| Chapter 1 Establishment of DEX-induced mouse thymocyte apoptosis model invitro and morphological observationAIM: To establishment of DEX-induced mouse thymocyte apoptosis model in vitro and to make morphological observation.METHOD: We studied on dexamethasone-induced mouse thymocyte apoptosis model, used flow cytometry (FCM) and transmission electronic (TEM) and fluorescence microscope analysis to determine the model feasibility. RESULTS: The apoptosis percentage of RPMI1640 control group was 2.90 + 0.62%, while by 2.5 X 10'6mol/L DEX treatment, it was 12.70 ?.44% (3h), 53.55+4.59% (6h), p<0.01, respectively. By lumol/L DEX treatment, the apoptosis rates at 3 h, 5 h and 7 h were (18.43 + 0.30)%, (37.85 + 1.46) % and (62.53 + 1.81)%, respectively; in control group, they were (4.05 + 0.29)%, (4.81 ?.24)% and (7.77 + 0.31)% (p<0.01). At same time intervals, the necrosis rates of DEX group were (1.20 ?.08)%, (3.99 + 0.31)% and (6.46+0.81)%; in control group, they were (0.93+0.10)%, (1.27 + 0.22)% and (1.55+0.19)% (p<0.0l). Typical apoptotic cells were observed in 6 h DEX group by TEM and fluorescence microscopy.CONCLUSION: We established DEX-induced mouse thymocyte apoptosis model in vitro, readily for quantitative study. We also proposed factors to affect model stability included animal, temperature, mechanical injury and operation period.Chapter 2 Study on the MAPK related signaling pathway in DEX-inducedmouse thymocyte apoptosis processAIM: To study on the MAPK related signaling pathway in DEX-induced mouse thymocyte apoptosis process and to explore inner mechanism.METHOD: To stimulate mouse thymocyte single with PD 98059 (10, 25, 50 umol/L), SB 203580 (10umol/L), respectively, or combined with 1 umol/L DEX; to detect apoptotic and necrotic cells by Annexin V-FITC/PI double staining flow cytometry at 3, 5, 7 h. To stimulate mouse thymocyte with lumol/L DEX, and dectect ERK1/2, p38, JNK1/2, c-Myc and caspase-3 signals by SDS-PAGE and Western blotat 0, 30, 60 and l80min.RESULTS: By the single stimulation of 10, 25, 50 umol/L PD, respectively,apoptotic rates of mouse thymocyte was significantly higher than control at 3, 5, 7h(p<0.01); and showed drug-dependent trend; necrotic rates also higher than controlat same time points.With the stimulation of 0, 25, 50 umol/L PD and combined with 1 umol/L DEX, theapoptosis to necrosis process showed faster trend than DEX group; results showed PDspeeds DEX-induced mouse thymocyte apoptosis.By the single stimulation of 10 umol/L SB, apoptotic rates of mouse thymocyte wassignificantly higher than control at 3, 5 and 7 h(p<0.01); necrotic rates also higherthan control at same time points. By 10 umol/L SB and 1 umol/L DEX combinedstimulation, apoptotic and necrotic rate was higher than DEX group at 3,5 and 7 h.results showed SB speeds DEX-induced mouse thymocyte apoptosis.Western blot results showed total ERK1/2 of control group was plenty and stable,almost not change with time lapse, pERKl/2 also stable following time lapse. WhileDEX existed, total ERK1/2, especially ERK1, decreased significantly; pERKl/2significantly decreased.In control group, total p38 remains nearly same level; while DEX existed, total p38showed decline trend.JNK1 showed increase trend and JNK2 remained stable in control group; while inDEX group, total JNK1/2 contents were lesser at all time points, and JNK2 showedsignificantly decrease trend following time lapse.Obvious expression of c-Myc was detected at 0 min in control group, then c-Myccontent increased at 30 min and finely reduced at 60 and 180 min; in DEX group,c-Myc expression was more than control group, and got a peak expression at 30 min,then significantly reduced at 60 and 180 min.Caspase-3 increased following culture time lapse in control group; while its contentwas more in DEX group at 0 min, peak content was detected at 30 min, andsignificant reduction also observed at Ih and 3h.CONCLUSION: DEX-induced apoptosis may have direct relationship with itsinhibitory e... |
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