【作者】 王莉；

【导师】 归绥琪； 葛锡锐； 陈松华； 徐丛剑；

【作者基本信息】 复旦大学 ， 妇产科学， 2006， 博士

【摘要】 子宫颈癌是最常见的妇科恶性肿瘤之一，近年来随着子宫颈人乳头状瘤病毒(HPV)感染发病率的升高，子宫颈癌的发病率呈上升趋势，高发年龄趋于年轻化，成为严重危害妇女健康的疾病。β-谷甾醇是一种植物固醇，多个在体和离体实验已证实β-谷甾醇对结肠癌、前列腺癌和乳腺癌有保护作用，我院于上世纪七十年代曾单独外用β-谷甾醇栓片治疗115例子宫颈癌，有效率达75-65％，但对该药的药理机制未进行过深入研究。本文旨在研究β-谷甾醇对子宫颈癌细胞的增殖抑制作用及其可能的作用机制，以期寻求一种新的子宫颈病变的治疗选择。本论文共分三部分：1．β-谷甾醇对子宫颈癌细胞株生物学行为的影响；2．β-谷甾醇对子宫颈鳞癌细胞株SiHa细胞微管系统的影响；3．β-谷甾醇对SiHa细胞干扰素(IFN)及其下游基因的作用。第一部分β-谷甾醇对子宫颈癌细胞株细胞生物学行为的影响目的了解β-谷甾醇对子宫颈癌细胞形态学、增殖、细胞周期和凋亡的影响。方法在子宫颈癌细胞SiHa／HeLa的培养基中加入不同浓度的β-谷甾醇和／或掌叶半夏总蛋白，采用MTT比色法检测不同浓度β-谷甾醇对SiHa／HeLa细胞增殖的影响。采用倒置相差显微镜、扫描和透射电镜、流式细胞术检测SiHa细胞经20umol／L的β-谷甾醇处理不同时间，细胞形态学和超微结构的变化，及β-谷甾醇对细胞周期和细胞凋亡的影响。结果β-谷甾醇能抑制子宫颈癌细胞SiHa／HeLa的增殖，且具有时间、剂量依赖关系，β-谷甾醇对SiHa的生长抑制作用较强，掌叶半夏总蛋白对SiHa细胞的生长无显著影响。与对照组比较，20umol／L的β-谷甾醇使SiHa细胞出现S期阻滞，凋亡和坏死细胞增加，细胞形态和超微结构发生显著改变。结论β-谷甾醇能抑制人子宫颈癌细胞增殖，对子宫颈鳞癌细胞株的抑制作用大于子宫颈腺癌，使细胞周期阻滞在S期。第二部分β-谷甾醇对子宫颈癌细胞SiHa微管系统的影响目的探讨β-谷甾醇对子宫颈癌细胞SiHa细胞内微管系统的影响。方法应用激光共聚焦显微技术观察β-谷甾醇处理的SiHa细胞内微管和微管相关蛋白2的表达、分布，采用蛋白免疫印迹法检测微管蛋白、微管相关蛋白2的表达，及聚合和未聚合微管蛋白含量的变化。结果激光共聚焦分析表明，20umol／L的β-谷甾醇作用5天的SiHa细胞微管网络异常，微管相关蛋白2表达显著下调，蛋白免疫印迹结果进一步证实β-谷甾醇能抑制微管蛋白α和微管相关蛋白2的表达，同时β-谷甾醇能抑制SiHa细胞内微管蛋白的聚合，并随着作用时间的延长而逐渐增强。结论β-谷甾醇能降低SiHa细胞微管蛋白α和微管相关蛋白2的表达，并抑制SiHa细胞内微管的聚合，提示β-谷甾醇具有一定的抗微管作用，这一作用可能是造成SiHa细胞生长抑制的重要原因。第三部分β-谷甾醇对子宫颈癌细胞株SiHa干扰素及其下游基因的作用目的探讨β-谷甾醇对子宫颈癌细胞SiHa细胞内源性干扰素及其下游基因和HPV E7蛋白表达的影响。方法采用RT-PCR法检测β-谷甾醇作用于SiHa细胞不同时间后，IFNα、IFNβ、ISG15、OAS(p39／41)、OAS(p69／71)、RNase L、MxA、PKR基因mRNA的表达变化。采用western blot法检测β-谷甾醇作用于SiHa细胞不同时间后STATl和HPV E6、E7蛋白的表达水平。结果RT-PCR结果表明20umol／Lβ-谷甾醇作用于SiHa细胞3天和5天后，IFNp、ISGl5、OAS(p40／46)、OAS(p69／71)和MxA的mRNA表达水平显著增加(P＜0.01)，并具有时间依赖效应：而RNase L、PKR的mRNA水平在药物作用前后没有明显的变化。western blot结果显示药物作用3天和5天后，STAT1蛋白表达水平显著增加，E7蛋白的表达水平在药物作用第一天即显著下降。结论β-谷甾醇可诱导IFNβ及下游系列基因的mRNA表达水平增加，这些基因表达产物可能直接或间接参与β-谷甾醇对SiHa细胞的作用，引起SiHa细胞生长抑制，同时β-谷甾醇降调了E7蛋白表达，该作用可能是β-谷甾醇的直接引起或是IFN及其下游基因产物抗病毒作用的结果。更多还原

