转染CD19-CAR的人原代T淋巴细胞的体外抗白血病作用研究

发布时间：2017-12-31 09:32

本文关键词：转染CD19-CAR的人原代T淋巴细胞的体外抗白血病作用研究　出处：《第二军医大学》2015年硕士论文　论文类型：学位论文

【摘要】：研究目的随着基因重组技术日益进步,细胞免疫治疗被认为是新世纪肿瘤治疗中最有前景的一种治疗方式,其中嵌合抗原受体(chimeric antigen receptor, CAR)因免除了MHC限制性等优势更被寄予厚望。本课题以MigRl质粒为表达载体,构建包括CD19单抗的单链可变区片段、CD28共刺激分子、T细胞受体-ζ链的二代靶向CD19 CAR结构(MigR1-CD19-CAR)。同时建立高表达CD19基因的CD19-K562稳定转染细胞株及其稳定成瘤NOD-SCID小鼠皮下移植瘤模型。进一步优化MigR1-CD19-CAR逆转录病毒对人T淋巴细胞(CAR-T)的转染效率,并以CD19-K562为靶细胞,对CAR-T细胞体外抗白血病作用进行初步研究。研究方法合成CD19单抗的单链可变区片段、CD28共刺激分子、T细胞受体-ζ链的目的基因序列。上述目的基因经电泳后行胶回收,利用酶切及连接反应将其构入MigRl质粒,转化DH5α感受态细胞,菌落挑取阳性克隆后经测序验证序列准确性。同上方法以扩增后CD19基因片段构建MigRl-CD 19重组质粒并测序验证。将构建的MigR1-CD19重组质粒转入Plat-A包装细胞,收集病毒上清重复转染K562细胞系,采用流式细胞仪检测CD19基因表达情况后体外反复传代获得稳转株,并以细胞计数、Annexin V/PI双重染色法分别检测其细胞增殖与凋亡特性。应用稳转株皮下接种NOD-SCID小鼠经体内、体外传代后建立移植瘤亚系CD19-K562-a并建立NOD-SCID小鼠皮下移植瘤模型。利用RT-PCR,瑞氏染色、免疫组化等验证CD19-K562-a细胞性质。将构建的MigR1-CD 19-CAR转入plat-A包装细胞,收集病毒上清离心转染经CD3/CD28磁珠及rhIL-2(重组人白介素-2)活化扩增后的人原代T淋巴细胞及K562细胞系,流式细胞仪检测转染效率,RT-PCR检测CD19-CAR目的基因转录。酶联免疫吸附法(ELISA)检测转染后CAR-T细胞与靶细胞(CD19-K562)共培养后干扰素-γ(IFN-γ)/肿瘤坏死因子α(TNF-α)释放量。结果1.测序验证二代MigR1-CD19-CAR及MigR1-CD19重组质粒分别与目的基因序列(仅MigR1-CD19一处碱基突变,但为无义突变)一致。2.应用MigR1-CD19重组载体经Plat-A包装细胞高效产毒,病毒滴度为(4.33-7.02)×107CFU/ml,离心转染法重复转染K562细胞并体外反复传代后流式细胞仪检测CD19阳性率为(99.80±0.17)%,获得稳转株,RT-PCR检测其CD19基因相对转录水平与转染空载K562、未转染K562细胞存在明显差异(P0.01),而细胞增殖、凋亡未受转染及传代过程影响。3.稳转株经皮下接种NOD-SCID小鼠,瘤体制成细胞悬液后体外结合体内传代后建立移植瘤亚系CD19-K562-a,其CD19阳性率为(99.78±0.04)%,且CD19基因与K562细胞原有bcr-ab1(210)基因均高效转录,瑞氏染色见CD19-K562-a, CD19-K562、未转染K562细胞均呈现未分化阶段细胞形态。CD19-K562-a皮下接种NOD-SCID小鼠建立移植瘤模型后行瘤体免疫组化,证实CD19-K562-a瘤体组CD19基因表达强阳性。4. MigRl-CD19-CAR重组载体转入Plat-A包装细胞后病毒滴度5.43-7.34×107CFU/ml,分离健康供者及化疗后缓解的B系血液系统肿瘤患者的外周血单个核细胞,经CD3/CD28磁珠联合rhIL-2活化扩增后,T淋巴细胞比例可高达(96.1±4.8)%。5.相同转染条件(32℃、1800 r/min、离心半径18.76cm、病毒滴度一定)时,K562细胞系转染效率高于T淋巴细胞(80.05±4.35)%VS(25.1±5.77)%,(P(0.01)。对活化扩增后的人原代T淋巴细胞,上述转染条件下,离心转染120min的转染效率为(54.5±14.62)%,相较未离心组(26.6±6.15)%、离心30min组(25.1±5.77)%、离心60min组(30.8±5.54)%均明显提高,后三组无统计学差异；根据个体T淋巴细胞体外活化扩增倍数选择合适的转染时机从而提高转染效率,健康供者转染效率最高达(68.7±0.6)%,B系恶性血液系统肿瘤患者(化疗后缓解)转染效率最高达(59.8±0.5)%。6.RT-PCR检测证实CD19-CAR目的基因在CAR-T细胞中高效特异性转录。针对scFv片段、跨scFv末端酶切位点至CD28片段、跨CD28末端酶切位点至TCR片段的3组引物,三组片段相对转录水平分别为(2057±549)%、(3737±1101)%、(1001±110)%,对各自阴性对照存在显著统计学差异(P值均小于0.01)。7. CD19-CAR-T细胞与CD19-K562细胞共培养组IFN-γ(13229.93±1542.99) pg/ml、TNF-α(4466.72±210.77)pg/ml释放量均增加,与阴性对照组统计学差异均显著(P0.01)。结论1.MigR1-CD19-CAR及MigRl-CD19重组载体均构建成功。2.成功建立CD19阳性率高达(99.80±0.17)%的CD19-K562稳定转染细胞株。3.采用体外结合体内传代建立稳定成瘤的NOD-SCID小鼠移植瘤亚系CD19-K562-a并成功建立NOD-SCID小鼠皮下移植瘤模型。4.转染条件为32℃、1800 r/min、离心半径18.76cm时,通过离心转染120min及根据体外活化扩增的人原代T淋巴细胞扩增倍数选择个体转染时机,成功优化MigRl-CD19-CAR重组载体对人T淋巴细胞转染效率,RT-PCR检测验证CD19-CAR目的基因在转染后T淋巴细胞中高效特异性转录。5.转染后CAR-T细胞特异性识别靶细胞引发细胞因子IFN-γ、TNF-α释放显著增加。
[Abstract]:Objective to study with gene recombination technology, cell immunotherapy is a treatment for the most promising new century tumor treatment, the chimeric antigen receptor (chimeric antigen, receptor, CAR) exempt from the advantage of MHC restricted more high hopes. This research on MigRl plasmid expression vector construction, including CD19 monoclonal antibody single chain variable fragment CD28, costimulatory molecules, T cell receptor zeta chain two generation targeting CD19 CAR structure (MigR1-CD19-CAR). At the same time to establish the high expression of CD19 in CD19-K562 