【作者】 周娜静；

【导师】 闫蕴力；

【作者基本信息】 河北医科大学 ， 人体解剖与组织胚胎学， 2010， 博士

【摘要】 第一部分genistein对HeLa细胞生长的抑制作用目的：子宫颈癌是常见的妇女癌症,根据统计显示：子宫颈癌发生率的排名为女性癌症第一位,各个年龄层的女性都有可能发生子宫颈癌,但以25岁到45岁的妇女最为常见。死亡率的排名则为女性癌症的第四位,死亡人数占全部癌症死亡人数的4%。genistein具有雌激素作用、抗氧化作用、防突变、防射线损伤、抗感染、抗心脑血管病、抗骨质疏松等多种生理功效,近年来,genistein的抗癌效应及其应用前景受到普遍关注。基础研究表明,genistein能够抑制肿瘤细胞的生长、侵袭、并可加强其它抗癌药物的疗效、以及逆转肿瘤细胞的耐药性、诱导肿瘤细胞凋亡、诱导肿瘤细胞分化等。本研究将检测genistein对宫颈癌细胞株HeLa细胞生长的抑制作用,并测定其对HeLa细胞的半数抑制浓度,同时检测genistein对HeLa细胞细胞周期及凋亡的影响。方法：先用MTT法测定不同浓度genistein对HeLa细胞作用24 h和48h的吸光度值,计算不同浓度genistein对HeLa细胞的抑制率,并绘制抑制率曲线,确定ICso值。然后以genisteinIC5o作用后,Hoechst 33258荧光染色检测凋亡时细胞核形态变化,Annexin V-FITC和PI标记后采用流式细胞术进一步定量检测凋亡细胞比例,同时检测细胞周期分布情况。结果：genistein可以抑制HeLa细胞生长,并具有剂量和时间依赖性,24 h半数抑制浓度为126 uM,48h半数抑制浓度为75uM。采用75uMgenistein作用48 h后,Hoechst 33258染色可见凋亡细胞形态改变,且凋亡细胞比率较对照组增加；Annexin V-FITC和PI标记后采用流式细胞术进一步定量检测凋亡细胞,genistein作用组凋亡细胞比率明显增加；细胞周期分析显示genistein作用后G2/M细胞明显增加,G0/G1期和S期细胞比例没有明显改变,提示genistein使HeLa细胞周期阻滞于G2/M期。结论：genistein能够抑制HeLa细胞增殖,且具有时间和剂量依赖性。以75uM genistein作用48 h,能够引起细胞凋亡及细胞周期G2/M期阻滞。第二部分genistein对HeLa细胞拓扑异构酶Ⅱα抑制作用及相关机制研究目的：拓扑异构酶Ⅱ(topoisomeraseⅡ,TopoⅡ)是普遍存在的细胞核酶,能够解开S期和G2/M期紧凑缠绕的DNA超螺旋结构,催化DNA的超螺旋状态与解旋状态之间的相互转换,以便DNA复制、转录、修复、重组、染色体分离等,使细胞生命活动得以顺利进行。TopoⅡ有两种亚型,即TopoⅡα和TopoⅡβ,分子量分别为170kDa and 180 kDa. TopoⅡα和TopoⅡβ在细胞中具有不同的功能,TopoⅡα蛋白水平存在明显细胞周期特异性,表现为G1期较低,S期开始升高,G2/M期达顶峰。临床研究表明,在如肺癌、乳腺癌及头颈部肿瘤组织内,TopoⅡα基因表达明显高于正常组织,因此TopoⅡα被认为是肿瘤细胞增殖的一个特异性标志,在恶性肿瘤的发生、诊断,以及治疗中具有重要作用。自上世纪八十年代末开始注意到,genistein对DNA TopoⅡ的抑制作用是其发挥抗癌效应的重要方面。并且逐渐认识到,genistein对TopoⅡ的作用与VP-16、米托蒽醌、安吖啶等临床常用抗癌药物相似,即参与形成TopoⅡ-DNA断裂复合体,诱导DNA断裂和细胞凋亡,从而达到抑制或杀灭肿瘤细胞的目的。但目前的研究多集中于genistein对TopoⅡ-DNA断裂复合体影响,而缺乏genistein对TopoⅡ表达调控的研究。有研究显示,TopoⅡα表达与mRNA稳定性无关。在TopoⅡα基因启动子区存在2个GC盒,其表达主要受Sp家族调控。Sp1和Sp3作为广泛存在的转录调控因子,可以调控基因的表达,Sp1通过其三个“锌指结构”与TopoⅡα启动子区的GC盒结合,增强TopoⅡαmRNA的转录,上调TopoⅡα蛋白的表达。而Sp3同样含有三个“锌指结构”,可与Sp1竞争性结合GC盒位点。近期有研究表明,在人消化系肿瘤Caco-2细胞株中,GC盒与genistein对金属结合蛋白metallothionein IIA表达影响作用有关,genistein能够阻断反式维甲酸对血管内皮生长因子(VEGF)的转录刺激作用,而VEGF启动子中Sp1和Sp3结合位点起到关键作用。这些研究都预示,genistein可能可以通过Sp1和Sp3调节TopoⅡα表达。本研究旨在研究genistein对TopoⅡα基因表达的影响,并探讨转录因子Sp1和Sp3是否参与genistein对TopoⅡα的表达调控。方法：RT-PCR检测genistein作用后TopoⅡα及其上游转录因子Spl、Sp3 mRNA表达改变,western-blotting的方法检测TopoⅡα及转录因子Sp1、Sp3蛋白表达变化。TopoⅡα的启动子区存在2个GC盒,其中一个靠近转录起始位点,另一个远离转录起始位点,为测定两个GC盒中哪个在TopoⅡα表达调控中发挥作用,针对两个GC盒,分别跨越GC序列设计两对扩增引物,用染色体免疫共沉淀的方法检测TopoⅡα表达改变是否与转录因子Sp1、Sp3相关,并确定Sp1和Sp3分别通过哪个GC盒发挥作用。结果：genistein作用后,TopoⅡα与Sp1 mRNA及蛋白表达降低,而Sp3 mRNA和蛋白表达增加,且TopoⅡα表达变化与表达改变有关,两个GC盒在Sp1和Sp3参与genistein对TopoⅡα表达调控中都发挥作用。结论：genistein能够降低TopoⅡαmRNA及蛋白表达,降低Sp1 mRNA及蛋白表达,增加Sp3表达；且TopoⅡα表达变化与Sp1、Sp3表达改变有关；两个GC盒在Sp1和Sp3参与genistein对TopoⅡα表达调控中都发挥作用。第三部分genistein对HeLa细胞SIRT1的抑制作用及相关机制研究目的：蛋白质赖氨酸侧链能够被乙酰化、甲基化、泛素化,及ADP核糖基化,这些竞争性、可逆性翻译后修饰由不同的酶及与其相互作用的一些复合体调节。在真核细胞,乙酰化是最普遍的共价修饰,且目前认为乙酰化比磷酸化有更广泛的底物。现已知的有数百种的蛋白都被乙酰化修饰,乙酰化能够明显改变蛋白的特性和功能,mRNA剪接、运输、整体性、翻译、蛋白活性、定位、稳定性和相互作用都受乙酰化调节,因此乙酰化能够影响从信号转导到转录到蛋白降解调节过程的每一步,可逆的赖氨酸基团乙酰化对蛋白功能和细胞网络的调节至关重要。调节蛋白乙酰化状态的酶,包括乙酰化酶和脱乙酰化酶。相对于乙酰化酶,作为药物靶点,脱乙酰化酶受到研究者的更多关注。至今研究表明,脱乙酰化酶抑制剂在各种疾病中的应用更有希望,如白血病和其他的血液系统疾病。脱乙酰化酶分为三类,一、二类属于经典的脱乙酰化酶,催化反应需要Zn离子作为辅因子,sirtuins蛋白属于第三类,与经典的脱乙酰化酶作用有所不同,它的催化作用不需要锌指的参与,而是烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide, NAD)依赖性的,因而新陈代谢则可调节其活性,这也使其刚刚问世便成为了众多科学研究的对象。