Fas/Fas ligand (FasL)-mediated apoptosis plays a critical role in deletion of activated T cells. This study aimed to construct the lentivirus encoding FasL gene induced by deoxycycline and evaluate its effects on apoptosis of Th1 cells. A plasmid expression system encoding FasL was constructed through utilizing the controlling gene and target gene of the Tet-on system controlled by the PBI plasmid containing the bidirectional promoter. The lentiviral vector that consisted of the vector plasmid, the packaging plasmid and the envelop plasmid was isolated and purified. 293 T cells were cotransfected with three plasmids using Lipofectamine 2000. 48 h post transfection, the viral supernatant was collected to determine the virus titre. The FasL protein expression in 293T cells was detected by Western blotting. Spleen lymphocytes of rats were transfected with the lentiviral vector system encoding FasL gene and 5 days later were induced by doxycycline for 24 h, followed by detection of FasL mRNA. The apoptosis index of Th1 cells was measured through both Annexin V-FITC flow cytometry and TUNEL. Additionally flow cytometry was adopted to determine changes of the quantity of Th1 cells. The FasL protein was expressed in 293T cells transfected by the expressing FasL lentiviral vector following deoxycycline induction, while it was not expressed in 293T cells without deoxycycline induction. The virus titre was 4 × 108 u/l for 293T cells. The deoxycycline induced lentiviral vector system encoding FasL gene was successfully transfected into spleen lymphocytes of rats. The FasL RNA expression significantly increased in the deoxycycline plus lentivirus group, compared to that in the control group and the deoxycycline group. A higher apoptosis index and an obviously smaller quantity of Th1 cells were obtained in the deoxycycline plus lentivirus group than in the other two groups. The lentivirus system encoding FasL induced by deoxycycline promotes apoptosis of Th1 cells.
Key words: FasL, lentiviral vector, Th1 cell, apoptosis.
RA, Rheumatoid arthritis; MS, multiple sclerosis; DM, diabetes mellitus; RT-PCR, reverse transcriptase-polymerase chain reaction; SD, Sprague Dawley; AGE, agarose gel electrophoresis; DMEM, Dulbecco's modiﬁed Eagle's medium; PBS, phosphate-buffered saline; TNF, tumor necrosis factor; NGF, nerve growth factor; FasL, Fas ligand; AICD, activation induced cell death; DISC, death-inducing signal complex.
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