【作者】 李睿；

【导师】 陈燕；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 第一部分藤黄酸抑制类固醇激素受体共活化因子3及Akt通路介导慢性粒细胞白血病急变K562细胞株G0／G1期阻滞和凋亡目的：研究藤黄酸抗白血病的影响及其分子机制，为其临床应用提供理论基础。方法：采用MTT比色法检测K562细胞的增殖活性，Annexin V-FITC／PI双标法检测细胞凋亡及透射电镜分析细胞凋亡形态学的改变，流式细胞术分析细胞周期改变，Realtime-PCR检测藤黄酸对K562细胞内SRC-3和Bcl-2基因表达的影响，Western blot法检测藤黄酸对K562细胞内SRC-3，Akt，pAkt，核糖体蛋白S6激酶1（S6K1），pS6K1，糖原合成激酶3β（GSK-3β），pGSK-3β和Bcl-2蛋白表达的影响。结果：（1）藤黄酸以剂量、时间依赖性方式抑制K562细胞的增殖，其24 h的IC50是1．9±0．1μmol／L。（2）藤黄酸以剂量依赖性方式诱导K562细胞凋亡，伴有典型的凋亡细胞形态学改变。（3）藤黄酸阻滞K562细胞周期于G0／G1期。（4）藤黄酸能以剂量依赖性方式抑制SRC-3，pAkt，pS6K1，pGSK-3β和Bcl-2表达水平，而Akt，S6K1和GSK-3β总蛋白水平不受影响。结论：藤黄酸发挥其抗白血病效应可能与下调类固醇激素受体共活化因子3（SRC-3）的表达有关。第二部分鱼藤素抑制类固醇激素受体共活化因子3及NF-κB通路介导急性淋巴细胞白血病Jurkat细胞凋亡目的：研究鱼藤素抗白血病的影响及其分子机制，为其临床应用提供理论基础。方法：采用MTT比色法检测Jurkat细胞的增殖活性，原位细胞凋亡检测技术（TUNEL）及透射电镜分析细胞凋亡形态学的改变，流式细胞术分析细胞周期改变，RT-PCR和Westem blot法检测鱼藤素对白血病细胞内SRC-3，NF-κB，Bcl-2，Bcl-xL基因和蛋白表达的影响。结果：（1）鱼藤素以剂量、时间依赖性方式抑制Jurkat细胞的增殖，其24 h的IC50是43．73±0．35 nmol／L，48h的IC50为28．96±0．29 nmol／L。（2）鱼藤素以剂量依赖性方式诱导Jurkat细胞凋亡。40 nmol／L的鱼藤素处理24 h后透射电镜观察到细胞染色质浓缩，核碎裂，典型凋亡小体形成。TUNEL法检测到TUNEL阳性细胞，40 nmol／L的鱼藤素处理24 h后TUNEL阳性细胞百分比为12．8±0．85％，与对照组（1．1±0．09％）相比有统计学意义（P＜0．01）；（3）鱼藤素阻滞Jurkat细胞周期于G0／G1期。（4）鱼藤素能同时在蛋白和基因水平，以剂量依赖性方式抑制SRC-3及其相关基因NF-κB和其下游的抗凋亡蛋白Bcl-2，Bcl-xL的表达。结论：鱼藤素能明显抑制Jurkat白血病细胞的增殖，并诱导其凋亡，其抗白血病效应可能与下调类固醇激素受体共活化因子3（SRC-3）的表达有关。第三部分鱼藤素对多发性骨髓瘤细胞类固醇激素受体共活化因子3的调节作用目的：研究鱼藤素对人多发性骨髓瘤细胞株RPMI-8226细胞抗增殖影响及其分子机制。方法：采用MTT比色法检测鱼藤素对RPMI-8226细胞增殖活性的影响，Annexin-VFITC／PI双标法及Hoechst33258分析细胞凋亡形态学的改变，肿瘤细胞侵袭实验检测鱼藤素对RPMI-8226细胞侵袭作用的影响，RT-PCR法检测鱼藤素对SRC-3和NF-κB基因表达的影响，免疫组化法检测鱼藤素对SRC-3和NF-κB蛋白的影响和分布情况，明胶酶法用来检测RPMI-8226细胞分泌MMP-2和MMP-9酶的活性。结果：（1）鱼藤素能明显抑制RPMI 8226细胞的增殖，其24 h的IC₅₀是54．55±0．40nmol／L。（2）鱼藤素以剂量依赖性方式诱导RPMI-8226细胞凋亡，伴有典型的凋亡细胞形态学改变。（3）鱼藤素能以剂量依赖性方式抑制RPMI-8226细胞侵袭。（4）鱼藤素能同时在蛋白和基因水平抑制SRC-3及其相关基因NF-κB，从而下调MMP-2和MMP-9酶的活性。结论：鱼藤素发挥其抗多发性骨髓瘤效应可能与下调SRC-3的表达有关。更多还原

【Abstract】 PartⅠGambogic acid induces G0/G1 arrest and apoptosisinvolving inhibition of SRC-3 and inactivation of Aktpathway in K562 leukemia cellsObjective:To investigate the anticancer effects and the molecular mechanisms ofGambogic acid（GA） on K562 cells and to provide more theories for clinical application.Methods:The effects of Gambogic acid on the growth ofK562 cells were studied by MTTassay.Annexin V/PI double-labeled cytometry and transmission electron microscopy wereused to detect cell apoptosis.A propidium iodide method was used to study cell cycledistribution.Quantitative real-time PCR was employed to assess the expression levels ofsteroid receptor coactivator-3 （SRC-3） and apoptosis related gene Bcl-2.Western blottingwas employed to assess the expression levels of SRC-3,Akt,pAkt,S6K1,pS6K1,GSK-3β,pGSK-3βand Bcl-2.Results:（1） GA was able to inhibit cell proliferation in K562 cells in a time- and dose-dependent manner,with 24h IC₅₀ value of 1.9±0.1μmol/L.（2） Gambogic acid inducedapoptosis of K562 cells in a dose-dependent manner,which was accompanied by typicalapoptotic morphological changes.（3） GA induced cell-cycle arrest in G0/G1 phase in K562cells.