cells stably transfected into tumor and its stable model of NOD-SCID mice subcutaneous transplantation tumor. Further optimization of MigR1-CD19-CAR retrovirus on human T lymphocytes (CAR-T) transfection efficiency, and with CD19-K562 as the target cell, preliminary study on the anti leukemia effect of CAR-T cells in vitro. Methods of synthesis of CD19 single chain variable antibody Fragment of CD28, costimulatory molecules, T cell receptor gene sequence. The zeta chain gene by electrophoresis after digestion and gel recovery, the reaction will be connected into MigRl plasmid by enzyme, DH5 alpha transformation competent cells, colony positive clones after sequencing accuracy. In the same way the amplified CD19 gene fragment to construct MigRl-CD 19 recombinant plasmid and sequencing. The recombinant plasmid MigR1-CD19 was constructed into Plat-A packaging cells, the viral supernatant was collected by repeated transfection of K562 cells, detect the expression of CD19 gene after repeated passage to obtain stable strain by flow cytometry, and the cell count, detect cell proliferation and apoptosis characteristics of Annexin V/PI double staining method. The application of stable strain NOD-SCID mice by subcutaneous inoculation in vivo and in vitro after establishment of transplanted tumor sublines CD19-K562-a and NOD-SCID mice to establish subcutaneous transplantation tumor Model. Using RT-PCR, Wright staining and immunohistochemistry to verify CD19-K562-a cell properties. The constructed MigR1-CD 19-CAR into plat-A packaging cells, the viral supernatant was collected by centrifugation with CD3/CD28 beads and rhIL-2 (recombinant human interleukin -2) activation after amplification of primary human T cells and K562 cells, the transfection efficiency of detection flow cytometry, RT-PCR detection of CD19-CAR gene transcription. Enzyme linked immunosorbent assay (ELISA) detection of transfected CAR-T cells and target cells (CD19-K562) after co culture of interferon gamma (IFN- y) / tumor necrosis factor alpha (TNF- alpha) release. Results 1. of two generation MigR1-CD19-CAR sequencing and MigR1-CD19 recombinant plasmid with the gene sequence (MigR1-CD19 only one mutation, but nonsense mutation).2. using recombinant MigR1-CD19 vector was packaged in Plat-A cells, toxin production, virus titer for (4.33-7.02) * 107CFU/ml, centrifugal rotor Repeat staining of transfected K562 cells in vitro and repeated passage after flow cytometry was used to detect the positive rate of CD19 (99.80 + 0.17)%, to obtain stable strain, RT-PCR detection of CD19 gene transcription and transfection of K562, no obvious differences in transfected K562 cells (P0.01), and cell proliferation and apoptosis by.3. effect of transfection and passage of stable strain by subcutaneous inoculation of NOD-SCID mice, the tumor cell suspension after in vitro binding in vivo passage established after transplantation tumor sublines CD19-K562-a, the positive rate of CD19 (99.78 + 0.04)%, and the CD19 gene of K562 cells and the original bcr-ab1 (210) genes were highly transcribed, Wright staining see CD19-K562-a, CD19-K562, K562 transfected cells showed undifferentiated cell morphology.CD19-K562-a subcutaneously inoculated NOD-SCID mice to