【作者】 孙迪；

【导师】 沈宜；

【作者基本信息】 重庆医科大学 ， 病理学与病理生理学， 2010， 硕士

【摘要】 目的:研究Ras及其下游PI3K和MAPK信号通路对人类宫颈癌细胞株HeLa细胞增殖及细胞周期素E(cyclin E)表达的影响。在此基础上,进一步探讨介导cyclin E泛素化降解的抑癌蛋白F-盒和含蛋白7的WD重复结构域(FBW7)在HeLa细胞中的表达特点,并观察调控cyclin E表达的Ras下游信号通路对FBW7表达的影响。方法:1.以宫颈癌细胞株HeLa细胞为研究对象,观察Ras信号通路对HeLa细胞增殖、细胞周期分布及cyclin E表达的影响:不同浓度Ras信号通路阻断剂FTI-277处理HeLa细胞,噻唑兰比色(MTT)法检测FTI-277对HeLa细胞的增殖抑制率;流式细胞术(FCM)检测FTI-277对HeLa细胞细胞周期分布的影响;RT-PCR和Western blot检测FTI-277对HeLa细胞cyclin E的mRNA和蛋白水平表达的影响。2.观察Ras下游PI3K和MAPK信号通路对HeLa细胞增殖及cyclin E表达的影响:不同浓度PI3K信号通路阻断剂LY-294002和MAPK信号通路阻断剂UO-126分别处理HeLa细胞,MTT法分别检测两者对HeLa细胞的增殖抑制率;RT-PCR和Western blot分别检测两者对HeLa细胞cyclin E的mRNA和蛋白水平表达的影响。3.观察Ras下游调控cyclin E表达的MAPK信号通路对HeLa细胞FBW7表达的影响:RT-PCR和Western blot检测UO-126对HeLa细胞FBW7的mRNA和蛋白水平表达的影响;免疫荧光法观察HeLa细胞FBW7的分布及UO-126对HeLa细胞FBW7表达的影响。结果:1. MTT实验显示,Ras信号通路阻断剂FTI-277具有抑制HeLa细胞增殖的作用,且具有时效量效依赖性。结果显示10μmol/L的FTI-277药物浓度处理组线性相关性最好,因此,在后续的实验中我们采用10μmol/L的FTI-277作为干扰因素,探讨FTI-277对HeLa细胞周期及cyclin E表达的影响。FCM检测细胞周期结果显示,FTI-277作用后细胞周期滞留于G1/S期,且具有时间依赖性。RT-PCR和Western blot检测结果显示FTI-277作用后,HeLa细胞cyclin E mRNA水平无明显改变,cyclin E蛋白表达水平减弱。2.PI3K信号通路阻断剂LY-294002和MAPK信号通路阻断剂UO-126均显示出抑制HeLa细胞增殖的作用,10μmol/L的LY-294002药物浓度处理组和10μmol/L的UO-126药物浓度处理组分别显示出最好的线性相关性,因此,实验中采用10μmol/L的LY-294002和10μmol/L的UO-126处理HeLa细胞,观察不同时间梯度下cyclin E表达的变化。LY-294002对cyclin E的蛋白表达无明显影响,相对之下,UO-126能下调cyclin E的蛋白表达。3. RT-PCR和Western blot检测结果显示UO-126作用后,HeLa细胞FBW7 mRNA及蛋白表达均增多。免疫荧光结果显示,FBW7在HeLa细胞的细胞质和细胞核中有表达,细胞核部位表达较多。UO-126处理HeLa细胞后FBW7阳性表达增强,并具有时间依赖性。结论:1. HeLa细胞的异常增殖与Ras信号通路的激活有关,阻断Ras信号通路激活可以减慢HeLa细胞周期G1/S期的进程并下调cyclin E蛋白的表达。Ras-MAPK信号通路而不是Ras-PI3K信号通路在HeLa细胞cyclin E蛋白的表达调控中发挥重要作用。2. HeLa细胞中泛素化降解cyclin E的抑癌蛋白FBW7表达也与Ras-MAPK信号通路有关。FBW7位于Ras-MAPK信号通路的下游。更多还原

