成熟t淋巴细胞分离培养及其慢病毒介导的基因转染的分析-isolation and culture of mature t lymphocytes and analysis of lentivirus mediated gene transfection.docxVIP

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成熟t淋巴细胞分离培养及其慢病毒介导的基因转染的分析-isolation and culture of mature t lymphocytes and analysis of lentivirus mediated gene transfection.docx

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成熟t淋巴细胞分离培养及其慢病毒介导的基因转染的分析-isolation and culture of mature t lymphocytes and analysis of lentivirus mediated gene transfection

第二军医大学硕士学位论文 As a result, ～ 2× 107 mononuclear cells could be isolated from each 3-week old mouse. Applyied to a 96-well round-bottom plate pre-coated by 2 μg / mL anti-mouse CD3 plus 4 μg / mL anti-mouse CD28, 2× 105 cells/well could rapidly proliferate to 1.6 ×106 cells / well after 3 days. Moreover, CD4+CD44high subset accounted for (74±1.8)% of the total cells and occurred an abruptly decline of percentage of CFSEhigh group from (92.3±2.8)% down to (16.34±1.3)%. The survival of activated T lymphocytes could be maintained in CTL medium in presence of 30 ng/mL IL-2 (with 1.5×106 cells /well at 14 days post activated). Comparatively, the lymphocytes stimulated by ConA need to arrive the top level of 1.4×106 cells / well at seventh day, and subsequently progressed to die with longer incubation time.The first part of our experiment evaluated the availability of extending the lifespan of mature T lymphocytes activated in vitro, so as to render its tranfection by lentivirus further. 2. Lentivirus packaging and mature T lymphocytes on tranfection efficiency. The recombinant GFP and lentiviral vector packaging plamids were co-transfected 293T cells using Lipofectamine 2000 transfection agents and then collected the cell supernatant after 48 hrs followed purified by ultra-centrifuge. The titer of the purified virus with the GFP as a reporter was measured by tranfected LEPC via FACS. Then, the mature T lymphocytes activated by anti-mouse CD3 plus anti-mouse CD28 were tranfected by the purified lentiviral GFP at MOI of 1, 5, 10 IU/cell, respectively and detected their tranfection efficiencies by FACS. As a result, all the 293T packaging cells were GFP positive 48 hrs posttransfection, leading to 95% above transfection efficiency. Moreover, (74.3±1.8)% of LEPC tranfection by purified lentivirus ( 50 μL/well) were GFP positive, comparable to the efficiency of those (85.9±3.6)% tranfection by unpurified lentivirus ( 500 μL/well ). Similarly, the MOI-dependent tranfection effi