沉默信息调节因子2(Silent information regulator 2, Sir 2)是由Sinclair和同事在麻省理工学院的Leonard Guarente实验室于酵母中发现的,它是一种NAD依赖的组蛋白脱乙酰化酶。酵母Sir2与染色体末端的失活及rDNA的转录沉默有关。酵母Sir2含量加倍,可以延缓复制衰老,延长寿命。Sir2是一个高度保守的基因家族。Sir2相关酶类(sirtuins, SIRT)是Sir2的同源物,它不仅存在于酵母、果蝇、秀丽杆菌等细胞内,还广泛存在于哺乳动物各种细胞内。caloric restriction (CR)试验表明,在酵母、秀丽杆菌、蠕虫、小鼠及人均可以通过增加SIRT1的活性而延长寿命,因而SIRT被认为是一个长寿基因。至今为止,人类细胞内已经发现了7种不同类型的SIRT蛋白,分别命名为SIRT1-SIRT7。SIRT1是目前研究最多最清楚的一个成员,研究发现,哺乳动物SIRT1与宿主一些已知与癌症有关的因子相互作用,可能在一些情况下促进肿瘤细胞的生存,而目前尚缺乏关于genistein对肿瘤细胞中SIRT1的作用的研究报道。因此,本研究就genistein对HeLa细胞SIRT1表达的影响及其作用机制进行探讨。方法：HeLa细胞经SIRT1选择性抑制剂splitocimin预处理后,用不同浓度genistein作用,MTT法测定不同浓度genistein对HeLa细胞作用,比较与非经抑制剂处理组之间细胞细胞生长抑制情况；Hoechst 33258荧光染色检测细胞凋亡情况的差别；流式细胞术检测细胞周期分布情况。并比较splitocimin预处理对genistein在细胞周期G2/M期阻滞作用中的影响；应用Western blot印迹检测genistein对细胞内SIRT1蛋白表达水平的变化及经splitocimin预处理后genistein对SIRT1蛋白表达的影响。结果：splitocimin预处理后,genistein对HeLa细胞生长抑制作用增强；Hoechst 33258染色可见凋亡细胞形态改变,单纯splitocimin处理组与对照组细胞凋亡率没有明显区别。但经splitocimin预处理后,genistein的促凋亡作用比未经splitocimin预处理的单纯genistein作用时细胞凋亡率增加。细胞周期分析显示,单纯splitocimin的作用可以使S期细胞增加,经splitocimin预处理后再经genistein作用,S期细胞数减少,而G2/M期阻滞增加。genistein可以抑制HeLa细胞内SIRT1蛋白的表达,且呈剂量依赖性；经splitocimin预处理后,再经genistein作用时,SIRT1蛋白的表达较只用splitocimin预处理时有所增加,但仍低于对照组水平。结论：genistein可通过下调SIRT1表达而抑制HeLa细胞的生长,诱导细胞凋亡。SIRT1抑制剂可以增强genistein对HeLa细胞的抑制作用。更多还原

【Abstract】 Part 1 genistein inhibits HeLa cell growthObjective:Cervical cancer is a common cancer for women, according to statistics, the incidence of cervical cancer ranks as the first female cancer. Cervical cancer is possible for all ages women, but the 25-year-old to 45-year-old women most common. Mortality of cervical cancer ranks fourth in that of female cancer and the number of deaths accounted for 4% in that of cancer deaths.Genistein is an isoflavone compound from natural plants and has many physiological functions, such as estrogen action, anti-oxidation, preventing mutation, anti-infection, preventing and therapy of heart-cerebrovascular disorders, which provide its usage in health care or in treating related diseases. For genistein anticancer property, recently studies have shown that genistein could inhibit a wide range of cancer cells, it can inhibit cancer cells growth,, induce cell deferience, reverse cell drug resisdence, do advantage for other anti-cancer drugs effect. This research will investage if genistein inhibite HeLa cell growth, and to know the effect of genistein on inducing cell apoptosis and arresttingt the cell cycle.Methods:The effect of genistein on HeLa cell growth was assessed using the MTT assay to measure the cell viability and calculate the half inhibition concentration (IC50) of genistein on the cell. To verify whether the decreased cell viability of HeLa cells treated with genistein was related to apoptosis, Hoechst 33258 staining was used to detect the nuclear morphological changes. Cell cycle distribution was examined with flow cytometry. To assess the capability of genistein inducing HeLa cell apoptosis and distinguish different types of cell death, HeLa cells were double-stained with annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry.Results:MTT results showed that