mCRT-vGPCR膜表达融合蛋白应用于肿瘤免疫治疗的基础研究
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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。
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.
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。
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.
引文
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[3] Reiman JM, Kmieciak M and Manjili MH, et al. Tumor immunoediting and immuno-sculpting pathways to cancer progression [J]. Semin Cancer Biol. 2007, 17: 275–287
[4]Prestwich RJ, Errington F and Hatfield P, et al. The Immune System, is it Relevant to Cancer Development, Progression and Treatment [J]? Clin Oncol. 2008, 20(2):101-12
[5] Gaipl US, Sheriff A, Herrmann M, et al. Inefficient clearance of dying cells and autoreactivity [J]. Curr Top Microbiol Immunol, 2006, 305:161–176
[6] Casares N, Pequignot MO, Kroemer G, et al. Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death [J]. J Exp Med, 2005, 202(12):1691–1701
[7] Blachere NE, Darnell RB, Albert ML. Apoptotic cells deliver processed antigen to dendritic cells for cross-presentation [J]. PLoS Biol, 2005, 3(6):185
[8] Steinman RM, Mellman I. Immunotherapy bewitched, bothered, and bewildered no More [J]. Science, 2004, 305(9):197–200
[9] Lake RA, van der Most RG. A better way for a cancer cell to die [J]. N Engl J Med, 2006, 354(8):2503-2504
[10] Zitvogel L, Tesniere A, Kroemer G. Cancer in spite of immunosurveillance: immunoselection and immunosubversion [J]. Nat Rev Immunol, 2006, 6(10):715–727
[11] Zitvogel L, Casares N, Kroemer G, et al. Immune response against dying tumor cells [J]. Adv Immunol, 2004, 84:131–179
[12] Albert ML. Death-defying immunity: do apoptotic cells in?uence antigen processing and presentation [J]? Nat Rev Immunol, 2004, 4:223–231
[13] Apetoh L, Ghiringhelli F, Zitvogel L, et al. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotehrapy and radiotherapy [J]. Nat Med, 2007, 13(9):1050–1059
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[19] Gardai SJ, McPhillips KA, Henson PM, et al. Cell-Surface Calreticulin Initiates Clearance of Viable or Apoptotic Cells through trans-Activation of LRP on the Phagocyte [J]. Cell Vol, 2005, 123:321-334
[20] Martins I, Kepp O, Kroemer G, et al. Surface-exposed calreticulin in the interaction between dying cells and phagocytes [J]. Ann N Y Acad Sci, 2010, 1209:77-82
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[2] Rescigno M, Avogadri F and Curigliano G. Challenges and prospects of immunotherapy as cancer treatment [J]. Biochim Biophys Acta. 2007, 1776: 108–123
[3] Reiman JM, Kmieciak M and Manjili MH, et al. Tumor immunoediting and immuno-sculpting pathways to cancer progression [J]. Semin Cancer Biol. 2007, 17: 275–287
[4]Prestwich RJ, Errington F and Hatfield P, et al. The Immune System, is it Relevant to Cancer Development, Progression and Treatment [J]? Clin Oncol. 2008, 20(2):101-12
[5] Gaipl US, Sheriff A, Herrmann M, et al. Inefficient clearance of dying cells and autoreactivity [J]. Curr Top Microbiol Immunol, 2006, 305:161–176
[6] Casares N, Pequignot MO, Kroemer G, et al. Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death [J]. J Exp Med, 2005, 202(12):1691–1701
[7] Blachere NE, Darnell RB, Albert ML. Apoptotic cells deliver processed antigen to dendritic cells for cross-presentation [J]. PLoS Biol, 2005, 3(6):185
[8] Steinman RM, Mellman I. Immunotherapy bewitched, bothered, and bewildered no More [J]. Science, 2004, 305(9):197–200
[9] Lake RA, van der Most RG. A better way for a cancer cell to die [J]. N Engl J Med, 2006, 354(8):2503-2504
[10] Zitvogel L, Tesniere A, Kroemer G. Cancer in spite of immunosurveillance: immunoselection and immunosubversion [J]. Nat Rev Immunol, 2006, 6(10):715–727
[11] Zitvogel L, Casares N, Kroemer G, et al. Immune response against dying tumor cells [J]. Adv Immunol, 2004, 84:131–179
[12] Albert ML. Death-defying immunity: do apoptotic cells in?uence antigen processing and presentation [J]? Nat Rev Immunol, 2004, 4:223–231
[13] Apetoh L, Ghiringhelli F, Zitvogel L, et al. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotehrapy and radiotherapy [J]. Nat Med, 2007, 13(9):1050–1059
[14] Obeid M, Tesniere A, Kroemer G, et al. Calreticulin exposure dictates the immunogenicity of cancer cell death [J]. Nat Med, 2006, 13(1):54-61
[15] Gelebart P, Opas M, Michalak M. Calreticulin, a Ca2+-binding chaperone of the endoplasmic reticulum[J], Int J Biochm Cell Biol, 2005, 37(2):260–266.
[16] Michalak M, Groenendyk J, Opas M, et al. Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum [J]. Biochem J, 2009, 417 (3):651-666
[17] Johnson S, Michalak M, Opas M, et al. The ins and outs of calreticulin:from the ER lumen to the extracellular space [J], Cell Biology, 2001, 11(3):122-129.
[18] Michalak M, Corbett EF, Mesaeli N, et al. Calreticulin: one protein, one gene, many functions [J], Biochem J, 1999, 344(Pt2):281-292.
[19] Gardai SJ, McPhillips KA, Henson PM, et al. Cell-Surface Calreticulin Initiates Clearance of Viable or Apoptotic Cells through trans-Activation of LRP on the Phagocyte [J]. Cell Vol, 2005, 123:321-334
[20] Martins I, Kepp O, Kroemer G, et al. Surface-exposed calreticulin in the interaction between dying cells and phagocytes [J]. Ann N Y Acad Sci, 2010, 1209:77-82
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