本文關(guān)鍵詞：轉(zhuǎn)染CD19-CAR的人原代T淋巴細(xì)胞的體外抗白血病作用研究　出處：《第二軍醫(yī)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 嵌合抗原受體 質(zhì)粒 MigR1 基因轉(zhuǎn)染 K562細(xì)胞 T淋巴細(xì)胞

【摘要】：研究目的隨著基因重組技術(shù)日益進(jìn)步,細(xì)胞免疫治療被認(rèn)為是新世紀(jì)腫瘤治療中最有前景的一種治療方式,其中嵌合抗原受體(chimeric antigen receptor, CAR)因免除了MHC限制性等優(yōu)勢(shì)更被寄予厚望。本課題以MigRl質(zhì)粒為表達(dá)載體,構(gòu)建包括CD19單抗的單鏈可變區(qū)片段、CD28共刺激分子、T細(xì)胞受體-ζ鏈的二代靶向CD19 CAR結(jié)構(gòu)(MigR1-CD19-CAR)。同時(shí)建立高表達(dá)CD19基因的CD19-K562穩(wěn)定轉(zhuǎn)染細(xì)胞株及其穩(wěn)定成瘤NOD-SCID小鼠皮下移植瘤模型。進(jìn)一步優(yōu)化MigR1-CD19-CAR逆轉(zhuǎn)錄病毒對(duì)人T淋巴細(xì)胞(CAR-T)的轉(zhuǎn)染效率,并以CD19-K562為靶細(xì)胞,對(duì)CAR-T細(xì)胞體外抗白血病作用進(jìn)行初步研究。研究方法合成CD19單抗的單鏈可變區(qū)片段、CD28共刺激分子、T細(xì)胞受體-ζ鏈的目的基因序列。上述目的基因經(jīng)電泳后行膠回收,利用酶切及連接反應(yīng)將其構(gòu)入MigRl質(zhì)粒,轉(zhuǎn)化DH5α感受態(tài)細(xì)胞,菌落挑取陽性克隆后經(jīng)測(cè)序驗(yàn)證序列準(zhǔn)確性。同上方法以擴(kuò)增后CD19基因片段構(gòu)建MigRl-CD 19重組質(zhì)粒并測(cè)序驗(yàn)證。將構(gòu)建的MigR1-CD19重組質(zhì)粒轉(zhuǎn)入Plat-A包裝細(xì)胞,收集病毒上清重復(fù)轉(zhuǎn)染K562細(xì)胞系,采用流式細(xì)胞儀檢測(cè)CD19基因表達(dá)情況后體外反復(fù)傳代獲得穩(wěn)轉(zhuǎn)株,并以細(xì)胞計(jì)數(shù)、Annexin V/PI雙重染色法分別檢測(cè)其細(xì)胞增殖與凋亡特性。應(yīng)用穩(wěn)轉(zhuǎn)株皮下接種NOD-SCID小鼠經(jīng)體內(nèi)、體外傳代后建立移植瘤亞系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、免疫組化等驗(yàn)證CD19-K562-a細(xì)胞性質(zhì)。將構(gòu)建的MigR1-CD 19-CAR轉(zhuǎn)入plat-A包裝細(xì)胞,收集病毒上清離心轉(zhuǎn)染經(jīng)CD3/CD28磁珠及rhIL-2(重組人白介素-2)活化擴(kuò)增后的人原代T淋巴細(xì)胞及K562細(xì)胞系,流式細(xì)胞儀檢測(cè)轉(zhuǎn)染效率,RT-PCR檢測(cè)CD19-CAR目的基因轉(zhuǎn)錄。酶聯(lián)免疫吸附法(ELISA)檢測(cè)轉(zhuǎn)染后CAR-T細(xì)胞與靶細(xì)胞(CD19-K562)共培養(yǎng)后干擾素-γ(IFN-γ)/腫瘤壞死因子α(TNF-α)釋放量。結(jié)果1.測(cè)序驗(yàn)證二代MigR1-CD19-CAR及MigR1-CD19重組質(zhì)粒分別與目的基因序列(僅MigR1-CD19一處堿基突變,但為無義突變)一致。2.應(yīng)用MigR1-CD19重組載體經(jīng)Plat-A包裝細(xì)胞高效產(chǎn)毒,病毒滴度為(4.33-7.02)×107CFU/ml,離心轉(zhuǎn)染法重復(fù)轉(zhuǎn)染K562細(xì)胞并體外反復(fù)傳代后流式細(xì)胞儀檢測(cè)CD19陽性率為(99.80±0.17)%,獲得穩(wěn)轉(zhuǎn)株,RT-PCR檢測(cè)其CD19基因相對(duì)轉(zhuǎn)錄水平與轉(zhuǎn)染空載K562、未轉(zhuǎn)染K562細(xì)胞存在明顯差異(P0.01),而細(xì)胞增殖、凋亡未受轉(zhuǎn)染及傳代過程影響。3.穩(wěn)轉(zhuǎn)株經(jīng)皮下接種NOD-SCID小鼠,瘤體制成細(xì)胞懸液后體外結(jié)合體內(nèi)傳代后建立移植瘤亞系CD19-K562-a,其CD19陽性率為(99.78±0.04)%,且CD19基因與K562細(xì)胞原有bcr-ab1(210)基因均高效轉(zhuǎn)錄,瑞氏染色見CD19-K562-a, CD19-K562、未轉(zhuǎn)染K562細(xì)胞均呈現(xiàn)未分化階段細(xì)胞形態(tài)。