肿瘤射频消融裂解物负载的DC—CIK细胞体外抗肿瘤活性实验研究

The research of antitumor activities in vitro of DCs loading antigen prouced by radiofrequency ablation of tumor combined with CIK cells

目的研究负载射频消融肿瘤原位裂解物的树突状细胞（Dc）联合细胞因子诱导杀伤活性细胞（CIK）体外抗肿瘤活性。方法制备BALB／C小鼠脾脏来源的CIK细胞及骨髓来源的Dc。建立射频消融灭活小鼠皮下结肠癌的实验模型，将其原位裂解的肿瘤组织反复冻融后取上清，lowry蛋白定量法定量，以终浓度5μg／ml载培养第5天的Dc（即Ag-Dc），2d后再与培养第7天的CIK细胞共培养48h，流式细胞术分析其表面共刺激分子的表达，细胞增殖与毒性检测试剂盒（CCK-8试剂盒）检测其体外杀伤活性。结果DC表面分子表达共刺激分子CD86＋ CD11c＋、MHCⅡ＋ CD11c＋、MHCⅡ＋ CD80＋双阳性细胞百分含量分别为9．50％、42．4％、53．4％；Ag-DC表面分子表达共刺激分子双阳性细胞百分含量明显提高，分别为19．2％、74．2％、61．1％。CIK细胞培养第1天，CD3＋ NK1．1＋双阳性的百分含量为1．45％，第7天CD3＋ NK1．1＋双阳性表达明显提高为36．9％。Ag—DC—CIK细胞对结肠癌细胞C26的杀伤活性明显高于DC-CIK、CIK细胞，且相同效靶比下前者的杀伤活性明显高于后者。效靶比为5：1时，Ag-DC-CIK细胞杀伤率为（74．9±3．5）％，DC-CIK细胞杀伤率为（71．2±2．1）％，CIK细胞杀伤率为（68．7±2．9）％，差异有统计学意义（F=7．007，P=0．007）；效靶比为10：1时，Ag—DC-CIK细胞杀伤率为（82．3±4．5）％，DC-CIK细胞杀伤率为（77．1±5．1）％，CIK细胞杀伤率为（72．7±2．8）％，差异有统计学意义（F=7．727，P=0．005）；效靶比为20：1时，Ag-DC-CIK细胞杀伤率为（83．2±1．9）％，DC-CIK细胞杀伤率为（77．2±4．2）％，CIK细胞杀伤率为（73．0±2．6）％，差异有统计学意义（F=16．594，P=0．000）。结论负载肿瘤射频消融原位裂解产物的DC联合CIK细胞可以提高体外细胞毒活性，为肿瘤综合治疗提供新策略。

Objective To study the in vitro anti-tumor activity of dendritic cells （DCs） loading with antigen produced by radiofrequency ablation of tumor lysate in situ combined with cytokine-induced killer cells （CIK）. Methods CIK cells derived from BALB/C mouse spleen and DCs derived from bone marrow were pre- pared, and experimental model of murine colon carcinoma were established for radiofrequency ablation. The supernatant of tumor tissue in situ lysis after repeated freezing and thawing were tested by lowry protein quantita- tive statutory, amounting to a final concentration of 5 μg/ml, then load to the first 5 days of culture DCs （ Ag-DC）, 2 days later, co-cultured with CIK cells after the first seven days of culture 48 h （Ag-DC-CIK）. Flow cytometry was used to analyze costimulatory molecules on the surface of the cells, and CCK-8 assay to detect in vitro cytotoxic activity. Results The DCs loading with antigen resulted in an increase in the proportion of CD86＋CD11c＋ , MHC Ⅱ＋ CD11c＋ and MHC Ⅱ＋ CD80＋ cells . The main effector cells of CIK ceils wereCD3 ＋ NK1.1 ＋cells. The percentage of CD3 ＋ NK1.1＋cells was 1.45% on the first day of the culture;while when they had been cultured for 7 days, the percentage CD3 ＋ NK1.1 ＋ significantly increased to 36.9%. The cytotoxicity of Ag-DC-CIK cells toward C26 cells was much more efficient than that of DC-CIK, CIK cells. The cytotoxic activity of the former was significantly lower than the latter and the same target ratio. When the ratios of effeetor cells to target cells were 5 ： 1, the cytotoxic activity of Ag-DC-CIK cells against C26 cells was （74.9 ± 3.5 ） %, ; while the DC-CIK was （71.2± 2.1 ） % and the CIK cells was （68.7 ±2.9） %. The differ- ence was statistically significant（ F = 7. 007, P = 0.007）. When the ratios of effector cells to target cells were 10 ： 1, the cytotoxic activity of Ag-DC-CIK cells against C26 cells was （ 82.3 ± 4.5 ） %, while the DC-CIK cells was （77.1 ± 5.1 ） %, and the CIK cells was （72.7± 2.8） %. The difference was statistically significant （F = 7. 727,P = 0.005）. When the ratios of effector cells to target ceils were 20 ： 1, the cytotoxic activity of Ag-DC-CIK cells against C26 cells was （83.2 ±1.9） %, while the DC-CIK cells was （77.2 ± 4.2）%, and the CIK cells was （73.0 ±2.6）%. The difference was statistically significant（ F = 16. 594,P =0.000）. Con- clusion DCs loading with antigen produced by radiofrequency ablation of tumor in situ pyrolysis products can improve in vitro cytotoxic activity combined with CIK ceils, which can provide a new comprehensive cancer treatment strategy.