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目的: 鉴定CTL识别的HLA A2限制性人卵巢癌相关抗原OVA66表位。方法: 以细胞因子从外周血单个核细胞(PBMC)中诱导树突状细胞(DC), 通过形态学观察和流式细胞术进行鉴定。用表位预测法选取并合成两种肽分子, 分别脉冲成熟的DC, 并刺激HLA- A2+健康人自体CD8+ T细胞。1wk后, 用脉冲肽的自体PBMC以每 7d的间隔刺激该CD8+T细胞 3次。以共接受 4次抗原肽刺激的T细胞作为CTL,用乳酸脱氢酶(LDH)释放试验, 检测CTL对靶细胞的杀伤效应。用酶联免疫斑点法 (ELISPOT), 检测CTL中抗原特异性分泌IFN- γ的T细胞数。结果: 形态学和流式细胞术的结果显示, PBMC可诱生成熟的DC。肽L235(FLPDHINIV)诱导的CTL, 可特异性杀伤L235脉冲的T2细胞和OVA66+、HLA A2+的SW480细胞, 且L235诱导的特异性分泌IFN- γ的T细胞数增加。结论: 卵巢癌相关抗原OVA66的HLA A2限制性CTL表位L235, 能激发对肿瘤抗原的特异性免疫应答, 为制备肿瘤特异性肽疫苗奠定了实验基础。
OBJECTIVE: To identify the HLA A2-restricted human ovarian cancer-associated antigen OVA66 epitope recognized by CTL. Methods: Dendritic cells (DCs) were induced from peripheral blood mononuclear cells (PBMCs) by cytokines and identified by morphological observation and flow cytometry. Two kinds of peptide molecules were selected and synthesized by epitope prediction method, respectively, pulsed with mature DC and stimulated autologous CD8 + T cells of HLA-A2 + healthy individuals. After 1 week, the CD8 + T cells were stimulated with pulsed peptide autologous PBMC three times every 7 days. T lymphocytes stimulated with 4 times antigen peptides were used as CTLs. The killing effect of CTLs on target cells was detected by lactate dehydrogenase (LDH) release test. The number of antigen-specific IFN-γ-secreting T cells in CTLs was detected by ELISPOT. Results: Morphology and flow cytometry results show that PBMC can induce mature DCs. CTL induced by peptide L235 (FLPDHINIV) specifically killed L235-pulsed T2 cells and OVA66 +, HLA A2 + -expressing SW480 cells, and L235-induced increase in the number of T cells secreting IFN-γ. CONCLUSION: L235, an HLA A2-restricted CTL epitope of ovarian cancer-associated antigen OVA66, can stimulate specific immune responses to tumor antigens and lay the foundation for the preparation of tumor-specific peptide vaccines.