热休克凋亡食管癌细胞抗原负载的树突状细胞联合放疗对食管癌的疗效

Efficacy of immunotherapy of DC loaded with heat-shocked apoptotic esophageal cancer cell antigen combined with radiotherapy for treatment of esophagus cancer

目的研究热休克凋亡自体食管癌肿瘤细胞抗原负载的树突状细胞(dendritic cell,DC)对激发食管癌患者特异性免疫应答的影响及治疗的临床疗效。方法选择2010年11月至2013年6月就诊的手术治疗后食管鳞癌患者40例,随机分为研究组(28例)与对照组(12例)。对照组行常规放疗,研究组采用放疗联合DC免疫治疗:(1)留取自体食管癌组织,经热休克处理(42℃,3 h)后获得热休克凋亡食管癌抗原;(2)分离、培养外周血单个核细胞,其中贴壁细胞经过重组人粒细胞-单核细胞集落刺激因子(rh GM-CSF)、白细胞介素(IL)-4联合诱导为DC;(3)负载热休克抗原,培养1周后经患者腹股沟皮内注射,1周注射1次,共注射4次。在治疗后1周进行迟发型超敏反应(DTH)检测;在患者治疗前后进行血清中细胞因子和抗原特异性IFN-γ+CD8+T细胞检测,并进行免疫学疗效评价。治疗后跟踪随访2年,对远期疗效进行评价。结果 28例研究组食管癌患者对DC治疗耐受良好,治疗后患者血清中Th1型细胞因子(IL-2、IL-12、INF-γ)水平较治疗前和对照组治疗后均有显著提高(P均<0.01)。治疗后研究组IFN-γ+CD8+T细胞比例较对照组有显著提高(P<0.01),其中10例患者IFN-γ+CD8+T细胞比例升高2倍以上。12例食管癌患者DTH试验呈阳性反应,其中9例IFN-γ+CD8+T细胞比例升高2倍以上。定期随访2年,两组均无脱落病例。研究组1年内死亡5例,存活82.14%;2年内共死亡9例,存活67.86%。对照组1年内死亡6例,存活50.00%;2年内共死亡8例,存活33.33%。研究组随访2年时生存比例大于对照组(P<0.05)。结论负载食管癌肿瘤细胞热休克凋亡抗原的DC能激发患者的特异性免疫应答、诱导Th1型细胞因子的分泌,联合放疗远期疗效良好,是一种安全的治疗方法。

Objective To investigate the influence of autologous dendritic cells（ DC） loaded with heat-shocked apoptotic esophageal cancer cell antigen on stimulating specific immune response of esophageal cancer patients and its clinical therapeutic effects. Methods Forty patients with esophageal squamous cell carcinoma after surgical treatment between June2010 and November 2013 were selected and randomly divided into research group（ n = 28） and control group（ n = 12）.The conventional radiotheraphy was given in control group. In research group,the radiotherapy combined with DC immunotherapy were given：（ 1） Self esophageal cancer tissues were collected,and the esophageal cancer cell antigen was acquired after treatment of heat shock（ 42℃,3 h）.（ 2） The mononuclear cells were isolated from peripheral blood and cultured in which the adherent cells were induced to DC by recombinant human granulocyte monocyte colony stimulating factor（ rh GMCSF） combined with interleukin（ IL）-4.（ 3） After being loaded with heat-shocked antigen and cultured for one week,theDC were administered by intradermal injection in inguinal region（ one time a week,a total of 4 times）. Delayed type hypersensitivity（ DTH） was detected one week after treatment. The cytokines in serum and antigen-specific interferon（ IFN）-γ^＋CD8^＋ cells were detected before and after the treatment,and the evaluation of immunological efficacy was performed. The patients were followed up for 2 years after treatment,and evaluation of long term efficacy was performed. Results All patients in research group had well-tolerance to DC therapy. In research group,The serum Th1 type cytokines（ IL-2,IL-12,INF-γ） levels after treatment were all significantly higher than those before treatment and those after treatment in control group（ all P〈0. 01）. After treatwent,IFN-γ^＋CD8^＋ cells level in research group increased significantly compared with control group（ P〈0. 01）,and the proportion of IFN-γ^＋CD8^＋ cells in 10 cases increased by more than three times. Positive DTH test was seen in 12 cases,in whom the proportion of IFN-γ^＋CD8^＋ cells increased by more than three times in9 cases. No case lost in both two groups during regular follow up for 2 years. In research group,5 cases died within one year（ 1-year survival rate was 82. 14%）,and 9 cases died within 2 years（ 2-year survival rate was 67. 86%）. In control group,6 cases died within one year（ 1-year survival rate was 50. 00%）,and 8 cases died within 2 years（ 2-year survival rate was33. 33%）. The 2-year survival proportion in research group was significantly more than that in control group（ P〈0. 05）.Conclusion DC loaded with heat-shocked apoptotic esophageal tumor cell antigen can stimulate specific immune response of esophagus cancer patients and induce the secretion of Th1 type cytokines. DC immunotherapy combined with radiotherapy is a safe treatment method with good long-term curative effect.