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首页 > 过刊浏览>2024年第40卷第2期 >458-472. DOI:10.13345/j.cjb.230468
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稳定表达CD19-FLUC-GFP的CT26细胞系的构建及鉴定
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国家自然科学基金(32270969，82001758)


Construction and identification of a stable CT26 cell line expressing CD19-FLUC-GFP
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    摘要:

    实体瘤缺乏明确的嵌合抗原受体T细胞(chimeric antigen receptor T-cell, CAR-T)治疗靶点。因此，通过慢病毒将已经明确的靶点分子CD19带入实体瘤细胞系，研究CD19 CAR-T细胞对其的杀伤，能够为CAR-T细胞针对实体瘤的治疗提供潜在的支撑。本研究利用三质粒慢病毒系统构建了稳定表达CD19、萤火虫荧光素酶(firefly luciferase, FLUC)和绿色荧光蛋白(green fluorescent protein, GFP)的结肠癌CT26细胞系CT26-CD19-FLUC-GFP。该细胞系与CT26细胞系的生长活性一致。通过流式细胞术检测不同代次CT26-CD19-FLUC-GFP细胞，证实了CT26-CD19-FLUC-GFP细胞连续传代至第5、10、22代后CD19及GFP的稳定表达。进一步证实，连续传代至第22代的CT26-CD19-FLUC-GFP细胞中的CD19 mRNA及FLUC表达水平显著高于对照组CT26细胞。与T细胞相比，CD19 CAR-T细胞能够显著杀伤CT26-CD19-FLUC-GFP细胞及MC38-CD19细胞。CT26-CD19-FLUC-GFP细胞腹腔植入小鼠体内1周后，通过活体成像仪可以检测到腹腔区域的FLUC表达。上述结果表明，成功构建了稳定表达CD19-FLUC-GFP的CT26细胞系，且该细胞系能够被CD19 CAR-T细胞特异性杀伤。

    Abstract:

    Solid tumors lack well-defined targets for chimeric antigen receptor T-cell (CAR-T) therapy. Therefore, introducing a known target molecule, CD19, into solid tumor cell lines via lentiviral transduction to investigate the cytotoxicity of CD19 CAR-T cells can potentially support CAR-T cell therapy against solid tumors. In this study, a stable colon cancer CT26 cell line, CT26-CD19-FLUC-GFP, expressing CD19, firefly luciferase (FLUC), and green fluorescent protein (GFP), was constructed using a triple-plasmid lentiviral system. The growth characteristics of this cell line were consistent with those of the CT26 cell line. Subsequent flow cytometry analysis confirmed stable expression of CD19 and GFP in CT26-CD19-FLUC-GFP cells after serial passaging up to the 5th, 10th, and 22nd generations. Further validation revealed significantly higher levels of CD19 mRNA and FLUC expression in CT26-CD19-FLUC-GFP cells continuously passaged up to the 22nd generation compared to the control CT26 cells. In comparison to T cells, CD19 CAR-T cells demonstrated substantial cytotoxicity against CT26-CD19-FLUC-GFP cells and MC38-CD19 cells. One week after intraperitoneal implantation of CT26-CD19-FLUC-GFP cells into mice, FLUC expression in the peritoneal region could be detected. These results indicate the successful establishment of a stable CT26 cell line expressing CD19-FLUC-GFP, which can be specifically targeted by CD19 CAR-T cells.

    参考文献
    [1] ALLEMANI C, MATSUDA T, di CARLO V, HAREWOOD R, MATZ M, NIKŠIĆ M, BONAVENTURE A, VALKOV M, JOHNSON CJ, ESTÈVE J, OGUNBIYI OJ, SILVA GAE, CHEN WQ, ESER S, ENGHOLM G, STILLER CA, MONNEREAU A, WOODS RR, VISSER O, LIM GH, et al. Global surveillance of trends in cancer survival 2000-14(CONCORD-3):analysis of individual records for 37513025 patients diagnosed with one of 18 cancers from 322 population-based registries in 71 countries[J]. Lancet (London, England), 2018, 391(10125):1023-1075.
    [2] XIE YM, SHI LS, HE XS, LUO YX. Gastrointestinal cancers in China, the USA, and Europe[J]. Gastroenterology Report, 2021, 9(2):91-104.
    [3] JIMENEZ-RODRIGUEZ RM, QUEZADA-DIAZ F, HAMEED I, KALABIN A, PATIL S, SMITH JJ, GARCIA-AGUILAR J. Organ preservation in patients with rectal cancer treated with total neoadjuvant therapy[J]. Diseases of the Colon & Rectum, 2021, 64(12):1463-1470.
