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目的:以AFP和HBsAg为双靶标,以树突状细胞(DCs)为抗原载体与佐剂,探讨其激发特异性(CTL)反应的能力及其在HBV阳性肝癌(HCC)患者应用的可行性。方法:采集20例HLA-A201+HBV+AFP+HCC患者外周血,分离单核细胞(Mo)和外周淋巴细胞(PBLs),将Mo诱导为成熟DCs(mDC);人工合成AFP和HBsAg多肽,负载mDC后体外致敏,检测其体外杀伤活性;同时于患者皮内注射负载AFP和HBsAg混合多肽的DCs,监测接种前后患者外周血中细胞因子和特异性CTL水平变化,并进行DTH试验,治疗后跟踪检测患者AFP水平和乙肝两对半水平的变化。结果:患者外周PBLs经DCs致敏后,对负载AFP和HBsAg多肽的T2靶细胞杀伤率明显增高,P<0.05。接种负载AFP和HBsAg多肽的DCs后,血清中IL-2、IL-12、IFN-γ水平较治疗前显著升高,P<0.05,TNF-α和IL-10水平较治疗前均差异不大,P>0.05;DTH试验阳性率为41.7%(5/12),患者外周血中特异性CTLs比例明显上升的有4例(4/12)。治疗后有4例患者AFP水平下降,9例HBeAg阳性的患者中有3例HBeAg下降,2例患者出现血清学应答。结论:负载AFP和HBsAg多肽的DCs体内、体外均具有激发特异性CTLs反应能力,可诱导Th1型细胞因子的分泌,有望成为一种HCC治疗的新方法。
OBJECTIVE: To investigate the ability of CTL and its application in patients with HBV-positive hepatocellular carcinoma (HCC) by using double-labeled AFP and HBsAg as targets and dendritic cells (DCs) as antigen carriers and adjuvants . Methods: Peripheral blood samples from 20 patients with HLA-A201 + HBV + AFP + HCC were collected and monocytes (Mo) and peripheral lymphocytes (PBLs) were isolated and Mo was induced to mature DCs (mDC). Artificial AFP and HBsAg- DCs loaded with AFP and HBsAg were injected intradermally, and the levels of cytokines and CTLs in peripheral blood were monitored before and after inoculation, and the DTH test was performed to treat DCs. Follow-up detection of patients with AFP levels and hepatitis B two and a half levels change. Results: After the peripheral PBLs were sensitized by DCs, the killing rate of T2 target cells loaded with AFP and HBsAg polypeptides was significantly increased (P <0.05). After inoculation of DCs loaded with AFP and HBsAg peptides, the levels of IL-2, IL-12 and IFN-γ in serum were significantly higher than those before treatment, P <0.05, while the levels of TNF-α and IL-10 were not significantly different , P> 0.05. The positive rate of DTH was 41.7% (5/12). There were 4 cases (4/12) in which the proportion of specific CTLs in peripheral blood increased significantly. AFP levels decreased in 4 patients after treatment, decreased in 3 of 9 HBeAg-positive patients, and serologically in 2 patients. CONCLUSION: DCs loaded with AFP and HBsAg polypeptides have the ability to stimulate specific CTLs in vitro and in vivo and induce the secretion of Th1-type cytokines, which is expected to become a new treatment for HCC.