（4） GA treatment downregulated the expression of SRC-3 and inhibited the activity ofAkt kinase and its downstream targets p70 S6 kinase 1 （S6K1） and glycogen synthasekinase 3β（GSK-3β） without changes in total protein levels of these three proteins in K562cells,accompanied by decreased expression of the apoptosis related gene Bcl-2.Conclusion:GA might exhibit its strong antitumor effects via the interruption of SRC-3.PartⅡDeguelin induces apoptosis involving inhibition of SRC-3 andNF-κB pathway in Jurkat cellsObjective:To investigate the anticancer effects and the molecular mechanisms of deguelin onJurkat cells and to provide more theories for clinical application.Methods:The effects of deguelin on the growth of Jurkat cells were studied by MTT assay.Terminal deoxynucleotide transferase-mediated dUTP nick end labeling （TUNEL） assay andtransmission electron microscopy were used to detect cell apoptosis.A propidium iodidemethod was used to study cell cycle distribution.RT-PCR and western blotting were employedto assess the expression levels of steroid receptor coactivator-3 （SRC-3）,nuclear factor-κB（NF-κB） and some apoptosis related genes,including Bcl-2 and Bcl-xL.Results:（1） Deguelin was able to inhibit cell proliferation in Jurkat cells in a time- anddose-dependent manner,with 24h IC₅₀ value of 43.73±0.35 nmol/L and 48h IC₅₀ value of28.96±0.29 nmol/L.（2） Deguelin induced apoptosis in Jurkat cells in a dose-dependentmanner in vitro.After treatment with 40nmol/l deguelin for 24h,apoptotic bodies containingnuclear fragments and the chromatin condensed were found by Transmission electronmicroscopy.Jurkat cells treated with 40nmol/l deguelin for 24h also showed positive TUNELstaining.The percentage of TUNEL positive cells was 12.8±0.85%,compared with only 1.1±0.09% TUNEL positive cells in the untreated samples （P＜0.01）.（3） Deguelin induced cell-cycle arrest in G0/G1 phase in Jurkat cells.（4） Deguelin could downregulate theexpression of SRC-3 and its downstream transcription factor NF-κB,which thus influencedthe expression of apoptosis related genes Bcl-2 and Bcl-xL.Conclusion:Deguelin presents potent effects on growth-arrest and apoptosis-induction inJurkat cells in vitro via the interruption of SRC-3.PartⅢEffects of deguelin on the regulation of SRC-3 in MM cells in vitroObjective:To investigate the anticancer effects and the molecular mechanisms of deguelin onRPMI-8226 cells.Methods:The effects of deguelin on the growth of RPMI-8226 cells were studied by MTTassay.Hoechst 33258 staining and Annexin-V FITC/PI double-labeled cytometry wereused to detect cell apoptosis.Transwell invasion assay was used to assess the role ofdeguelin on RPMI-8226 cell invasion.RT-PCR was employed to assess the mRNAexpression levels of SRC-3 and NF-κB,whereas,SRC-3 and NF-κB protein expression andlocalization were determined by using immunohistochemistry method.GelatinZymography was used to detect the gelatinolytic activity of secreted MMP-2 and -9.Results:（1） Deguelin presented striking proliferation inhibition potency on RPMI-8226cells in vitro,with 24h IC50 value of 54.55±0.40nmol/L.（2） Deguelin induced apoptosis inRPMI-8226 cells in a dose-dependent manner in vitro,which was accompanied by typicalapoptotic morphological changes.（3） Deguelin could inhibit RPMI-8226 cell invasion.（4）Deguelin could downregulate the expression of SRC-3 and its downstream transcriptionfactor NF-κB,which thus influenced the gelatinolytic activity of secreted MMP-2 and -9.Conclusion:Deguelin exhibit its strong antitumor effects via the interruption of SRC-3.更多还原