自体抗原负载DC联合CIK对肾癌免疫治疗的实验研究

Immune efficacy of dendritic cells pulsed by autologous tumor antigen combined with CIK cells on renal cell carcinoma

目的:评价自体抗原负载DC联合CIK体外治疗肾癌的杀瘤活性差异,为临床上肿瘤抗原不明确的肾癌免疫治疗提供一种新的研究思路。方法:收集2010年8月至2013年1月在中国医科大学附属第一医院泌尿外科接受手术治疗的20例肾癌患者肾癌组织及其外周血,10例健康成人志愿者外周静脉血。分为无抗原DC组、自体肿瘤全细胞抗原递呈DC组、异体肿瘤细胞裂解物抗原递呈DC组,与CIK混合培养后检测杀瘤率,并用流式细胞仪分别检测DC的CIK细胞表面成熟标志,统计两者的相关性。结果:加入肿瘤裂解物抗原及诱导成熟因子后,DC表面成熟标志CD83、CD80、CD86、HLA-DR的表达明显增加。与DC共培养的CIK表面抗原CD3^+CD4^+、CD3^+CD56^+、CD3^+CD8^+均高于单独培养CIK;自体全抗原递呈的CIK-DC-自体A组与异体全抗原递呈的CIK-DC-异体A组杀瘤活性最大,两者之间无差异;未经抗原刺激的CIK-DC组次之,单纯CIK组杀瘤活性最低;四组效应细胞组间杀伤率相关性分析显示,除CIK-DC-自体A组与CIK-DC-异体A组之间差异无统计学意义外,各组间差异均显著,各组之间杀伤率比较具有正相关关系;肾癌患者外周血杀瘤活性比健康供者杀瘤活性低,有统计学意义;肾癌细胞患者自体效应细胞与异体健康效应细胞差别无统计学意义;此外,各实验组间两两比较,差异显著,有统计学意义(P<0.05)。结论:自体/异体全抗原负载可促进DC成熟,提高CIK细胞免疫表型及增殖能力,增强其对肾癌细胞的杀伤活性。

Objective：To evaluate the difference of tumor killing activity of autologous antigen loaded DC com- bined with CIK on renal cell carcinoma in vitro ,which provides a new research idea for immunotherapy of renal cancer with unclear tumor antigen. Methods：From August 2010 to January 2013, we collected 20 cases of renal cell carcino- ma patients＇tissue and peripheral blood in the First Affiliated Hospital of China Medical University urology, 10 healthy adult volunteers＇peripheral venous blood. The DCs were divided into three groups, including DCs without antigen, au- tologous tumor cell antigen presenting DC, allogeneic tumor cell lysate antigen presenting DC, then combined with CIK to detect the killing rate. Flow cytometry was used to detect DC and CIK cells＂ surface maturation signs, and statistic the correlation between the them. Results： The expression of CD83, CD80, CD86j and HLA - DR were significantly increased after adding the tumor lysate antigen and induced maturation factor. The expression of CD3 ＋ CD4＋ , CD3 ＋ CD56 ＋ and CD3 ＋ CD8 ＋ of CIK co - cultured with DC were higher than that of singlef, cultured CIK. Autologous anti gen presenting DC - CIK and allogeneic antigen presenting DC - CIK had the largest killing activity, and there was no difference between them. No antigen presenting DC - CIK was lower than them,but higher than the single CIK. Analy- sis of the correlation between the four groups of effector cells showed that the difference between the groups was signif icant, the killing rate between the groups had a positive correlation, except no difference between autologous antigen presenting DC - CIK and allogeneic antigen presenting DC - CIK. The anti - tumor activity of renal cancer patients peripheral blood was lower than that of the healthy donors, and had statistically significance （P 〈 0.05 ）. There was no significant difference between autologous effector cells and allogeneic healthy effector cells in renal cancer patients （P 〉 0.05 ）. Conclusion：Autologous/allogeneic tumor antigen presenting could promote DC maturation, improve CIK cell immune phenotype and proliferation ability, and enhance its cytotoxic activity.