CD19-K562-a皮下接種NOD-SCID小鼠建立移植瘤模型后行瘤體免疫組化,證實(shí)CD19-K562-a瘤體組CD19基因表達(dá)強(qiáng)陽性。4. MigRl-CD19-CAR重組載體轉(zhuǎn)入Plat-A包裝細(xì)胞后病毒滴度5.43-7.34×107CFU/ml,分離健康供者及化療后緩解的B系血液系統(tǒng)腫瘤患者的外周血單個(gè)核細(xì)胞,經(jīng)CD3/CD28磁珠聯(lián)合rhIL-2活化擴(kuò)增后,T淋巴細(xì)胞比例可高達(dá)(96.1±4.8)%。5.相同轉(zhuǎn)染條件(32℃、1800 r/min、離心半徑18.76cm、病毒滴度一定)時(shí),K562細(xì)胞系轉(zhuǎn)染效率高于T淋巴細(xì)胞(80.05±4.35)%VS(25.1±5.77)%,(P(0.01)。對(duì)活化擴(kuò)增后的人原代T淋巴細(xì)胞,上述轉(zhuǎn)染條件下,離心轉(zhuǎn)染120min的轉(zhuǎn)染效率為(54.5±14.62)%,相較未離心組(26.6±6.15)%、離心30min組(25.1±5.77)%、離心60min組(30.8±5.54)%均明顯提高,后三組無統(tǒng)計(jì)學(xué)差異；根據(jù)個(gè)體T淋巴細(xì)胞體外活化擴(kuò)增倍數(shù)選擇合適的轉(zhuǎn)染時(shí)機(jī)從而提高轉(zhuǎn)染效率,健康供者轉(zhuǎn)染效率最高達(dá)(68.7±0.6)%,B系惡性血液系統(tǒng)腫瘤患者(化療后緩解)轉(zhuǎn)染效率最高達(dá)(59.8±0.5)%。6.RT-PCR檢測(cè)證實(shí)CD19-CAR目的基因在CAR-T細(xì)胞中高效特異性轉(zhuǎn)錄。針對(duì)scFv片段、跨scFv末端酶切位點(diǎn)至CD28片段、跨CD28末端酶切位點(diǎn)至TCR片段的3組引物,三組片段相對(duì)轉(zhuǎn)錄水平分別為(2057±549)%、(3737±1101)%、(1001±110)%,對(duì)各自陰性對(duì)照存在顯著統(tǒng)計(jì)學(xué)差異(P值均小于0.01)。7. CD19-CAR-T細(xì)胞與CD19-K562細(xì)胞共培養(yǎng)組IFN-γ(13229.93±1542.99) pg/ml、TNF-α(4466.72±210.77)pg/ml釋放量均增加,與陰性對(duì)照組統(tǒng)計(jì)學(xué)差異均顯著(P0.01)。結(jié)論1.MigR1-CD19-CAR及MigRl-CD19重組載體均構(gòu)建成功。2.成功建立CD19陽性率高達(dá)(99.80±0.17)%的CD19-K562穩(wěn)定轉(zhuǎn)染細(xì)胞株。3.采用體外結(jié)合體內(nèi)傳代建立穩(wěn)定成瘤的NOD-SCID小鼠移植瘤亞系CD19-K562-a并成功建立NOD-SCID小鼠皮下移植瘤模型。4.轉(zhuǎn)染條件為32℃、1800 r/min、離心半徑18.76cm時(shí),通過離心轉(zhuǎn)染120min及根據(jù)體外活化擴(kuò)增的人原代T淋巴細(xì)胞擴(kuò)增倍數(shù)選擇個(gè)體轉(zhuǎn)染時(shí)機(jī),成功優(yōu)化MigRl-CD19-CAR重組載體對(duì)人T淋巴細(xì)胞轉(zhuǎn)染效率,RT-PCR檢測(cè)驗(yàn)證CD19-CAR目的基因在轉(zhuǎn)染后T淋巴細(xì)胞中高效特異性轉(zhuǎn)錄。5.轉(zhuǎn)染后CAR-T細(xì)胞特異性識(shí)別靶細(xì)胞引發(fā)細(xì)胞因子IFN-γ、TNF-α釋放顯著增加。
[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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R733.7

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1 王揚(yáng);轉(zhuǎn)染CD19-CAR的人原代T淋巴細(xì)胞的體外抗白血病作用研究[D];第二軍醫(yī)大學(xué);2015年

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