【作者】 李进；

【导师】 杨罗艳；

【作者基本信息】 中南大学 ， 外科学， 2009， 博士

【摘要】 研究背景:前列腺癌是一种老年男性高发疾病,在西方国家,前列腺癌是男性第二大恶性肿瘤,仅次于肺癌。中国近年来发病率稳步升高,已列为泌尿系肿瘤的第三位,并且发病年龄也日趋年轻化。大部分前列腺癌病人在经历18～24个月的中位期后,原来对激素依赖的前列腺癌最终成为激素非依赖性前列腺癌。对激素、化疗、和放疗不敏感也是激素非依赖性前列腺癌治疗过程中面临的难题。因此,寻找新的治疗方法仍是当前研究的热点。冬凌草甲素是从冬凌草等植物中提取出来的一种天然活性物质,具有抗炎、抗DNA突变、抗氧化作用。目前研究证明,冬凌草甲素对多种肿瘤具有生长抑制作用,有望成为一种高效、低毒的新型抗肿瘤药物。本研究用冬凌草甲素作用于人前列腺癌细胞株——PC-3细胞株,观察冬凌草甲素对PC-3细胞是否具有抑制增殖、凋亡诱导和转移抑制作用,并对其分子机制进行部分揭示。第一章冬凌草甲素对PC-3细胞的增殖抑制效应目的:确定冬凌草甲素是否具有抑制PC-3细胞增殖的能力。方法:用不同浓度的冬凌草甲素干预PC-3细胞,通过台盼蓝拒染试验、MTT试验和细胞的药物浓度—时间生长曲线分析观察其对PC-3细胞活力的影响;通过流式细胞仪检测PC-3细胞的细胞周期变化。结果:（1）冬凌草甲素能以时间和浓度依赖性方式有效的抑制PC-3细胞增殖,药物抑制PC-3细胞活力的IC50约为10.29μmol/L。（2）冬凌草甲素能增加PC-3细胞G0/G1期百分率,降低PC-3细胞S期百分率,细胞增殖指数下降。结论:冬凌草甲素能够有效的抑制PC-3细胞的增殖,并呈时间和药物浓度依赖性。第二章冬凌草甲素对PC-3细胞的凋亡诱导效应目的:确定冬凌草甲素是否能诱导PC-3细胞凋亡。方法:用不同浓度的冬凌草甲素作用于PC-3细胞,用Hochest33258染色鉴定PC-3细胞凋亡的形态学特征;用流式细胞仪分析PC-3早期凋亡细胞的百分率;用Caspase-3蛋白印迹分析证明PC-3细胞凋亡发生的分子特征。结果:（1）凋亡细胞形态学鉴定、流式细胞仪检测均有力证明冬凌草甲素能以浓度依赖性方式诱导PC-3细胞凋亡。（2）冬凌草甲素诱导PC-3细胞凋亡伴有Caspase-3蛋白表达上调和裂解激活。结论:冬凌草甲素能以浓度依赖性方式诱导PC-3细胞凋亡。第三章冬凌草甲素对PC-3细胞增殖抑制和凋亡诱导效应的分子机制目的:（1）揭示冬凌草甲素抑制PC-3细胞增殖与细胞周期改变的关系;（2）部分揭示冬凌草甲素对PC-3细胞凋亡诱导的分子机制;（3）确定Caspase-3活化在冬凌草甲素诱导的PC-3细胞凋亡中的重要作用。方法:（1）流式细胞仪检测细胞周期变化;（2）Western印迹检测COX-2、Survivin、Bcl-2和Bax蛋白表达的变化;（3）实时荧光定量PCR方法检测PC-3细胞COX-2、Survivin、CyclinD₂、CyclinE、P27蛋白表达的变化;（4）Z-IETD-fmk阻断试验检测Caspase-3阻断后,冬凌草甲素对PC-3细胞凋亡影响的变化。结果:（1）冬凌草甲素增加G0/G1期PC-3细胞百分率,降低S期PC-3细胞百分率;（2）冬凌草甲素以浓度依赖性方式抑制PC-3细胞的COX-2、Survivin、CyclinD₂、CyclinE、和Bcl-2蛋白表达,而P27和Bax蛋白表达上调;（3）冬凌草甲素以浓度依赖性方式抑制PC-3细胞端粒酶的活性;（4）Z-IETD-fmk阻断Caspase-3活性后,能部分逆转和减弱冬凌草甲素对PC-3细胞的凋亡诱导作用。结论:冬凌草甲素通过影响细胞周期调节蛋白、阻断细胞周期G1/S期“稽查点”;抑制COX-2、Survivin、CyclinD₂、CyclinE和Bcl-2,上调P27和Bax以及活化Caspase-3等途径抑制PC-3细胞增殖及诱导PC-3细胞凋亡。第四章冬凌草甲素对PC-3细胞转移的抑制效应和分子机制目的:确定冬凌草甲素是否具有抑制PC-3细胞转移的能力及其分子机制。方法:用不同浓度的冬凌草甲素作用于PC-3细胞,用体外驱化运动试验检测PC-3细胞运动驱化能力的变化;用实时荧光定量PCR方法检测PC-3细胞中VEGF、MMP-2和MMP-9 mRNA表达的变化;用ELISA方法检测PC-3细胞上清VEGF、MMP-2和MMP-9浓度的变化。结果:（1）体外驱化运动试验证明冬凌草甲素能以浓度依赖性方式抑制PC-3细胞转移。（2）冬凌草甲素以浓度依赖性方式抑制PC-3细胞及其上清中VEGF、MMP-2和MMP-9的表达。结论:冬凌草甲素具有抑制PC-3细胞转移的能力,其机制可能与药物下调VEGF、MMP-2和MMP-9有关。更多还原