【Abstract】 Cervical cancer is one of the most frequently gynecological neoplasm. In recent years the prevalence of cervical HPV infection has raised, the incidence of cervical cancer increased. More and more young women were suffered with cervical cancer. Cervical cancer becomes one of severe diseases that could be harmful to women’s health.β-sitosterol is a phytosterol. Several in vitro and animal studies suggest that β-sitosterol offer protection from the most common cancers including colon, prostate and breast. In 1970’s, β-sitosterol suppository and β-sitosterol tablets were used on cervical os in vaginal to treat 115 cases of cervical cancer, the effective rate was 75.65%. The exact pharmacological mechanism by which β-sitosterol offers this treatment has not been determined. In this study, we have investigated the effect of β-sitosterol on the growth inhibition of cervical cancer cells and the associated mechanisms.There are three parts in the study: Part Ⅰ Effects of β-sitosterol on the cellular biological behavior in the cervical cancer cell lines. Part Ⅱ Effects of β-sitosterol on microtubular systems in cervical cancer SiHa cells. Part Ⅲ Effects of β-sitosterol on IFN and its downstream genes in cervical cancer SiHa cells.Part Ⅰ Effects of β-sitosterol on the biological behavior in the cervical cancer cell lines.Objective To study the effects of β-sitosterol on morphology, proliferation, cell cycle and apoptosis of human cervical cancer cell lines. Methods Theβ-sitosterol was incubated at different concentrations with cervical cancer cell line SiHa and HeLa. The effects of β-sitosterol on cell proliferation were tested by MTT assay. The effects of β-sitosterol on morphology, ultrastructure and cell cycle were studied by phase-contrast microscope, electron microscope and flowcytometry(FCM). Results β-sitosterol could obviously inhibit the proliferation of SiHa / HeLa cells in a time and dose dependent manner. Better results of growth inhibitory effects were got in SiHa cells than that in HeLa cells. The total protein of pinella has no effects on SiHa cells proliferation. 20 μmol/L β-sitosterol induced the accumulation of SiHa cells in S phase in the cell cycle. And the percent of apoptosis and necrosis increased. The morphology and the ultrastructure changed significantly after treated with 20 μmol/L β-sitosterol. Conclusions β-sitosterol can suppress proliferation of cervical cancer cells. Better results of growth inhibition were got in cervical squamous carcinoma than that in cervical adenocarcinoma. β-sitosterol induces the accumulation of cells in S phase.Part Ⅱ Effects of β-sitosterol on microtubule systems in cervical cancer cellsObjective To investigate β-sitosterol’s effects on microtubular system in SiHa cell line. Methods The expression and distribution of microtubule and microtubule associated protein 2 in SiHa cells were investigated by confocal microscopy. Immunoblotting analysis was used to determine tubulin α, microtubule associated protein 2, and the proportion of polymerization of tubulin. Results Confocal microscopy showed an abnormal microtubular network in SiHa cell treated with β-sitosterol for 5 days, and the expression of microtubule associated protein 2 was markedly down-regulated. Further analysis by immunoblotting confirmed the down-regulation of β-sitosterol on the expression of both microtubule associated protein 2 and tubulin α. Moreover, β-sitosterol reduced the proportion of polymerization of microtubule in a time-dependent manner. Conclusion β-sitosterol could down-regulate the expression of tubulin α and microtubule associated protein 2 in SiHa cells, and inhibit the microtubular polymerization. Our results suggested ananti-microtubule characteristic of β-sitosterol which might contribute to the proliferation inhibition of SiHa cells.Part Ⅲ Effects of β-sitosterol on IFN and its downstream genes in cervical cancer SiHa cellsObjective To study the effects of β-sitosterol on IFN, its downstream genes, and HPV E6/E7 protein in cervical cancer SiHa cells. Methods RT-PCR was used to estimate the relative mRNA amounts of IFNα, IFNβ, ISG15, OAS(p40/46), OAS(p69/71), RNase L, MxA, PKR in SiHa cells exposed to β-sitosterol. Western blot were used to measure the STAT1, E6 and E7 protein levels before and after β-sitosterol’s treatment. Results after treated with 20 μmol/L β-sitosterol, the mRNA’s expression of IFNβ, ISG15, OAS(p39/41), OAS(p69/71) and MxA increased significantly in a time-dependent manner. But the RNase L and PKR’s mRNA expression showed no change before and after β-sitosterol’s exposure. Through exposure to β-sitosterol also led to an increase in STAT1 and a decrease in E7 protein. Conclusion β-sitosterol could induce IFNβ and its downstream gene’s mRNA expression. The products of these genes could contribute to β-sitosterol’s effect on SiHa cells directly or indirectly, and then caused growth inhibition. β-sitosterol could also decrease HPV E7 protein directly of through IFN’s anti-virus pathway indirectly.更多还原