    [4] CADY B, STONE MD. The role of surgical resection of liver metastases in colorectal carcinoma[J]. Seminars in Oncology, 1991, 18(4):399-406.
    [5] XIE W, MEDEIROS LJ, LI SY, TANG GL, FAN G, XU J. PD-1/PD-L1 pathway:atherapeutictarget in CD30+ large cell lymphomas[J]. Biomedicines, 2022, 10(7):1587.
    [6] DENLINGER N, BOND D, JAGLOWSKI S. CAR T-cell therapy for B-cell lymphoma[J]. Current Problems in Cancer, 2022, 46(1):100826.
    [7] WOYACH JA, AWAN F, FLINN IW, BERDEJA JG, WILEY E, MANSOOR S, HUANG Y, LOZANSKI G, FOSTER PA, BYRD JC. A phase 1 trial of the Fc-engineered CD19 antibody XmAb5574(MOR00208) demonstrates safety and preliminary efficacy in relapsed CLL[J]. Blood, 2014, 124(24):3553-3560.
    [8] ZALEVSKY J, LEUNG IWL, KARKI S, CHU SY, ZHUKOVSKY EA, DESJARLAIS JR, CARMICHAEL DF, LAWRENCE CE. The impact of Fc engineering on an anti-CD19 antibody:increased Fcγ receptor affinity enhances B-cell clearing in nonhuman primates[J]. Blood, 2009, 113(16):3735-3743.
    [9] POE JC, MINARD-COLIN V, KOUNTIKOV EI, HAAS KM, TEDDER TF. A c-Myc and surface CD19 signaling amplification loop promotes B cell lymphoma development and progression in mice[J]. Journal of Immunology (Baltimore, Md:1950), 2012, 189(5):2318-2325.
    [10] IMURA Y, ANDO M, KONDO T, ITO M, YOSHIMURA A. CD19-targeted CAR regulatory T cells suppress B cell pathology without GvHD[J]. JCI Insight, 2020, 5(14):e136185.
    [11] ANDERSON KC, BATES MP, SLAUGHENHOUPT BL, PINKUS GS, SCHLOSSMAN SF, NADLER LM. Expression of human B cell-associated antigens on leukemias and lymphomas:a model of human B cell differentiation[J]. Blood, 1984, 63(6):1424-1433.
    [12] GINALDI L, de MARTINIS M, MATUTES E, FARAHAT N, MORILLA R, CATOVSKY D. Levels of expression of CD19 and CD20 in chronic B cell leukaemias[J]. Journal of Clinical Pathology, 1998, 51(5):364-369.
    [13] OLEJNICZAK SH, STEWART CC, DONOHUE K, CZUCZMAN MS. A quantitative exploration of surface antigen expression in common B-cell malignancies using flow cytometry[J]. Immunological Investigations, 2006, 35(1):93-114.
    [14] SCHEUERMANN RH, RACILA E. CD19 antigen in leukemia and lymphoma diagnosis and immunotherapy[J]. Leukemia & Lymphoma, 1995, 18(5/6):385-397.
    [15] KHALED WT, LIU PT. Cancer mouse models:past, present and future[J]. Seminars in Cell & Developmental Biology, 2014, 27:54-60.
    [16] HANGAUER MJ, VISWANATHAN VS, RYAN MJ, BOLE D, EATON JK, MATOV A, GALEAS J, DHRUV HD, BERENS ME, SCHREIBER SL, McCORMICK F, McMANUS MT. Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition[J]. Nature, 2017, 551(7679):247-250.
    [17] LÖSCHER W. Animal models of seizures and epilepsy:past, present, and future role for the discovery of antiseizure drugs[J]. Neurochemical Research, 2017, 42(7):1873-1888.
    [18] TIFFEN JC, BAILEY CG, NG C, RASKOJEJ, HOLST J. Luciferase expression and bioluminescence does not affect tumor cell growth in vitro or in vivo[J]. Molecular Cancer, 2010, 9:299.
    [19] NASRI M, KARIMI A, FARSANI MA. Production, purification and titration of a lentivirus-based vector for gene delivery purposes[J]. Cytotechnology, 2014, 66(6):1031-1038.