【Abstract】 Background:In China,the incidence of prostate cancer has been increasing in recent years.Hormone therapy has been the mainstay of the therapeutic options for metastatic diseases for many years.But many metastatic tumors progress at a median of 18 to 24 months and become hormonal refractory prostate cancer（HRPC）.The frequent acquisition of drug-resistant phenotypes and the occurrence of second malignancies often associated with chemotherapy remain serious problems.New therapeutic strategies must be evaluated to improve survival.Oridonin is a naturally active substance isolated from Rabdosia rubescens.The medicinal value of oridonin has been well recognized due to its anti-inflammation and anti-DNA-damaging effects.In recent years,many studies have shown that oridonin possesses anti-proliferative and apoptotic activities against various cancer cells with little toxicity.In our study,we observe whether Oridonin suppresses proliferation,induces apoptosis and inhibits metastasis of PC-3 cells and to explore its possible mechanisms. PartⅠProliferation-inhibiting effect of Oridonin on PC-3 cellsObjective:To determine proliferation-inhibiting capability of Oridonin on PC-3 cells.Methods:PC-3 cells were treated with different concentrations of Oridonin,then cell viability was analyzed with trypan blue exclusion test、MTT assay and drug concentration-time survival curve test;Cell cycle was tested by flow cytometry.Results:（1） Oridonin effectively inhibited the proliferation of PC-3 cells in a concentration-time dependent way,and the IC50 of PC-3 cells was 10.29μmol/L.（2） Oridonin increased the percentage of G0/G1 phase and decreased S phase of PC-3 cells.Conclusion:Oridonin can inhibit proliferation of PC-3 cells effectively with a concentration-time dependent manner. PartⅡApoptosis-inducing effect of Oridonin on PC-3 cellsObjective:To ensure whether Oridonin can induce the apoptosis of PC-3 cells.Methods:After PC-3 cells were incubated with different concentrations of Oridonin,（1） morphologic character of apoptosis was evaluated by Hochest33258 staining;（2） the percentage of earlier apoptosis cell was analyzed by flow cytometry;（3） the molecular character of apoptosis was analyzed by Western blot detecting Caspase-3 expression.Results:（1） Hochest33258 staining and flow cytometry detection powerfully testify Oridonin can induce the apoptosis of PC-3 cells in a concentration-dependent way;（2） The apoptosis of PC-3 cells,which was induced by Oridonin,kept company with up-regulation and cleavage of Caspase-3.Conclusion:Oridonin can induce the apoptosis of PC-3 cells with a concentration-dependent manner. PartⅢMolecular mechanisms of the proliferation-inhibiting and apoptosis-inducing effects of Oridonin on PC-3 cellsObjective:（1） To reveal the relation between the proliferation-inhibiting effect of Oridonin and the change of PC-3 cells cycle;（2） To partly explore the mechanism of apoptosis-inducing effects of Oridonin on PC-3 cells;（3） To determine the important role Caspase-3 plays in the apoptosis of PC-3 cells induced by Oridonin.Methods:（1） The change of cell cycle was analyzed by flow cytometry;（2） The protein expression of COX-2,Survivin,Bcl-2 and Bax in PC-3 cells were detected by Western blot;（3） The protein expression of COX-2、Survivin、CyclinD₂、CyclinE、P27in PC-3 cells were detected by fluorescent quatititive PCR;（4） After the activation of Caspase-3 was inhibited,the changes of proliferation-inhibiting and apoptosis-inducing effects of Oridonin were detected by Z-IETD-fmk blocking tests.Results:（1） Oridonin increased the percentage of G0/G1 phase and decreased S phase of PC-3 cells;（2） Oridonin down-regulated CyclinD₁,Survivin and Bcl-2,and up-regulated P21 and Bax protein in a concentration-dependent way in PC-3 cells;（3） Oridonin down-regulated telomerase activities in a concentration-dependent way in PC-3 cells;（4） The apoptosis-inducing effects of Oridonin were partly suppressed after Z-IETD-fmk blocking the Caspase-3 activation of PC-3 cells.Conclusion:Oridonin inhibits proliferation and induces apoptosis of PC-3 cells through regulating cell cycle protein,blocking "checkpoint" of G1/S phase,down-regulating COX-2、Survivin、CyclinD2、CyclinE and Bcl-2,up-regulated P27and Bax and activating Caspase-3. PartⅣMetastasis-inhibiting effect of Oridonin on PC-3 cells and its mechanismObjective:To determine metastasis-inhibiting capability of Oridonin on PC-3 cells and to explore its mechanisms.Methods:After PC-3 cells were incubated with different concentrations of Oridonin,（1） the metastasis ability was analyzed with experiment of chemotaxic migration;（2） the mRNA expressions of VEGF, MMP-2 and MMP-9 were analyzed by fluorescent quatititive PCR;（3） the expressions of VEGF,MMP-2 and MMP-9 were analyzed by ELISA.Results:（1） Oridonin effectively inhibited the metastasis of PC-3 cells in a concentration-time dependent way.（2） Oridonin effectively inhibited the expressions of VEGF and MMP-9 in a concentration-time dependent way.Conclusion:Oridonin can inhibit metastasis of PC-3 cells effectively with a concentration dependent manner and the molecular mechanism may relate to Oridonin down-regulating VEGF,MMP-2 and MMP-9 expression.更多还原