【Abstract】 Objective:To study the effects of Ras and its downstream PI3K and MAPK signaling pathway on the proliferation and the expression of cyclin E in human cervical cancer HeLa cells.On this base,to further explore the expression characteristics of tumor suppressor protein F-box and WD repeat domain-containing protein7(FBW7) which mediated the ubiquitination degradation of cyclin E and to observe the effect of Ras downstream signaling pathway which regulated the expression of cyclin E on the expression of FBW7.Methods:1. Cervical cancer HeLa cells were used as the research model,the effects of Ras signaling pathway on the proliferation,cells cycle distribution and the expression of cyclin E were investigated in the following experiment.HeLa cells were treated with different concentrations of FTI-277 ,a Ras inhibitor,MTT assay was used to observe the proliferation of HeLa cells.Flow cytometry(FCM) was used to examine the growth cycles of the HeLa cells.The mRNA and protein expression of cyclin E were detected by RT-PCR and Western blot before and after Ras signal was blocked by FTI-277.2. To observe the effects of PI3K signaling pathway and MAPK signaling pathway that lie downstream of Ras on the proliferation and the expression of cyclin E in HeLa cells. HeLa cells were treated with different concentrations of LY-294002 ,a PI3K inhibitor ,and UO-126,a MAPK inhibitor,HeLa cells proliferation inhibition rate was analysed by MTT test.Expression of cyclin E was detected by RT-PCR and Western blot.3. To investigate the effect of Ras-MAPK signaling pathway regulating the expression of cyclin E on the expression of FBW7 in HeLa cells.The MAPK signaling pathway was blocked by UO-126,the mRNA and protein expression of FBW7 were detected by RT-PCR and Western blot.Immunofluorescence showed the location and expression of FBW7 in HeLa cells.Results:1. MTT test showed HeLa cells proliferation was inhibited by FTI-277,a Ras signaling pathway inhibitor,in time-dependent and dose-dependent way.The results indicated that FTI-277 were in 10μmol/L treatment groups which had the best linear relationship.Therefore,we have chosen 10μmol / L as the interfering factors to observe FTI-277 on cells cycle and cyclin E expression at different time. Flow cytometry showed that FTI-277 blocked Hela cells in G1/S phase in a time dependent way. Cyclin E mRNA expression in HeLa cells treated with FTI-277 had no significant change, but the level of cyclin E protein expression was significantly reduced.2. MTT test demonstrated that both PI3K pathway inhibitor LY-294002 and MAPK pathway inhibitor UO-126 inhibited the proliferation of HeLa cells,10μmol/L LY-294002 treatment group and 10μmol/L UO-126 treatment group had the approximat best linear correlation.Then cyclin E protein expressions were observed at various intervals of 10μmol / L LY-294002 and 10μmol/L UO-126 treatment.The protein level of cyclin E was not affected by LY-294002.In contrast,UO-126 reduced the level of cyclin E protein expression.3. RT-PCR and Western blot exhibited that the FBW7 mRNA and protein expression had significantly increased after HeLa cells were treated with UO-126. Immunofluorescence showed that the positive FBW7 was mainly found in cytoplasm and nucleus of HeLa cells and nuclear FBW7 expression was more.The expression of positive FBW7 had increased after HeLa cells were treated with UO-126 in a time dependent way.Conclusion:1. The abnormal proliferation of HeLa cells were associated with the activation of Ras signaling pathway. Blocking Ras signaling pathway could slow down HeLa cells cycle G1/S phase transition and decrease the expression of cyclin E. Ras-MAPK signaling pathway not Ras-PI3K signaling pathway should be essential for regulation of cyclin E expression in HeLa cells.2. The expression of tumor suppressor protein FBW7 which mediated the ubiquitination degradation of cyclinE could have some relevance to the Ras-MAPK signaling pathway. FBW7 lied downstream of Ras-MAPK signaling pathway.更多还原