    [20] CARTIER N, HACEIN-BEY-ABINA S, BARTHOLOMAE CC, VERES G, SCHMIDT M, KUTSCHERA I, VIDAUD M, ABEL U, DAL-CORTIVO L, CACCAVELLI L, MAHLAOUI N, KIERMER V, MITTELSTAEDT D, BELLESME C, LAHLOU N, LEFRÈRE F, BLANCHE S, AUDIT M, PAYEN E, LEBOULCH P, et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy[J]. Science, 2009, 326(5954):818-823.
    [21] CAVAZZANA-CALVO M, PAYEN E, NEGRE O, WANG G, HEHIR K, FUSIL F, DOWN J, DENARO M, BRADY T, WESTERMAN K, CAVALLESCO R, GILLET-LEGRAND B, CACCAVELLI L, SGARRA R, MAOUCHE-CHRÉTIEN L, BERNAUDIN F, GIROT R, DORAZIO R, MULDER GJ, POLACK A, et al. Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia[J]. Nature, 2010, 467(7313):318-322.
    [22] BIFFI A, MONTINI E, LORIOLI L, CESANI M, FUMAGALLI F, PLATI T, BALDOLI C, MARTINO S, CALABRIA A, CANALE S, BENEDICENTI F, VALLANTI G, BIASCO L, LEO S, KABBARA N, ZANETTI G, RIZZO WB, MEHTA NAL, CICALESE MP, CASIRAGHI M, et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy[J]. Science, 2013, 341(6148):1233158.
    [23] AIUTIA, BIASCO L, SCARAMUZZA S, FERRUA F, CICALESE MP, BARICORDI C, DIONISIO F, CALABRIA A, GIANNELLIS, CASTIELLO MC, BOSTICARDO M, EVANGELIO C, ASSANELLI A, CASIRAGHI M, Di NUNZIO S, CALLEGARO L, BENATI C, RIZZARDI P, PELLIN D, Di SERIO C, et al. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome[J]. Science (New York, NY), 2013, 341(6148):1233151.
    [24] SESSA M, LORIOLI L, FUMAGALLI F, ACQUATI S, REDAELLI D, BALDOLI C, CANALE S, LOPEZ ID, MORENA F, CALABRIA A, FIORI R, SILVANI P, RANCOITA PMV, GABALDO M, BENEDICENTI F, ANTONIOLI G, ASSANELLI A, CICALESE MP, del CARRO U, SORA MGN, et al. Lentiviral haemopoietic stem-cell gene therapy in early-onset metachromatic leukodystrophy:an ad-hoc analysis of a non-randomised, open-label, phase 1/2 trial[J]. Lancet (London, England), 2016, 388(10043):476-487.
    [25] de RAVIN SS, WU XL, MOIR S, ANAYA-O'BRIEN S, KWATEMAA N, LITTEL P, THEOBALD N, CHOI U, SU L, MARQUESEN M, HILLIGOSS D, LEE J, BUCKNER CM, ZAREMBER KA, O'CONNOR G, McVICAR D, KUHNS D, THROM RE, ZHOU S, NOTARANGELO LD, et al. ematological Oncology, 2019, 37(suppl 1):48-52. for X-linked severe combined immunodeficiency[J]. Science Translational Medicine, 2016, 8(335):335ra57.
    [26] McGARRITY GJ, HOYAH G, WINEMILLER A, ANDRE K, STEIN D, BLICK G, GREENBERG RN, KINDER C, ZOLOPA A, BINDER-SCHOLL G, TEBAS P, JUNE CH, HUMEAU LM, REBELLO T. Patient monitoring and follow-up in lentiviral clinical trials[J]. The Journal of Gene Medicine, 2013, 15(2):78-82.
    [27] POOREBRAHIM M, SADEGHI S, FAKHR E, ABAZARI MF, POORTAHMASEBI V, KHEIROLLAHI A, ASKARI H, RAJABZADEH A, RASTEGARPANAH M, LINĒ A, CID-ARREGUI A. Production of CAR T-cells by GMP-grade lentiviral vectors:latest advances and future prospects[J]. Critical Reviews in Clinical Laboratory Sciences, 2019, 56(6):393-419.
    [28] PALFI S, GURRUCHAGA JM, LEPETIT H, HOWARD K, RALPH GS, MASON S, GOUELLO G, DOMENECH P, BUTTERY PC, HANTRAYE P, TUCKWELL NJ, BARKER RA, MITROPHANOUS KA. Long-term follow-up of a phase I/II study of ProSavin, a lentiviral vector gene therapy for parkinson's disease[J]. Human Gene Therapy Clinical Development, 2018, 29(3):148-155.
