGM—CSF），重组人白细胞介素4（rhIL-4）诱导和负载癌细胞抗原，制成不同的DC瘤苗，悬浮的单个核细胞经细胞因子处理成为细胞因子诱导的杀伤细胞（CIK）。采用二苯基溴化四氮唑蓝（MTT）法测定DC激活同源CIK的相对增殖率和CIK对肝癌细胞的杀伤效果。结果肝癌患者术中失血来源的单个核细胞在体外能够诱导分化为具有典型形态和表型的DC。术中失血来源的DC表面标志物表达水平低于脐血来源的DC，但二者均能有效地激活CIK，并增强其对肝癌细胞的杀伤活性。负载患者自身癌细胞抗原的术中失血DC和脐血DE，其激活的CIK增殖率分别为（388．9±137．3）％和（315．1±44．5）％，对肿瘤细胞的杀伤率分别为（87．1±8．0）％和（90．0±5．1）％；而负载SMMC-7721抗原的术中失血DC和脐血DC，其激活的CIK增殖率分别为（239．9±48．7）％和（226．3±32．3）％，对肿瘤细胞的杀伤率分别为（76．4±7．9）％和（81．1±4．3）％。在激活CIK和增强对肝癌细胞杀伤能力方面，相同抗原负载的两种DC差异无统计学意义，但负载自身抗原优于负载SMMC-7721抗原。结论肝癌患者术中失血来源的DC可有效激活CIK，并增强其对肝癌细胞的杀伤效应，为临床研究和应用DC瘤苗提供了一个新的来源。

Objective To investigate the practical possibility of inducing dendritic cells （DCs） from mononuelear cells in the lost blood during operation of hepatocellular carcinoma （HCC） patients, and attempted to find a new source of precursor cells for the personalized immunotherapy based on DCs. Methods Collected lost blood during hepatectomy from 9 HCC patients and human cord blood from 8 cases of healthy donors undergoing caesarean section. Their mononuelear cells were divided into monoeytes and nonadherent lymphoeytes. RhGM-CSF and rhIL-4 were administered to induce the monoeytes differentiation into DCs, and then loaded with different antigens （ lysate antigen of autologous liver cancer cells and cell llne SMMC-7721 cells）. The lymphoeytes were induced into cytokine-induced killer cells （CIK） with IL-2, CD3-Ab, γ-IFN and PHA. MTT assay was performed to detect the proliferation rate of T lymphoeytes mediated by DC and the cytotoxicity of CIK to liver cancer cells. Results DCs induced from monocytes of the intra-operative lost blood possessed typical morphology and phenotypes. Compared with the DCs from cord blood, the DCs from intra-operative lost blood expressed lower level of surface markers, but both could effectively induce proliferation of CIK and enhance the cytotoxieity of activated CIK against liver cancer cells at similar levels. When the DCs from lost blood and their counterpart from cord blood were both loaded with autologous tumor cell antigen, the proliferation rates of CIK were （388.9 ± 137. 3 ）% and （315. 1 ± 44.5） % , respectively, and the killing rates against tumor cells were （87. 1 ± 8.0 ）% and （90. 0 ± 5.1）%, respectively. When the two similar DC groups were loaded with lysate antigen of SMMC-7721 cells, the proliferation rates of CIK were （239.9 ± 48.7） % and （226.3 ± 32.3 ） %, respectively, and the killing rates against tumor cells were （76.4 ±7.9）% and （81.1 ±4.3）%, respectively. There were no significant differences between those two DC groups. The data also showed that the proliferation and cytotoxicity of CIK induced by DCs loaded with autologous antigen were higher than that of DCs loaded with SMMC-7721 antigen. Conclusion Mononuclear cells separated from intra-operative lost blood of HCC patients can be induced into mature DCs, which can effectively activate CIK and significantly increase its killing effect on the liver cancer cells, and may become a new source of DCs to study and develop vaccines for clinical application.