【作者】 宋晓红；

【导师】 马丁；

【作者基本信息】 华中科技大学 ， 妇产科学， 2009， 博士

【摘要】 第一部分PI3K／AKT／GSK3β信号通路在紫杉醇诱导卵巢癌细胞凋亡中作用的研究目的通过应用糖原合酶激酶-3β的特异性抑制剂LiCl和PI3K抑制剂LY294002，探讨PI3K／AKT／GSK3β信号通路在紫杉醇诱导卵巢癌细胞凋亡和周期阻滞中的作用。方法1、蛋白质印迹法检测抑制剂LiCl对GSK3β磷酸化水平的影响及对Bcl-2家族抗凋亡蛋白Bcl-2、Bcl-x1和Mcl-1表达的影响。2、细胞凋亡的检测：AnnexinV-FITC／PI凋亡检测试剂盒检测紫杉醇和LiCl作用下卵巢癌细胞的凋亡率；Hochest33342染色检测各组细胞凋亡形态学变化；蛋白质印迹法检测凋亡执行分子Caspase 9和Caspase 3的剪切情况。3、应用线粒体跨膜电位指示剂JC-1对各组细胞进行染色，荧光显微镜观察不同的凋亡程度下荧光的变化情况。4、应用线粒体提取试剂盒提取各组细胞的线粒体，蛋白质印迹法检测抑制剂LiCl对Bax线粒体转位的影响。5、细胞免疫荧光法检测紫杉醇对Bax蛋白表达和分布的影响。6、PI染色流式细胞仪检测LY294002和LiCl对紫杉醇诱导周期阻滞的影响。结果1、蛋白印迹实验结果显示，浓度增加的紫杉醇90 nM、300 nM和1μM作用OV2008细胞24h后，可上调GSK3β的磷酸化水平，而总的GSK3β蛋白表达无明显变化；LiCl可明显增加GSK3β的磷酸化水平，而总体GSK3β表达无明显变化。2、细胞凋亡的检测结果：各组细胞分别进行AnnexinV-FITC／PI凋亡染色，流式检测结果显示，LiCl预孵育后，梯度浓度紫杉醇作用细胞24h后的细胞凋亡率由26．77％，27．35％，32．28％下降为17．82％，20．17％，18．96％；Hochester33342染色结果显示，LiCl可抑制紫杉醇导致的细胞核形态学变化；蛋白质印迹结果显示，LiCl可抑制Caspase 9和Caspase 3的分子剪切，从而抑制凋亡的发生。3、线粒体跨膜电位指示剂JC-1染色结果显示，抑制剂LiCl可维持卵巢癌细胞OV2008线粒体的极化状态下红绿荧光的强度，阻抑紫杉醇诱导的线粒体去极化所导致的红色荧光减弱。4、细胞免疫荧光和线粒体提取试剂盒提取各组卵巢癌细胞线粒体后的蛋白质印迹结果显示，抑制剂LiCl可抑制紫杉醇导致的Bax蛋白的线粒体转位，并且抑制紫杉醇诱导的抗凋亡分子Bcl-2、Bcl-x1和Mcl-1的降解，上调其表达。5、流式细胞仪周期分析显示，紫杉醇作用后可引起明显的G₂M期阻滞（69．33％），而LiCl预孵育的细胞G₂M期阻滞明显增加（88．13％），紫杉醇与PI3K抑制剂LY294002（20μM）合用后则可明显降低其G₂M期阻滞（44．06％）。6、蛋白质印迹结果显示，应用RNA干扰降低AKT2表达或应用LY294002降低AKT活性均可增强紫杉醇诱导的卵巢癌细胞凋亡。结论1、GSK3β高水平磷酸化可抑制紫杉醇诱导的卵巢癌细胞凋亡。2、磷酸化GSK3β可抑制紫杉醇诱导的Bax蛋白的线粒体转位和抗凋亡分子Bcl-2、Bcl-x1和Mcl-1的降解，从而维持线粒体的稳定，抑制凋亡。3、GSK3β可通过调节Cyclin B1和CDC2影响紫杉醇诱导的G₂M期阻滞。第二部分PI3K／AKT／GSK3β信号通路在顺铂诱导卵巢癌细胞凋亡中作用的研究目的探讨增加GSK3β的磷酸化状态在顺铂诱导的卵巢癌细胞凋亡中的作用。方法运用GSK3β特异性抑制剂LiCl增加GSK3β磷酸化水平，应用流式细胞仪检测浓度梯度增加的顺铂诱导的两株卵巢癌细胞凋亡情况，以及LY294002和LiCl对顺铂诱导细胞周期阻滞的影响；Western Blot检测GSK3β及磷酸化GSK3β蛋白的表达。结果Western Blot结果显示，梯度浓度增加的顺铂（40μM、80μM及120μM）可导致GSK3β磷酸化水平不断增加；Western Blot结果显示，抑制剂LiCl作用后，磷酸化GSK3β水平明显增加；流式结果显示，梯度增加的顺铂作用于0V2008细胞24h，其凋亡率不断增加，分别为3．34％、23．48％和55．17％，而应用LiCl抑制剂预孵育的细胞，不同浓度顺铂作用24h后凋亡率分别为1．21％、16．09％和21．73％；而在另一株卵巢癌细胞SKOV3中，40μM、80μM及120μM作用24h后凋亡率分别为24．98％、53．34％和64．93％，而LiCl预孵育后，细胞凋亡率明显降低为13．94％、20．65％和35．75％。流式细胞周期分析结果显示，顺铂作用后可引起OV2008明显的S期阻滞（83．1 8％），而LY294002则可明显改善细胞S期阻滞（9．96％），且呈现轻度G₂M期阻滞（22．75％）；LiCl也可部分改善细胞的S期阻滞（39．55％）。结论GSK3β磷酸化水平增加可抑制顺铂诱导的卵巢癌细胞的凋亡。更多还原

