Study On In Vitro Culture Of Mature T Lymphocytes And Its Gene Transfection Mediated By Lentivirus System

Induced pluripotent stem cells, commonly abbreviated as iPS cells or iPSCs, are a type of non-pluripotent cell, such as an adult somatic cell, derived by viral transfection of certain stem cell-associated genes and reprogrammed to induce a "forced" expression of specific genes. With potencies of self-renewing and multi-direction differentiation, IPS cells are similar to pluripotent stem cells in many respects, such as the morphology of cell, the expression of certain stem cell gene and proteins, chromatin methylation patterns, the capability of embryoid bodies and teratoma formation. IPS cell was first generated by Shinya Yamanaka's team in 2006 that successfully reprogrammed the mouse fibroblasts transduced by the recombinant retroviuses carrying four key pluripotency genes ( Oct-3/4, SOX2, c-Myc, and Klf4 ), respectively. As for iPS cells, sufficiency and readily availability of the somatic cells reprogrammed are very critical. However, mature T lymphocyte was limited to be one candidate due to its short lifespan and very low efficiency of transduction.Certain Lentivirus derived from the human immunodeficiency virus typeⅠ( HIV-1 ) , replicating in both dividing and non-dividing cells whereas a standard retroviral vector does, can introduce a gene into abnormal cells and treat various disease as a gene therapy tool. Another common application of the lentivirus is to deliver a reporter gene into some cells, such as primary-, stem- and other undifferentiated cells, to improve its transduction efficiency greatly and achieve high expression level of target gene in the above cell transiently. In addition, the lentivirus can also increase the frequency of extra gene integrated with the genome of host cell.This study established for the activation of mature T lymphocytes in mice cultured in vitro for a long time, and lentiviral transfection technology platform, the main results obtained are as follows:1.Mature T lymphocyte proliferation and activation.The mononuclear cells were separated by the Ficoll gradient centrifugation and were then treated with concanavalin A (ConA) and anti-mouse CD3 plus anti-mouse CD28 to activate the mature T lymphocytes, respectively. The activated T cells were confirmed by mononuclear cell surface marker via flow cytometry (FACS) as CD4+CD44high. The lymphocyte proliferation were monitored by Carboxy fluorescein diacetate succinimidyl ester (CFSE) labelling. As a result,～2×10~7 mononuclear cells could be isolated from each 3-week old mouse. Applyied to a 96-well round-bottom plate pre-coated by 2μg / mL anti-mouse CD3 plus 4μg / mL anti-mouse CD28, 2×105 cells/well could rapidly proliferate to 1.6×10~6 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 1.5×10~6 cells /well at 14 days post activated). Comparatively, the lymphocytes stimulated by ConA need to arrive the top level of 1.4×10~6 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 ( 50μL/well) were GFP positive, comparable to the efficiency of those (85.9±3.6)% tranfection by unpurified lentivirus ( 500μL/well ). Similarly, the MOI-dependent tranfection efficiency was found in the mature activated T lymphocytes: after 3 days, the percentages of GFP positive cells were (25.8±1.75)%, (36±9.5)% and (43±4.75)% at MOI of 1, 5, 10 IU/cell, respectively. The research to further improve the lentiviral tranfection efficiency of T lymphocytes is ongoing.Establishment and optimization of the technique will be useful in the study of inducing the mature T lymphocytes by liver-specific transcription factors and reprogramming into liver cells directly in future.