【作者】 秦烨；

【导师】 王艳林； 韩钰；

【作者基本信息】 三峡大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 目的:用基因重组技术在病毒G蛋白偶联受体(vGPCR)N端融合小鼠钙网蛋白(mCRT),获得真核表达质粒pcDNA3.1(+)-mCRT/vGPCR。将此质粒稳定转染至小鼠黑色素瘤细胞B16-F1中,获得膜上高表达融合蛋白mCRT/vGPCR的B16-F1细胞株,然后体外检测高表达融合蛋白的细胞被吞噬效应。用凋亡药物处理这种细胞株后作为免疫原免疫Balb/C小鼠,研究其激发机体抗肿瘤免疫应答的效用。本研究的目的在于探索抗肿瘤免疫预防和治疗的新途径。方法:(1)应用RT-PCR技术从小鼠黑色素瘤B16-F1细胞总RNA中克隆获得含mCRT ORF全长序列的DNA片段。(2)从质粒pSG5/vGPCR中克隆获得vGPCR ORF全长片段。(3)构建融合基因mCRT/vGPCR并克隆入真核表达载体pcDNA3.1(+)中。(4)用脂质体转染法将重组质粒转染至小鼠黑色素瘤B16-F1细胞株中,用RT-PCR和Western blotting检测基因的表达,并用流式细胞术和细胞免疫荧光鉴定该融合蛋白在细胞膜上的定位。(5)直接将活的对照B16-F1细胞、转染vGPCR的B16-F1细胞(B16-vGPCR)和转染mCRT/vGPCR的B16-F1细胞(B16-mCRT/vGPCR)接种于Balb/C小鼠皮下,观察肿瘤生长情况。( 6 ) MTT法检测稳定转染质粒pcDNA3.1(+)-vGPCR和pcDNA3.1(+)-mCRT/vGPCR对B16-F1细胞株生长的影响。(7)多胺类似物(BENS)和蒽环类药物(米托蒽醌)分别诱导小鼠黑色素瘤细胞B16-F1和B16-mCRT/vGPCR细胞株凋亡,经皮下注射免疫Balb/C小鼠,免疫8天后,用活的B16-F1皮下接种免疫后的Balb/C小鼠,观察并纪录肿瘤生长状况。(8)ELISA法检测小鼠血清中细胞因子的含量。结果:(1)成功构建了重组真核表达质粒pcDNA3.1(+)-mCRT/vGPCR,pcDNA3.1(+)-vGPCR。(2)获得膜稳定表达融合蛋白的细胞株B16-mCRT/vGPCR。(3)体外吞噬实验表明,B16-F1细胞膜上高表达融合蛋白mCRT/vGPCR可增强其被吞噬效应。(4)膜上高表达mCRT的B16-F1细胞在小鼠体内生长受抑制,但体外MTT检测发现mCRT的高表达促进B16-F1细胞的生长。(5)动物实验表明,包被有融合蛋白mCRT/vGPCR的凋亡B16-F1细胞作为免疫原可刺激小鼠产生抗同种肿瘤的免疫效应。结论:细胞膜上包被有融合蛋白mCRT/vGPCR的凋亡肿瘤细胞能作为细胞抗原,在动物体内诱导出特异性抗同种肿瘤的免疫杀伤活性。本研究提示了CRT在抗肿瘤研究中的重要的潜在应用价值,为肿瘤预防和治疗提供了新的思路。更多还原

【Abstract】 Objective In order to develop an universal technique that could make CRT-coating more efficiently in the tumor cells, in this study, a mouse CRT recombinant gene with virus G-protein coupled receptor (vGPCR) was constructed and cloned into vector pcDNA3.1(+). And then the plasmids pcDNA3.1(+)-mCRT/vGPCR were stably transfected into the mouse melanoma B16-F1 cells. Proliferation assay for B16-F1 cell lines transfected with exgenous genes were performed in vivo and in vitro. mCRT/vGPCR mediated phagocytosis was also investigated. When mCRT-vGPCR coated B16-F1 cells were used as a cell-antigen to immunize mice, the specific anti-tumor immune response against the homologous tumor cells was tested. This novel approach may provide a new possibility for CRT-mediated tumor immune prevention and treatment.Methods The full length CDS of vGPCR was amplified from plasmid pSG5/vGPCR by PCR technique, and the full-length CDS of mouse calreticulin (mCRT) was amplified by RT-PCR using total RNA derived from mouse B16-F1 cells as the template. Two PCR products were ligated first and then inserted into pcDNA3.1(+). The resulted plasmid pcDNA3.1(+)-mCRT/vGPCR was transfected into B16-F1 cells by LipofectamineTM 2000 reagent. Expression and localization of mCRT/vGPCR fusion protein in the transfected cells was identified by RT-PCR, Western blotting, flow cytometry (FCM) and cell immunofluorescrence (CIF). The possible implication of mCRT/vGPCR in the phagocytosis was investigated by FCM. The different cell lines were inoculated into Bbal/C mouse to test their proliferation rate in vivo. We also assayed the proliferation rate of different cell lines by MTT in vitro. Three different apoptotic B16-F1 cells were prepared and used as the cell antigens to inoculate animals, they are BENS-treated B16-F1, BENS-treated B16-mCRT/vGPCR and mitoxantrone-treated B16-F1. And then the inhibition effects of immuno-inoculation on the growth of B16-F1 live cells inoculated latterly were observed. ELISA assay was used to detect the cytokines in the experimental mouse serum.Results 1) Eukaryotic expression plasmid pcDNA3.1(+)-mCRT/vGPCR and pcDNA3.1(+)-vGPCR were constructed succeffuly. 2) A cell line (B16-mCRT/vGPCR) with recombinant mCRT/vGPCR coated on the cell surface was obtained. 3) mCRT/vGPCR on the surface of B16-F1 cells enhanced phagocytosis in vitro. 4) Overexpression of mCRT/vGPCR can decrease proliferation of B16-F1 in vivo while increase proliferation of B16-F1 in vitro. 5) Apoptotic B16-F1 cells coated with mCRT/vGPCR induced a specific antitumor immunological effect against homogeneous tumor cells in mice.Conclusion When mCRT-vGPCR coated B16-F1 cells were used as a cell-antigen to immunize mice, the specific anti-tumor immune response against the homologous tumor cells was initiated efficiently. This novel approach may provide a new possibility for CRT-mediated tumor immune prevention and treatment.更多还原