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Objective To investigate the secrection of Polytuftsin, a macrophage activator in Pichia pastoris expression system. Methods The recombinant plasmid encoding polytuftsin was transformed into the yeast by chemical method, and the positive clones were screened and expressed under the induction of methanod. It was observed that the influence of polytuftsin on macrophage in vivo and in vitro. Results It was verified that the recombinant products was confirmed to be our target protein by SDS-PAGE analysis and PCR assay. The expressed product could activate macrophage to secret cytokine and enhance the capacity of macrophages to kill L1210 tumor cells, the maximum killing rate is up to 40 %. In vivo the leukemia therapeutic trial, the time of tumor arise was delayed; the survival time of L1210 tumor-bear mice treated with Polytuftsin was extended, which is about 20 day. Conclusion The recombination protein polytuftsin, which could activate macrophage and has anti-tumor activity.
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Objective To establish a human adrenocortical carcinoma (ACC)cell line ACC-LWL, and investigate its cell phenotypes and expression of tumor associated antigens, to detect sensitivity of ACC-LWL against adoptive immune cells. Methods Fresh tumor tissues from the resection of a human ACC were primary cultured and passed generation to generation so as to achieve stable growth in vitro. For analyzing its biological characteristic, the cell cloning formation in soft agar, chromosome and tumorigenesis were tested. Flow cytometric analysis was carried out for phenotype analysis and RT-PCR examination for MN/CA9 and HLA-A2 genes expression. PBMC from 4 healthy donors (2 HLA-A2+ or 2 HLA-A3+) co-cultured with IL-2 (200 U/ml)and the tumor cell lysate of ACC-LWL (20 μg/ml) in vitro to generate CTL. After 14 day stimulation, CTLs were incubated with ACC-LWL with a E/T ratio of 10:1 for 4 hours at 37 ℃. Cytotoxicity was measured with MTr assay. Results One human ACC cell line had been established that showed the characteristics of malignant cells. The cells grew a solid tumor in nude mice. ACC-LWL had a high expression of MHC-Ⅰ, weakly expression of Her2/neu and MHC-Ⅱ, no expression of CD80 or CD86. RT-PCR showed that ACC-LWL expressed MN/CA9 and HLA-A2 genes. CTL, either HLA-A2+ or HLA-A3+, had the capability of killing ACC-LWL in vitro. Conclusion This newly established ACC would provide a useful target in vitro and in vivo for investigations related to human ACC.
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ObjectiveTo provide experimental proof for the specific immunotherapy in patients with renal cell carcinoma (RCC).MethodsThe expression of c-erbB-2 and HLA-A2 molecules in RCC were examined by flow cytometery and HLA-A2 cDNA RT-PCR.Tumor lysatic antigens (Tuly) was used to load DCs (DC-Tuly) to induce the generation of c-erbB-2 specific CTLs derived from peripheral blood mononuclear cells of RCC patients in vitro and CTLs was used to kill RCC cells which express or not express c-erbB-2 and HLA-A2.The cytotoxic activity of induced CTLs was further studied by antibodies (anti-c-erbB-2 and anti-CD 8) blocking assay.Resultsc-erbB-2 protein is tumor associated antigens for RCC.DC-Tuly induced specific CTLs highly killed c-erbB-2+ HLA-A2+ autologous and allogenic RCC,but non-specific ones lowly killed c-erbB-2- HLA-A2+ or c-erbB-2+ HLA-A2-RCC cells. The cytotoxic activity against tumor cells was blocked by anti-c-erbB-2 and anti-CD 8 monoclonal antibodies.ConclusionsThese results suggest that this specific CTLs adoptive immunotherapy for RCC patients with c-erbB-2+ HLA-A2+ could be a novel approach for clinical use.
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Objective To establish human renal cell carcinoma (RCC) cell lines,and to investigate the cell phenotypes and expression of tumor associated antigens. Methods Fresh tumor tissues from pathologically proven human RCCs were primary cultured and passed generation to generation until a stably grow in vitro.The cell cloning form,chromosome and graft into nude mice in vivo were examined for cell lines.Immunofluorescent stain and flow cytometric analysis were carried out for cell's phenotype,RT PCR examination for MN/CA9 gene expression. Results Six human RCC cell lines have been established,including four of the cell lines derived from clear cells,one from mixed clear granular cells and one from papillary cells.All the cell lines showed the characteristics of malignant cells.All the lines highly expressed HLA ClassⅠ and HER2/neu.Three lines weakly expressed HLA ClassⅡ and one cell line highly expressed CD86 but all the lines did not express CD80.RT PCR showed that three cell lines have the expression of MN/CA9 gene. Conclusions These newly established RCC cell lines would provide a useful in vitro model for studies related to biological characteristics,tumor associated antigen,immunogenity and immunotherapy of human RCC.