【摘要】 目的　研究应用不同肿瘤睾丸抗原 (CTA)多肽诱导肝细胞肝癌 (HCC)患者特异性细胞毒反应的情况。方法　RT- PCR方法检测一例 HL A- A2阳性表达的 HCC患者肝癌组织中三种 CT抗原 (MAGE- 1、MAGE- 3和 NY- ESO- 1)基因m RNA的表达情况 ,用免疫磁珠从该患者外周血单个核细胞 (PBMC)中分离出 CD8+T细胞作为效应细胞 ;将其余自体 CD8-PBMC分别与三种 CT抗原肽共孵育 ,经 γ-射线照射后作为抗原呈递细胞 (APC) ,分别与 CD8+效应细胞进行混合淋巴细胞培养。 7天后同法再刺激患者的 CD8+T细胞 1次 ,诱导形成细胞毒 T淋巴细胞 (CTL)。同时取相同的 CD8+效应细胞 ,加入IL- 2培养作为对照组。培养两周后将三种 CT抗原肽刺激的效应细胞 ,分别与带有相应抗原肽的 T2靶细胞共孵育来检测杀伤效应 ,效应细胞 :靶细胞 (E:T)为 10 :1;用 IFN- γ细胞因子分泌检测法 ,通过流式细胞仪检测产生 IFN- γ的效应 CD8+T细胞的频数来反映杀伤活性。结果　该患者 MAGE- 3和 NY- ESO- 1表达阳性 ,MAGE- 1表达阴性。培养第 14天 ,效应细胞共增加至原细胞数的 4倍。 NY- ESO- 1抗原肽刺激的效应细胞对靶细胞 (T2细胞 +NY- ESO- 1抗原肽 )杀伤活性为 10 .0 % ;MAGE- 3抗原肽刺激的效应细胞对靶细胞 (T2细胞 +MAGE- 3抗原肽 )杀伤活性为 8更多还原

【Abstract】 Objective To study the induction of specific cytotoxic T Lymphocytes (CTL) response from hepatocellular carcinoma patient’s peripheral blood CD8 positive T cells with different peptides of Cancer Testis Antigen.Methods The mRNA of MAGE 1?MAGE 3 and NY ESO 1 genes in the tumor tissues from a HCC patient were detected by RT PCR, the CD8+ T cells were separated with MACS immunobeads from PBMC of this patients as effector cells. The irradiated autologous CD8 PBMC were loaded with three kind of peptides as APC respectively. The APC and some CD8+ T cells were mixed and co cultured for one week, APCs were added to stimulate CD8 lymphocytes again. Other CD8+ T cells as the control group were only added IL 2 and not APC. After 14 days’ co culture with IL 2, the effect cells were mixed with target cells,the frequency of CD8 positive T lymphocytes secreting IFN γ in response to three different peptides were monitored by MACS IFN γ assay with Flow Cytometry. The T2 cell lines loaded with different peptides were as target cells ,the E:T was10:1. Results After 2 weeks’ co culture, the CD8 cells increased by average 4 folds. When T2 cells loaded with NY ESO 1 peptide as the target cells, the frequency of CD8 positive T cells secreting IFN γ in response to NY ESO 1 peptide was 10% .When T2 cells loaded with MAGE 3 peptide as the target cells, which in response to MAGE 3 peptide was 8.5%. When T2 cells loaded with MAGE 1 peptide as the target cells, which in response to MAGE 1 peptide was 3.2%. Conclusion The results suggest that the peptides of CTA could induce and proliferate cytotoxic T lymphotocytes (CTL) with the ability of specifically killing target cells which express the same CTA mRNA. The responses are obviously stronger than which of with negative peptide or without peptide.更多还原