genistein inhibited HeLa cell growth in a dose and time dependent manner. The IC50 value of genistein on HeLa cell was calculated at 126μM for 24 h and 75μM for 48 h. By Hoechst 33258 staining, the control cells were well spread flatly and appeared uniform in chromatin density, while genistein treated cells displayed many examples of chromatin fragments or nuclear debris, which could be identified as apoptotic cells, and the percentage of apoptotic cells was 18.34±2.35% after exposure to genistein for 48 h, while that of control cells was only 1.52±0.56%. Double-stained with annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry reveal the ratio of apoptotic cells obviously increased in genistein treated cells compared to that of control groups. Cell cycle distribution was examined with flow cytometry showed that the percentage of G2/M cells increased significantly, while the percentage of G0/G1 and S cells have no significant difference with that of control group, which suggesting that the cell cycle was arrested at G2/M phase.Conclusions:genistein can inhibit HeLa cell growth and induce cell apoptosis and arrest the cell cycle at G2/M phase.Part 2 The effect of genistein on topoisomeraseⅡαexpression in HeLa cellObjective:TopoisomeraseⅡ(TopoⅡ) is a ubiquitous nuclear protein catalyzing the reaction of breakage and relinking of DNA, which plays an important role in maintenance of DNA topology required for DNA replication, transcription, and recombination of cellular genes. The two TopoⅡisoforms, TopoⅡαand TopoⅡβ, are 170 kDa and 180 kDa proteins, respectively, and have different roles in regulating cellular function. TopoⅡαshows cell-cycle specific expression during the S and G2/M phases and is essential for chromosome condensation. TopoⅡαis closely related to the occurrence, development, diagnosis, prognosis and treatment of cancer. Otherwise, TopoⅡβis associated with non-proliferating function and believed to maintain the integrity of nuclear chromatin.In recent years, TopoⅡhas become a popular target for cancer chemotherapy treatments. Genistein can inhibit a wide range of cancer cells, and the mechanism of genistein cytotoxicity is considered to involve an inhibitory effect on TopoⅡ. It is believed that genistein can bind to and stabilize the topoisomerase-DNA complex, resulting in DNA strand breaks and inducing cell apoptosis. However, the molecular events of genistein inhibiting TopoⅡexpression at the gene transcription level have not been clearly understood.It is known that the human TopoⅡαgene promoter has five functional CCAAT boxes and two GC boxes. The two GC boxes, GC1 and GC2 are located at the proximal and distal region of the TopoⅡαgene promoter, respectively. Sp1 and Sp3 can bind with similar affinities to both GC boxes by three zinc-fingers. It is believed that Sp1 and Sp3 have different functions in regulating TopoⅡαgene expression. Sp1 acts mainly as a transcriptional activator, while Sp3, consisting of three subtype proteins of 115 kDa,80 kDa, 78 kDa, respectively, can act as a transcriptional repressor binding the GC boxes competitively with