【Abstract】 Part oneStudy of the role and mechanism of PI3K/AKT/GSK3βcellsingnaling pathway in paclitaxel induced apoptosis in ovarian cancerObjective: To investigate the effects of PI3K/AKT/GSK3βcell signal pathway on theapoptosis and cell cycle arrest induced by paclitaxel in ovarian carcinoma cell lines.Methods :1. Western Blot was used to detect the expression of p-GSK3βand the anti-apoptoticproteins Bcl-2、Bcl-xl and Mcl-1 in ovarian carcinoma cell lines treated by differentconcentration paclitaxel or by paclitaxel and LiCl.2. The apoptotic ratio test : the AnnexinV-FITC apoptosis detection kit was use to measurethe apoptotic ratio of OV20008 induced by paclitaxel and LiCl; the change of cellmorphology was identified by the Hochest33342 staining; and the Western Blot was use toevaluate the expression of Caspase 9 and Caspase 3.3. The effects of LiCl on mitochondrial membrane potential of OV2008 treated bypaclitaxel was measured by mitochondrial membrane potential detection Kit, then observedthe fluorescence under fluorescence microscope.4. Immunofluorescence and the mitochondria isolation kit were used to detect thetransposition of Bax from cytoplasm to mitochondria.5. Cell cycle were detected by flow cytometry after stained with PI in ovarian cancer cellstreated by LY294002 and LiCl.Results:1. LiCl could enhance the expression of p-GSK3β.The results of FACS showed that theapoptotic rates of cells treated with paclitaxel （26.77%,27.35%,32.28%）was higher than the cells treated with paclitaxel and LiCl （17.82%,20.17%,18.96%）. Hochest33342 stainingshowed that LiCl could inhibit the apoptosis induced by pacitaxel, compared to the cellswithout inhibitor .And the Western Blot also showed that LiCl could inhibit the clip ofCaspase 9 and Caspase 3.2. The JC-1 staining showed that LiCl could inhibit the reduce of red fluorescence when thecells treated only by paclitaxel were detected stronger green fluorescence and lower redfluorescence.3. The results of immunofluorescence and Western Blot suggested that LiCl could block thetransposition of Bax from cytoplasm to mitochondria which induced by paclitaxel. TheWestern Blot also showed LiCl could suppress the down-regulation of Bcl-2、Bcl-xl andMcl-1.4. The results of FACS showed that LiCl could enhanced the G₂M arrest induced bypaclitaxel （88.13%） and in contrast LY294002 could improve the cell cycle arrest（ 44.06% ）5. Western Blot showed down-regulation of AKT by RNAi or by LY294002, both couldpromote the apoptosis induced by paclitaxel.Conclusions: The augment of p-GSK3βexpression could inhibit the apoptosisinduced by paclitaxel through up-regulating the function of Bax and down-regulating theanti-apoptotic proteins （Bcl-2,Bcl-xl,Mcl-1）; and could also enhanced the G₂M arrest inovarian carcinoma cell lines.Part twoStudy of the effects of PI3K/AKT/GSK3βcell singnaling pathway oncisplatin induced apoptosis in ovarian cancerObjective: To investigate the effects of enhanced p-GSK3βon the apoptosis induced bypaclitaxel in ovarian carcinoma cell lines.Methods: LiCl was used to enhance the expression of p-GSK3β.The apoptosis wasdetected by FACS. The expression of p-GSK3βand GSK3βwas measured by WesternBlot. Results: The results of Western Blot showed that after treated by different concentrationcisplatin 24h, the expression of p-GSK3βwas up-regulated in the ovarian cancer cellsOV2008 and SKOV3.The results of FACS showed that the apoptotic rates of OV2008cells treated with different concentration cisplatin was 3.34 %、23.48 % and 55.17%,when pretreated with LiCl, the apoptotic rates reduced to 1.21%、16.09% and 21.73 % ;in another ovarian cancer cell SKOV3 , the apoptotic rates treated with differentconcentration cisplatin was 24.98%、53.34% and 64.93%, when pretreated with LiCl, theapoptotic rates reduced to 13.94%、20.65% and 35.75%. The results of Western Blotshowed that LiC1 could enhance the expression of p-GSK3βand the cisplatin also couldup-regulated the expression ofp-GSK3β.Conclusions: The augment ofp-GSK3βexpression could inhibit the apoptosis inducedby cisplatin in ovarian carcinoma cell lines.更多还原