establish xenograft tumor model by immunohistochemistry confirmed that CD19-K562-a tumor group strong positive expression of CD19 gene of.4. MigRl The recombinant plasmid -CD19-CAR was transfected into packaging cell Plat-A after the virus titer of 5.43-7.34 * 107CFU/ml, B Department of hematological malignancies were isolated from healthy donors and after chemotherapy with peripheral blood mononuclear cells by CD3/CD28 magnetic beads combined rhIL-2 activation after amplification, T lymphocyte ratio can be as high as (96.1 + 4.8)%.5. transfection conditions (32 c r/min, 1800, centrifugal radius 18.76cm, the titer of virus, K562) a cell line transfection efficiency is higher than that of T lymphocytes (80.05 + 4.35)%VS (25.1 + 5.77)%, (P (0.01). The activation after amplification of primary human T cells, the transfection conditions, the transfection efficiency was transfected into 120min centrifuge (54.5 + 14.62)%, compared with non centrifugal group (26.6 + 6.15)%, group 30min (25.1 centrifugal + 5.77)%, (30.8 + 5.54 60min centrifugal group%) were significantly increased after no statistical difference between the three groups; according to the individual T cells activated in vitro amplification times appropriate transfection The machine can improve the transfection efficiency, the transfection efficiency of healthy donors up to (68.7 + 0.6)%, B patients with malignant hematological tumor (after chemotherapy) transfection efficiency up to (59.8 + 0.5)%.6.RT-PCR showed that CD19-CAR gene in CAR-T cells, specific transcription. For scFv fragment, cross scFv terminal restriction sites to the CD28 fragment, 3 sets of primers across CD28 terminal restriction sites to the TCR fragment, the relative transcription levels of the three groups were fragments (2057 + 549)% and (3737 + 1101)% and (1001 + 110)% of their negative control had statistically significant difference (P value less than 0.01) group IFN- gamma.7. co cultured CD19-CAR-T cells and CD19-K562 cells (13229.93 + 1542.99) pg/ml, TNF- alpha (4466.72 + 210.77) pg/ml release was increased, and the negative control group were significant different (P0.01). Conclusion 1.MigR1-CD19-CAR and MigRl-CD19 recombinant plasmid were successfully constructed.2. successfully established CD The positive rate of 19 (99.80 + 0.17) CD19-K562 stable transfected cell line.3.% by in vitro binding in vivo passage to establish stable NOD-SCID mice transplanted tumor sublines CD19-K562-a and successfully established NOD-SCID mice xenograft model.4. transfection conditions of 32 DEG C, 1800 r/min, from the heart of 18.76cm radius, by centrifugal 120min transfection and according to the ex vivo expansion of human primary T cells amplification factor selecting individual transfection time, the successful optimization of MigRl-CD19-CAR recombinant vector on the transfection efficiency of T lymphocyte, RT-PCR detection and verification of CD19-CAR gene in T lymphocytes after transfection in high specific transcription of.5. target cells after transfection of CAR-T cell specific recognition by cytokines IFN-, TNF- alpha release increased significantly.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R733.7

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