负载肿瘤抗原DC与CIK共培养对裸鼠肺腺癌移植瘤抑制作用观察

Observation of tumor antigen-loaded DC co-culture with CIK suppress the lung adenocarcinoma xenografts in nude mice

目的探讨负载肿瘤抗原的树突状细胞(dendritic cells,DC)与细胞因子诱导的杀伤细胞(cytokine induced killer,CIK)共培养后在体内的杀伤活性及分子机制。方法常规方法分离2011-05-06-2013-07-08南京军区福州总医院健康成年志愿者外周血单个核细胞,体外分别诱导培养为DC和CIK细胞;流式细胞仪测定负载抗原的DC与CIK的表型变化;建立人肺腺癌A549裸鼠(24只)移植瘤模型,应用随机数字表按完全随机分组原则分为PBS组、CIK组和AgDC-CIK组。治疗30d后摘取瘤块,称质量并计算抑瘤率;HE染色观察瘤体组织细胞的形态学变化,免疫组化法检测瘤体组织中VEGF、MMP-9和Bcl-2表达情况。结果负载抗原后DC细胞表面标志CD83+为(45.812±6.110)%,CD86+为(78.341±6.839)%,HLA-DR+为(75.103±5.913)%,与负载前比较均显著升高,t值分别为5.384、7.902和10.132,P值分别为0.009、0.003和<0.001;与CIK共培养CD3+CD8+为(55.417±8.428)%,CD3+CD56+为(39.823±7.215)%,均有显著增加,t值分别为6.185和9.904,P值分别为0.008和0.001;Ag-DC-CIK组能明显下调移植瘤VEGF、MMP-9和Bcl-2等蛋白的表达,t值分别为7.630、11.751和9.624,P值分别为0.002、<0.001和<0.001;且Ag-DC-CIK组对VEGF和Bcl-2表达的抑制作用均强于单纯CIK组,t值分别为5.832和6.214,P值分别为0.009和<0.001;在MMP-9的表达上与单纯CIK组差异无统计学意义,t=1.022,P=0.865。结论负载肿瘤抗原的DC与CIK共培养后可有效抑制VEGF、MMP-9和Bcl-2的表达,抑制裸鼠肺腺癌移植瘤的生长。

OBJECTIVE To investigate the in vivo killing activity and molecular mechanism of tumor antigen-load- ed DC co-culture with CIK. METHODS Peripheral blood mononuclear cells from healthy human were separated by con- ventional methods,and induced to DC and CIK cells in vitro. The phenotypic changes of antigen-loaded DC and CIK were detected by flow cytometry. Human lung adenocarcinoma A549 xenografts in nude mice were established and randomly di- vided into PBS group,CIK group and Ag-DC-CIK group. The tumor mass were removed,weighed and the inhibition rates were calculated 30 days after the treatment. HE staining and immunohistochemistry assay such as VEGF, MMP-9 and Bcl-2 were carried out with the tumor mass. RESULTS Surface markers CD83^＋ （45. 8124-6. 110） % ,CD86^＋ （78. 3414- 6. 839） % and HLA-DR＋ （75. 103 ± 5. 913） % of DC were significantly increased, with statistics value （t= 5. 384, P = 0. 009）,（t=7. 902,P=0.003） and （t= 10. 132,P〈0. 001） after antigen Loading,CD3^＋ CD8^＋ （55. 417 4-8. 428） % and CD3^＋ CD56^＋ （39. 823±7. 215）% were significantly increased after co-cultured with CIK,with statistics value （t=6. 185, P〈0. 008） and （t= 9. 904, P = 0. 001）. VEGF, MMP-9 and Bcl-2 protein expression were significantly decreased in the Ag-DC-CIK group with statistics value （t=7. 630,P=0. 002）,（t=11. 751,P〈0. 001） and （t=9. 624,P〈0. 001）. The VEGF and Bcl-2 expression inhibition was stronger in Ag-DC-CIK group than that in CIK group with statistics value （t= 5. 832,P=0. 009） and （t= 6. 214, P〈0. 001）. The expressions of MMP-9 were not significantly different between Ag- DC-CIK group and CIK group （t = 1. 022, P = 0. 865）. CONCLUSIONS The tumor antigen-loaded DC co-culture with CIK can effectively suppress the expression of VEGF, MMP-9 and Bcl-2, it also supress the lung adenocarcinoma xeno- grafts in nude mice.