CAJ | 학술논문

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5 μg/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48 h,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.
목적 연구부재사빈소융종류원위렬해물적수돌상세포(DC)연합세포인자유도살상활성세포(CIK)체외항종류활성.방법 제비BALB/C소서비장래원적CIK세포급골수래원적DC,건립사빈소융멸활소서피하결장암적실험모형,장기원위렬해적종류조직반복동융후취상청,lowry단백정량법정량,이종농도5 μg/ml재배양제5천적DC(즉Ag-DC),2d후재여배양제7천적CIK세포공배양48 h,류식세포술분석기표면공자격분자적표체,세포증식여독성검측시제합(CCK-8시제합)검측기체외살상활성.결과 DC표면분자표체공자격분자CD86+ CD11c+、MHCⅡ+CD11c+、MHCⅡ+CD80+쌍양성세포백분함량분별위9.50％、42.4％、53.4％;Ag-DC표면분자표체공자격분자쌍양성세포백분함량명현제고,분별위19.2％、74.2％、61.1％.CIK세포배양제1천,CD3+ NK1.1+쌍양성적백분함량위1.45％,제7천CD3+ NK1.1+쌍양성표체명현제고위36.9％.Ag-DC-CIK세포대결장암세포C26적살상활성명현고우DC-CIK、CIK세포,차상동효파비하전자적살상활성명현고우후자.효파비위5∶1시,Ag-DC-CIK세포살상솔위(74.9±3.5)％,DC-CIK세포살상솔위(71.2±2.1)％,CIK세포살상솔위(68.7±2.9)％,차이유통계학의의(F =7.007,P=0.007);효파비위10∶1시,Ag-DC-CIK세포살상솔위(82.3±4.5)％,DC-CIK세포살상솔위(77.1±5.1)％,CIK세포살상솔위(72.7±2.8)％,차이유통계학의의(F=7.727,P=0.005);효파비위20∶1시,Ag-DC-CIK세포살상솔위(83.2±1.9)％,DC-CIK세포살상솔위(77.2±4.2)％,CIK세포살상솔위(73.0±2.6)％,차이유통계학의의(F=16.594,P=0.000).결론 부재종류사빈소융원위렬해산물적DC연합CIK세포가이제고체외세포독활성,위종류종합치료제공신책략.
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 ceils derived from BALB/C mouse spleen and DCs derived from bone marrow were prepared,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 quantitative 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 + CD11 c +,MHC Ⅱ + CD11 c + and MHC Ⅱ + CD80 + cells.The main effector cells of CIK cells were CD3 + 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 effector 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 difference 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 cells 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).Conclusion DCs loading with antigen produced by radiofrequency ablation of tumor in situ pyrolysis products can improve in vitro cytotoxic activity combined with CIK cells,which can provide a new comprehensive cancer treatment strategy.