    [29] CAMPOCHIARO PA, LAUER AK, SOHN EH, MIR TA, NAYLOR S, ANDERTON MC, KELLEHER M, HARROP R, ELLIS S, MITROPHANOUS KA. Lentiviral vector gene transfer of endostatin/angiostatin for macular degeneration (GEM) study[J]. Human Gene Therapy, 2017, 28(1):99-111.
    [30] ELIAS HANSEN A, FLIEDNER FP, HENRIKSEN JR, JØRGENSEN JT, CLEMMENSEN AE, BØRRESEN B, ELEMA DR, KJÆR A, LARS ANDRESEN T. Liposome accumulation in irradiated tumors display important tumor and dose dependent differences[J]. Nanomedicine:Nanotechnology, Biology and Medicine, 2018, 14(1):27-34.
    [31] DEROOSE CM, de A, LOENING AM, CHOW PL, RAY P, CHATZIIOANNOU AF, GAMBHIR SS. Multimodality imaging of tumor xenografts and metastases in mice with combined small-animal PET, small-animal CT, and bioluminescence imaging[J]. Journal of Nuclear Medicine:Official Publication, Society of Nuclear Medicine, 2007, 48(2):295-303.
    [32] SHI Y, KUNJACHAN S, WU ZJ, GREMSE F, MOECKEL D, van ZANDVOORT M, KIESSLING F, STORM G, van NOSTRUM CF, HENNINK WE, LAMMERS T. Fluorophore labeling of core-crosslinked polymeric micelles for multimodal in vivo and ex vivo optical imaging[J]. Nanomedicine (London, England), 2015, 10(7):1111-1125.
    [33] BRAY F, FERLAY J, SOERJOMATARAM I, SIEGEL RL, TORRE LA, JEMAL A. Global cancer statistics 2018:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA:a Cancer Journal for Clinicians, 2018, 68(6):394-424.
    [34] WANG XY, McMANUS M. Lentivirus production[J]. Journal of Visualized Experiments:JoVE, 2009(32):1499.
    [35] SEGURA MM, MANGION M, GAILLET B, GARNIER A. New developments in lentiviral vector design, production and purification[J]. Expert Opinion on Biological Therapy, 2013, 13(7):987-1011.
    [36] 敖罗权, 敖翔, 郭韡, 邢伟, 胡雪停, 李战, 姚孟维, 吴晓凤, 徐祥. 稳定表达CD19的HeLa细胞株的构建及其作为CD19-CAR-T细胞的靶细胞功能鉴定[J]. 细胞与分子免疫学杂志, 2020, 36(12):1095-1101. AO LQ, AO X, GUO W, XING W, HU XT, LI Z, YAO MW, WU XF, XU X. Construction of HeLa cell line stably expressing CD19 and identification of its target cell function as CD19-CAR-T cells[J]. Chinese Journal of Cellular and Molecular Immunology, 2020, 36(12):1095-1101(in Chinese).
    [37] OTERODC, RICKERT RC. CD19 function in early and late B cell development. II. CD19 facilitates the pro-B/pre-B transition[J]. The Journal of Immunology, 2003, 171(11):5921-5930.
    [38] DIAMANT E, KEREN Z, MELAMED D. CD19 regulates positive selection and maturation in B lymphopoiesis:lack of CD19 imposes developmental arrest of immature B cells and consequential stimulation of receptor editing[J]. Blood, 2005, 105(8):3247-3254.
    [39] MILLSDM, STOLPA JC, CAMBIER JC. Modulation of MHC class II signaltransduction by CD19[M]//Advances in Experimental Medicine and Biology. Boston, MA:Springer US, 2007:139-148.
    [40] JUNE CH, O'CONNOR RS, KAWALEKAR OU, GHASSEMI S, MILONE MC. CAR T cell immunotherapy for human cancer[J]. Science, 2018, 359(6382):1361-1365.
    [41] NEELAPU SS. Managing the toxicities of CAR T-cell therapy[J]. H
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郭雨洁,段海潇,程奕宁,杨斌丰,胡翰,刘滨磊,汪洋. 稳定表达CD19-FLUC-GFP的CT26细胞系的构建及鉴定[J]. 生物工程学报, 2024, 40(2): 458-472

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  • 收稿日期:2023-06-27
  • 最后修改日期:2023-09-08
  • 在线发布日期: 2024-01-26
  • 出版日期: 2024-02-25
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