Sp1. Recently, it has been shown that the GC-rich sequence is necessary for the effect of genistein on metal-binding protein, metallothioneinⅡA expression in human intestinal Caco-2 cells. Moreover, genistein can block the stimulation of VEGF gene transcription by all trans-retinoic acid through Sp1 and Sp3 sites in a human bronchioloalveolar carcinoma cell. Thus, it is likely that genistein may be able to regulate TopoⅡαgene expression through Sp1 and Sp3.The aim of the present study is to investigate the effect of genistein on TopoⅡαgene expression and to determine whether the transcription factors Sp1 and Sp3 have a role in the regulation of TopoⅡαexpression in HeLa cells.Methods:To investigate the effect of genistein on TopoⅡαexpression at gene transcription level, HeLa cells were treated with genistein for 48 h and the expression of TopoⅡα, Spl and Sp3 mRNA in HeLa cells were examined with RT-PCR. TopoⅡα, Sp1 and Sp3 protein expression were examined by Western blot assay. There are two GC boxes, GC1 and GC2 in the TopoⅡαpromoter, which the transcription factors Sp1 and Sp3 can bind to. To assess the role of Sp1 and Sp3 in the inhibitory effect of genistein on TopoⅡαexpression, we analyzed their binding ability to the two GC boxes by ChIP experiment.Results:RT-PCR results showed that genistein down-regulated TopoⅡαand Spl mRNA expression, while Sp3 mRNA expression was up-regulated in HeLa cells Western blot assay results showed that genistein down-regulated TopoⅡαand Sp1 protein expression, while Sp3 protein expression was up-regulated in HeLa cells. ChIP experiment results showed that Sp1 occupancy was reduced and Sp3 occupancy was elevated in cells treated with genistein compared to those of controls for both GC1 and GC2.Conclusions:genistein can inhibit TopoⅡαexpression, which has relation with the regulation of Specificity protein 1 and Specificity protein 3 in HeLa cell.Part 3 The effect of genistein on SIRT1 expression in HeLa cellObjective:Lysine side chains can be acetylated, methylated, ubiquitinated, sumoylated and ADP-ribosylated. These rivalling and reversible post translational modifications are regulated by a complex interplay of different enzymes. Reversible acetylation of lysine-amino groups crucially modulates protein function und cellular networks. In eukaryotic cells, acetylation is among the most common covalent modifications and ranks similar to the important master switch phosphorylation. Acetylation apparently shows a broader substrate spectrum than phosphorylation. Hundreds of proteins are known to be modified by acetylation. Acetylation can change protein characteristics and functions enormously. In general, acetylation changes the electrostatic state of lysine from positive to neutral and increases the size of the amino acid side chain. Acetylation can equally affect enzymatic activities and determine protein function at multiple levels. Comparatively little attention has been drawn to inhibitors of acetyltransferases (HATi), as HATs are rarely considered as drug targets. A reason for this could be the promising use of inhibitor of histone deacetylases (HDACi) in various diseases like leukaemia and other haematological disorders. HDACs can be grouped into two distinct families. The "classical family" of zinc-dependent HDACs are structurally related to the yeast Hdal/Rpd3 proteins, and the second one consists of the NAD+-dependent yeast Sir2 homologues. Sirtuins make use of a different mechanism of catalysis. Instead of using an electrophilic Zn2+ ion to directly hydrolyse the amide bond with water, they transfer the acetyl group to the cosubstrate NAD+ yielding two products, nicotinamide and 2-O-acetyl-ADPribose. This reaction depends on the NAD+/NADH ratio. Metabolism thus may provide a mechanism to regulate SIRTs. Silent Information Regulator 2 (Silent information regulator 2, Sir 2) was found by the Sinclair and colleagues at the Massachusetts Institute of Technology’s Leonard Guarente lab in yeast, it is a NAD dependent histone deacetylase. Yeast Sir2 has relation with the chromosome end inactivation and the silencing of rDNA transcription. If content of yeast Sir2p double, it can delay replicative senescence and prolong life. Sir2 is a highly conservative gene family. Sir2-related enzymes (sirtuins, SIRT) is a Sir2 homologs, it not only in yeast, flies, but widely present in various mammalian cells. SIRT1 can delay replicative senescence, prolonging life, which is considered a longevity gene and the evidence is from caloric restriction (CR) test, in yeast, worms, mice and human CR have increased SIRT1 activity can prolong life.To date, seven human isoforms have been identified (SIRT1 to SIRT7), and SIRT1 is the most studied one. Recent reports revealed that hSIRT1 is up-regulated in tumor cell lines, suggesting that hSIRTl may be involved in contributing to tumorigenesis. Therefore, this study will research the action of genistein on SIRT1 in HeLa cell.Methods:After treatment with splitomicin, a special inhibitor of SIRT1, the effect of genistein on HeLa cell growth was assessed using the MTT assay, the capability of genistein inducing HeLa cell apoptosis was tested with Hoechst 33258 staining, and cell cycle distribute was analyzed by flow cytometry. The action of genistein on SIRT1 protein expression was detected by western blotting.Results:after pretreatment with splitomicin, MTT results showed that genistein inhibited more HeLa cells growth in a dose and time dependent manner. By Hoechst 33258 staining, after pretreatment with splitomicin the ratio of apoptotic cells obviously increased in genistein treated cells. Cell cycle distribution was examined with flow cytometry showed the percentage of S cells increased after treatment with splitomicin only, and the percentage of G2/M cells in genistein and splitomicin group is more than of splitomicin group. Genistein can inhibite SIRT1 expression, after pretreatment with splitomicin, genistein can increase SIRT1 expression, but SIRT1 protein level is still lower than that of control group.Conclusions:SIRT1 has relation with genistein inhibition on HeLa cell, and SIRT1 inhibitor can help genistein induce cell apoptosis and arrest the cell cycle at G2/M phase.更多还原