| Objective (1) To establish the model of apoptosis of activated human y8T cells induced by reatimulating with Mycobacterium tuberculosis antigen (Mtb-Ag). (2) To investigate effects of different concentrations of ethanol and dimethyl sulfoxide (DMSO) on the apoptosis and the viability of γδT cells and αβT cells. (3) To investigate the difference of ceramide (C2-Cer) in induction of apoptosis of γδT cells and αβT cells. (4) To investigate the roles of PKC, PI3K and MAPK pathways in the apoptosis of activated human γδT cells induced by restimualting wih Mtb-Ag.Methods (1) The peripheral blood mononuclear lymphocytes (PBMC) of healthy donors were stimulated with Mtb-Ag and CD3mAb, cultured in IL-2 containing media, to generate Mtb-Ag activated T cells (MtbAT) (γδT cells were predominant) and CD3mAb activated T cells (CD3AT) (αβT cells were predominant) respectively. (2) MtbAT which were cultured about 15d to 25d were restimulated with three concentrations of Mtb-Ag for 24 hours, and the apoptosis of roT cells were measured by flowcytometry (FCM) using Annexin-V-FITC/PI staining. (3) Mtb-AT were restimulating with Mtb-Ag (10ug/ml) for 3 hours, 6 hours, 12 hours, and 24 hours, the apoptosis of γδT cells were detected. (4) MtbAT and CD3AT were treated with four concentrations of ethanol and DMSO for 4 hours or 24 hours, the apoptosis of γδT cells and αβT cells were measured by FCM using Annexin-V-FITC/PI staining, and theviability of T cells were detected by MTT assay. (5) MtbAT and CD3AT were treated with four concentrations of C2-Cer for 3 hours, the apoptosis of γδT cells and αβT cells were measured, and the viability of T cells was detected. (6) Mtb-AT cells, were pretreated with SB203580 (an inhibitor for P38 pathway), LY294002 (an inhibitor for PI3K pathway) and Rottlerin (an inhibitor for PKC) respectively for 60 minutes, and restimulating with Mtb-Ag for 3 hours, the apoptosis of γδT cells were detected. Results (1) The percentages of γδT cells in MtbAT were from 70% to 90%, and aBT cells in CD3AT were above 90%. (2) The percentages of apoptosis of γδT cells restimulated with Mtb-Ag (5.0ug/ml) only increased 8.05%, appeared to be weak (P>0.05) in contrast to control. But both 10.0ug/ml and 20.0ug/ml Mtb-Ag significantly induced the apoptosis of γδT cells (P<0.01), the percentages of apoptosis of γδT cells increased respectively 35.63% and 38.83%, and there were not apparent difference between them (P>0.05) as compared with control. (3) Compared with control, the apoptosis of γδT cells could be significantly induced by restimulating MtbAT with Mtb-Ag (10.0ug/ml) for 3 hours, 6 hours, 12 hours or 24 hours (P<0.01), the percentages of apoptosis of γδT cells were among 44.21% to 51.76%. There weren't profound difference (P>0.05) in the percentages of apoptosis of γδT cells restimulated by Mtb-Ag (10.0ug/ml) between for 3 hours and for 24 hours, the percentages of apoptosis of the latter is higher than the former about 7.55%. (4) Besides 1.0% and 2.0% DMSO in media for 24 hours induced profoundly apoptosis (P<0.05) on αβT cells, and 2.0% DMSO in media for 4 hours inhibited significantly the viability of αβT cells (P<0.05), the apoptosis and the inhibition of the viability of γδT cells and αβT cells induced by lower concentration of ethanol and DMSO (< 2.0%) appeared to be weak (P>.05) in contrast to control. With the exception of inapparent apoptosis (P>0.05) of αβT cells induced by 5.0% ethanol in media for 4 hours and γδT cells induced by 5.0% DMSO in media for 4 hours, the apoptosis and the inhibition of the viability of γδT cells and αβT cells induced by higher concentration of ethanol and DMSO (5.0%) appeared to be significant increase (P<0.05) as compared with control. (5) The apoptosis and the inhibition of the viability of γδT cells and αβT cells induced by lower concentrationof C2-Cer (< 10umol/L) appeared to be weak (P>. 05) in contrast to control. There were significant differences in the apoptosis of yST cells and αβT cells induced by highe... |
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