人肺鳞癌胩瘤特异性抗原cDNA文库疫苗抗肿瘤作用研究

To clone and identify the LSCC-related antigen and investigate whether it is capable of inducing a specific cytotoxic T lymphocytes (CTL) response in vitro and provide the experimental foundation for future clinical trials of lung cancer immunotherapy. Methods: Through screening a subtracted cDNA library of human LSCC constructed by using suppression subtracted hybridization (SSH) method, the LSCC-related antigen gp96 was selected to continue the following experiment of antitumor immunity in one case of LSCC patient. To perform a cytotoxicity assay, DCs derived from the patient's bone marrow blood mononuclear cells (BMCs) in the presence of various cytokines were co-cultured with the patient's tumor-derived gp96-peptide complexes to be used as the tester, tumor cell lysate isolated from the autologous LSCC patient and pulsed by the BM-drived DC and gp96-peptide complexes vaccine alone were used as positive controls, DCs without pulsing any tumor antigen were used as negative control. Then, the cognate lymphocytes were added into the above DC-vaccines, after incubation, the supernate were collected and stored in -70℃.Interferon-γ (IFN-γ) secreting from activated lymphocytes was detected by using an IFN-γ ELISA Kit and the Cr51 release test were performed to evaluate the gp96-peptide specific CTL response in three kinds of target cells including the primary culturing LSSC cells, PG cells and K562 cells. Results: 10 differentially expressed gene cDNA fragments of LSSC were obtained by SSH. Among them six were already known genes; two sequences were already identified but their functions were still unknown (hypothetical protein); two were novel (GenBank accession number were AF363068 and AY032661 respectively). In combine with the analysis of these differentially expressed genes in LSCC, the gp96 was finally selected to be the LSCC-related antigen. The results from cytotoxicity showed that DC vaccines pulsed by tumor antigens from both tester and control effectively activated autologous T lymphocytes to proliferate and secret IFN-γ. The concentration of IFN-γ induced by gp96-peptide complexes/DC and gp96-peptide complexes vaccine were higher than that of inducing by tumor cell lysate/DC vaccine, while DCs pulsed by gp96-peptide complexes could stimulate effective immunological response than singly using gp96-peptide complexes vaccine. More importantly, the activated T lymphocytes effectively lysed the patient's primary tumor cells, but had a relatively low or totally non response to PG and K562 cells. Conclusion: gp96 was identified as a LSCC-related antigen by method of SSH. In vitro studies have shown that autologous tumor-derived gp96-peptide complexes could induce a peptide complexes specific CTL response, while it was used to pulse DCs, the CTL response was going to be more effective than that of stimulating by gp96 alone or tumor cell lysate/DC, which show the broaden field of possible gp96-peptide complexes/DC based clinical application.

运用SMART-PCR和差减杂交等方法筛选出人类肺鳞关细胞癌肿瘤特异性抗原cDNA文库。将此cDNA文库装入真核表达载体构建DNA疫苗，以此真核表达载体体外刺激树突状细胞构建树突状细胞疫苗。在导入人体免疫细胞的联合免疫缺陷小鼠（Scid小鼠）模型是，分析这两类疫苗诱导体内产生特异性CTL和抑制肿瘤生长的能力。