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Study on in Vitro Culture of Mature T Lymphocytes and Its Gene Transfection Mediated by Lentivirus System

Author: YeLiWei
Tutor: HuYiPing
School: Second Military Medical University
Course: Cell Biology
Keywords: Induced pluripotent stem cells Lentiviral transfection T lymphocytes Flow cytometry
CLC: R329
Type: Master's thesis
Year: 2011
Downloads: 154
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Abstract

Induced pluripotent stem cells (induced pluripotent stem cells, iPSc) is the use of viral vectors to specific combination of transcription factors into differentiated cells to reprogramming cells similar to embryonic stem cells. It not only has the self-renewal and differentiation potential, and cell morphology, gene and protein expression, epigenetic modification state embryoid body and teratoma generation capacity are similar to embryonic stem cells. The technique is to first create the recombinant 2006 by Yamanaka Oct3 / 4, Sox2, c-Myc, and Klf4 four transcription factor gene of the virus vector is introduced into mouse fibroblast cells, and successfully induce reprogramming. In subsequent studies, how to choose convenient sources, a sufficient number of mature somatic target cells for reprogramming, become one of the hotspots in IPS. Mature T lymphocytes of these selection conditions, but there is in vitro survival time is short, and very low viral transduction efficiency of technical problems. (Lentivirus) of lentivirus vector based on human immunodeficiency virus type I (HIV-1) is developed on the basis of the gene therapy vector. Distinguish between general retroviral vector, its dividing cells and non-dividing cells have the ability to infect. In a related cell experimental operation, some not even transferred cells by conventional methods is difficult to turn, such as primary cells, stem cells, etc. undifferentiated cells, mediated through lentivirus method can greatly improve the efficiency of gene transduction, and the objective gene is integrated into the host cell genome chance also increased significantly, which provides a favorable way for reporting efficient transient expression of genes within cells. Fit the mice mature activated T lymphocytes in vitro prolonged culture, as well as the lentiviral transduction technology platform, get the main findings of this study are as follows: 1, mature T lymphocyte proliferation and activation. Ficoll gradient centrifugation and mononuclear cells in mice with concanavalin A (ConA) and anti-mouse CD3 anti-mouse CD28 methods such as activation of mature T lymphocytes and flow cytometry assay T cell surface CD4 < sup> of CD44 ^{high activation markers; carboxy-fluorescein diacetate - succinimidyl ester (CFSE) tracer method detects the activation of cell proliferation split situation. The results show: the ~ 2 × 10 ^{7 can be isolated from the spleens of the 3 month old female Balb / c mice (SPF) to the mononuclear cells; plate of a 96-well \mouse CD3 anti-mouse CD28 (final concentration, respectively 2μg/mL, 4μg/mL) after the package is, enables 2.0 × 10 ^{5 cells / well in rapidly proliferating to the third day (D3) 1.6 × 10 ^{6 cells / well CD4 CD44high T lymphocyte activation after the anti-mouse CD3 anti-mouse CD28 stimulation share of the total number of live cells (74.0 ± 1.8)%, and CFSE strong positive cells original (92.3 ± 2.8)% reduced to (16.34 ± 1.3)%; the activation of T lymphocytes can be maintained in the culture medium containing IL-2 (final concentration of 30 ng / mL) CTL growth (D14 cell count was 1.5 × 10 ^{6 / holes); activated T lymphocytes stimulated with ConA D7 to achieve the highest value 1.4 × 10 ^{6 cells / well, and with the incubation time, apoptosis increased, until it is completely dead. Experimental evaluation of mature mouse T lymphocytes ability to maintain long-term proliferation and activation status of the feasibility of in vitro culture conditions, and to lay the foundation for the next step the GFP recombinant lentiviral transduction. 2, lentivirus packaging and the transfection efficiency of mature T lymphocytes. Using flow cytometry, using liposomes GFP recombinant the lentivirus plasmid was transfected into the packaging cell line 293T, cell supernatants were collected after 48 hours, and further with the ultracentrifugation method of purified the GFP recombinant lentiviral transduction LEPC cell D2 assay the transfection efficiency and virus titer. Finally, the purified GFP recombinant lentivirus at MOI = 1,5,10 and different titer transfection of activated T lymphocytes stimulated by anti-mouse CD3 anti-mouse CD28, and observing the GFP recombinant lentiviral of T lymphocytes The transfection efficiency. The results show: liposome GFP recombinant lentiviral plasmid transfected into packaging cells 293T, all with a green fluorescence after 48 hours, 293T transfection efficiency of more than 95%. Not purified GFP recombinant lentiviral particles (500μL virus / well) transduced the LEPC cells, flow cytometry analysis of GFP positive rate (85.9 ± 3.6)%; And by the super concentrated 10-fold from the purified recombinant lentiviral group (50 μL virus / well), the GFP-positive still reached (74.3 ± 1.8)%. The GFP recombinant lentiviral transfection purified anti-mouse CD3 anti-mouse CD28 stimulation activated T lymphocytes after 3 days, GFP-positive rate, respectively: MOI = 1 group (25.8 ± 1.75)%, MOI = 5 (36.0 ± 9.5)%, MOI = 10 group (43.0 ± 4.75)%; compared with the same titer of adenovirus, the transfection efficiency of T lymphocytes more significantly improved; With GFP recombinant lentivirus titer increased, the transfection efficiency on T lymphocytes is also higher. Lentiviral transduction efficiency of the T lymphocytes to further improve the experiment is still ongoing. The establishment of the technical system and continuous improvement induced liver stem cells to differentiate into mature T lymphocytes prepared the conditions for the next step with the liver-specific transcription factor.}}}}}}

Study on in Vitro Culture of Mature T Lymphocytes and Its Gene Transfection Mediated by Lentivirus System

Abstract

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