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Antitumor Immune Responses Induced by Gene Transfer of B7-H3 into Squamous Cell Carcinoma Tca8113 in Vitro

Author: ChuMei
Tutor: ZhouJian；YangHongYu
School: Anhui Medical University,
Course: Clinical Stomatology
Keywords: B7-H3 Costimulatory molecules Immunotherapy Squamous cell carcinoma of the tongue
CLC: R730.5
Type: Master's thesis
Year: 2008
Downloads: 46
Quote: 0
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Abstract


Objective: Tumor cells present antigens to T cells, since the surface expression of costimulatory molecules lacking, not the initial effective anti-tumor immune response. To costimulatory molecules into tumor cells to produce tumor vaccines, we will express B7-H3 gene eukaryotic expression vector pEGFP-C1-B7-H3 transfected into oral squamous cell Tca8113, the detection of B7-H3 gene transfection Tca8113 oral squamous cell carcinoma, and further study human B7-H3 gene transfer squamous cell vaccine induces anti-tumor immune responses. Methods: liposome-mediated method eukaryotic expression plasmid pEGFP-C1-B7-H3 transfected into human cancer cell Tca8113 in transfection 24h, 14d, 30d, the fluorescence microscopy of green fluorescent protein expression, be transfected successfully, using RT-PCR detection of gene B7-H3 in transfected cells; detected by Western blot protein expression by G418 selection to obtain stable expression clones. With mitomycin C (MMC) treatment, the tumor cell vaccine made by in vitro co-culture with human peripheral blood lymphocytes were measured after lymphocyte-specific cytotoxic activity and on lymphocytes produce cytokines. Results: Cell transfection kit Lipofectamine2000 the pEGFP-C1-B7-H3 vector into the oral squamous cell carcinoma after Tca8113 in 24h, 14d, 30d, the 10 × 20 times in an inverted microscope with a fluorescent cells were observed ( wavelength 488nm or 513nm), while non-transfected plasmid with oral squamous cell Tca8113 as controls. Transfected with B7-H3 of Tca8113 cells, through the cell subculture is still able to detect expression of the B7-H3 genes, using Trizol from transfected with pEGFP-C1-B7-H3 of Tca8113 cells extracting total RNA, and then RT -PCR, the expression of B7-H3, PCR products electrophoresis showed that the size of the amplification product of approximately 215bp, with the expected size. Transfected with the empty vector pEGFP-C1 Tca8113 cells, not detected 215bp PCR product. Western blot was extracted B7-H3/Tca8113 gene transfection, cell lysates have been transfected with pEGFP-C1-B7-H3's Tca8113 cells (B7-H3/Tca8113) membrane protein gene transfection relative molecular mass of about 84 ~ 90KD size of specific bands; while mock/Tca8113 no specific bands. After G418 selection were picked up B7-H3/Tca8113 monoclonal cell lines were established. B7-H3 transfected cells can Tca8113 high expression of B7-H3 protein. After MMC treatment, Tca8113 with wild-type cells compared to the vaccine on T cells in vitro tests show that the gene transfected cells co-cultured with T cells can promote their proliferation and induce lymphocytes to produce specific killing against Tca8113 role of lymphocytes secrete can significantly enhance the ability of IFN-γ. Conclusion: The liposome eukaryotic expression vector pEGFP-C1-B7-H3 transfected into oral squamous cell Tca8113, use the selection method to establish a stable monoclonal B7-H3 protein expression cell lines by RT-PCR and Western blot identified B7-H3 gene expression in the cells there. B7-H3/Tca8113 treated by MMC get TCV-hB7-H3, be it with T-cells in vitro tests show that the gene transfected cells co-cultured with T cells can promote their proliferation and induce lymphocytes specific for Tca8113 cytotoxicity, can significantly enhance the lymphocytes secrete IFN-γ capabilities. B7-H3 gene transfected human squamous cell vaccine can induce an effective immune response against squamous cell carcinoma. For future in vivo studies B7-H3 on the regulation of immune cells and tumor gene therapy foundation.

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