【作者】 王通；

【导师】 曾耀英；

【作者基本信息】 暨南大学 ， 生物医学工程， 2004， 博士

【摘要】 第一章 地塞米松诱导小鼠胸腺细胞凋亡体外模型建立及形态学研究 目的：建立地塞米松(DEX)诱导小鼠胸腺细胞凋亡体外模型并且进行凋亡形态学研究。方法：以地塞米松诱导小鼠胸腺细胞凋亡为模型，通过流式细胞术、电镜和荧光显微镜测定模型的稳定性。结果：仅在完全RMPI 1640培养基培养条件下小鼠胸腺细胞自发凋亡百分率为(2.90±0.62)％，在2.5 μmol／L DEX诱导下，3小时培养组(12.70±2.44)％、6小时培养组(53.55±4.59)％发生了细胞凋亡；在1μmol／L DEX诱导下，小鼠胸腺细胞在3h、5h和7h凋亡比率分别为(18.43±0.30)％、(37.85±1.46)％和(62.53±1.81)％，对照组分别为(4.05±0.29)％、(4.81±0.24)％和(7.77±0.31)％，组间差异显著(p＜0.01)；相同时点下，DEX组坏死比率分别为(1.20±0.08)％、(3.99±0.31)％和(6.46±0.81)％，对照组分别为(0.93±0.10)％、(1.27±0.22)％和(1.55±0.19)％，组间差异显著(p＜0.01)。形态学和荧光显微观察到典型的细胞凋亡现象。结论：建立了体外DEX诱导小鼠胸腺细胞凋亡定量模型，并且发现影响模型稳定性的主要因素包括实验动物、温度、机械损伤和操作时间等。 第二章 地塞米松诱导小鼠胸腺细胞凋亡过程中MAPK相关信息通路研究 目的：研究在DEX影响小鼠胸腺细胞MAPK相关信号变化特点，以进一步探讨DEX诱导小鼠胸腺细胞凋亡过程中的作用机制。 方法：分别用终浓度为10、25和50μmol／L的ERK1／2特异性阻断剂PD98059(PD)以及10μmol／L的p38特异性阻断剂SB203580(SB)单独和联合1μmol／L的DEX作用小鼠胸腺细胞，并通过Annexin V-FITC／PI双染流式细胞术测定3 h、5 h和7 h凋亡和坏死细胞比率；以终浓度1μmol／L的DEX诱导小鼠胸腺细胞凋亡，利用SDS-PAGE及Western blot检测0、30、60和180 min细胞内ERK1／2、p38、JNK1／2、c-Myc和caspase-3信号变化特点。 结果：分别在终浓度10、25和50μmol／L的PD单独刺激下，小鼠胸腺细胞凋亡比例在3、5和7 h时点均显著高于对照组(p＜0.01)，而且表现出PD剂量依赖型诱导小鼠胸腺细胞凋亡趋势；各组坏死比例均高于对照组。 在终浓度10、25和50μmol／L PD和1μmol／L DEX联合刺激下，小鼠胸腺细胞凋亡坏死进程明显提高，结果显示PD加速DEX诱导小鼠胸腺细胞凋亡。 SB10林mol/L单独刺激下，小鼠胸腺细胞凋亡比例在3、5和7h时点均显著高于对照组(p<0.01);同时小鼠胸腺细胞坏死比例在3在相同时点下均高于对照组。SB10林mol几和1林mol/L DEX联合刺激下，小鼠胸腺细胞凋亡和坏死比例在3、5和7h时点分别为均高于DEX组。结果显示SB加速DEX诱导小鼠胸腺细胞凋亡 W亡stem blot结果显示，对照组胸腺细胞ERKI/2总量相对丰富而且稳定，基本上不随时间变化而变化，不仅如此，磷酸化激活的ERKI/2(pERKI/2)含量随时间变化也相对恒定。在DEX存在的条件下，ERKI/2总量相对有所下降，尤其是ERKI比较明显;pERKI/2含量在DEX的刺激后立即显著下降。 对照组p38含量随着培养时间增加呈不明显变化趋势;而DEX组p38呈逐渐减少趋势。 对照组JNKI随着时间变化呈逐渐增加趋势，而爪K2含量变化不明显。DEX组每个时点州Kl/2含量均低于对照组，而且JNKZ呈明显的随培养时间延长而减少趋势。 对照组在omin即可检测到c一Myc的明显表达，30min略见增多，lh和3h呈微弱的下降趋势;而DEX组c一Myc在30 min明显增强，lh和3h显著减弱，但均高于对照组。 对照组caspase一3在O和30 min表达不明显，而在lh和3h有着较为明显的表达;DEX组easpase一3在30 min最多，lh和3h显著减少。 结论:本研究结果提示DEX对MAPK三种激酶表达和激活的阻断作用可能与其诱导凋亡有着密切的关系。在DEX诱导的小鼠胸腺细胞凋亡呈现c一Myc介导的细胞凋亡模式，而且在该过程中c一Myc、ERKI/2和casPase一3信号可能存在着反馈抑制调节的可能性。 第三章地塞米松诱导小鼠胸腺细胞凋亡过程中线粒体膜电势和 钙离子变化研究目的:研究DEX诱导小鼠胸腺细胞凋亡过程中线粒体膜电势(△平m)和钙离子([eaZ+]*)变化特点。方法:以终浓度1娜ol几的DEX刺激小鼠胸腺细胞，通过JC一1染色流式细胞术，在。、z、3、5、7、9、一l、24和27h时点分别检测小鼠胸腺细胞平均J一眼『egate(瓦2)和平均J一monomer(瓦l)含量，并计算线粒体膜电势(瓦2/瓦1)。利用激光扫描共聚焦分析测定细胞内[C扩勺i。利用cFDA一sE染色技术评价sh时点细胞骨架蛋白变化。 结果:本研究中，小鼠胸腺细胞离体之后元1所反映的线粒体质量迅速下降，至sh对照组达到平台期(913.38士70.11)，然后缓慢下降到27h的(622.80士22.81)。DEX组瓦l在lh和3h与对照组没有统计学差异(P>0.05)，而从sh (660.91土72.95)开始显著低于对照组(P<0.01)，并且随培养时间延长呈下降趋势，至27h的(309.70士53.35)。对照组和DEx组瓦2随培养延长而呈降低趋势。l一gh，对照组和DEX的△wm没有显著差异(P>0.05)。而在11、24和27h，对照组△wm分别为(256.41士21.59)、(214.14士23.21)和(146.14士17.97)，而更多还原

【Abstract】 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更多还原