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目的:探讨以HER-2/neu为靶抗原、树突状细胞(dendritic cell,DC)为抗原载体激发特异性细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTL)反应的能力及研制治疗型乳腺癌疫苗的可行性。方法:采集17例HLA-A201+HER-2/neu+乳腺癌患者外周血,分离单个核细胞与外周血淋巴细胞(peripheral blood lymphocyte,PBL),并诱导为成熟DC(mature dendriticcell,mDC);人工合成HER-2/neu多肽[E75(KIFGSLAFL)和GP2(IISAVVGIL)2条]负载mDC后体外反复致敏PBL(3次,每周1次),检测其激发HER-2/neu特异性CTL的能力与CTL的杀伤活性。同时于患者腹股沟淋巴结富集区皮内注射负载HER-2/neu多肽的DC,每周1次,共接种4次,检测接种前后患者外周血细胞因子和特异性的CTL水平变化,并进行DTH试验。结果:患者外周PBL经过负载HER-2/neu多肽DC共3轮致敏后,HER-2/neu多肽特异的CTL平均比例比对照组(未负载HER-2/neu多肽DC组)明显增高[(5.41±1.44)%vs(0.41±0.12)%,P<0.05];致敏后PBL对负载HER-2/neu多肽T2靶细胞的杀伤率明显高于对照组(未负载DC诱导的CTL)[效靶比为30∶1时,(35.5±4.7)%vs(11.2±1.4)%,P<0.05]。接种负载HER-2/neu多肽的DC后,患者体内血清中细胞因子IL-2、IL-12、IFN-γ水平较治疗前显著升高[(409.09±89.39)vs(148.79±28.32)ng/ml,(56.23±14.08)vs(24.49±56.23)ng/ml,(146.57±25.97)vs(67.77±39.35)ng/ml;均P<0.05],TNF-α和IL-10水平较治疗前变化不大(P>0.05)。患者DTH试验阳性率为47%(8/17),DTH阳性患者外周血中特异性CTL比例明显上升。结论:负载HER-2/neu多肽的DC体内、外均具有激发特异性CTL反应能力,可诱导Th1型细胞因子的分泌,未发生临床不良反应。
Objective: To explore the ability of using dendritic cells (DCs) as target antigen and HER-2 / neu as target antigen to stimulate specific cytotoxic T lymphocytes (CTL) reactions and to develop therapeutic breast cancer Vaccine feasibility. Methods: Peripheral blood samples from 17 HLA-A201 + HER-2 / neu + breast cancer patients were collected and isolated from peripheral blood lymphocytes (PBLs) and induced to mature dendritic cells (mDC) Synthesis of HER-2 / neu polypeptide [2 E75 (KIFGSLAFL) and GP2 (IISAVVGIL)] PBMCs were repeatedly sensitized in vitro (3 times a week) Ability and cytotoxic activity of CTL. At the same time, DCs carrying HER-2 / neu polypeptide loaded with HER-2 / neu polypeptide were intradermally injected into the inguinal lymph node enrichment area of the patient once a week for 4 times to determine the changes of peripheral blood cytokines and specific CTL levels before and after inoculation and DTH test . Results: The average proportion of CTL specific for HER-2 / neu polypeptide in PBL of patients with HER-2 / neu polypeptide DC sensitized by total 3 cycles of sensitization was significantly higher than that of the control group (DC group without HER-2 / neu polypeptide) (5.41 ± 1.44)% vs (0.41 ± 0.12)%, P <0.05]. The killing rate of PBL loaded with HER-2 / neu polypeptide T2 target cells after sensitization was significantly higher than that of the control group [(35.5 ± 4.7)% vs (11.2 ± 1.4)%, P <0.05] when the target ratio was 30: 1. After inoculation of DCs loaded with HER-2 / neu polypeptide, the levels of IL-2, IL-12 and IFN-γwere significantly increased in the serum of patients (409.09 ± 89.39 vs 148.79 ± 28.32 ng / ml, (56.23 ± 14.08) vs (24.49 ± 56.23) ng / ml, (146.57 ± 25.97) vs (67.77 ± 39.35) ng / ml, all P <0.05]. The levels of TNF-α and IL- Not (P> 0.05). The positive rate of DTH test in patients was 47% (8/17), and the proportion of specific CTL in peripheral blood of DTH-positive patients was significantly increased. Conclusion: The DCs loaded with HER-2 / neu polypeptide have the ability to stimulate specific CTLs both in vitro and in vivo, and induce the secretion of Th1 cytokines without clinical adverse reactions.