【作者】 王梁；

【导师】 孙颖浩；

【作者基本信息】 第二军医大学 ， 泌尿外科学， 2010， 博士

【摘要】 前列腺癌(prostate Cancer, PCa)是威胁男性健康的常见肿瘤之一。在美国,前列腺癌自1984年以来就是男性最常见的内脏恶性肿瘤。仅2005年就有232,090例新发病例,占所有男性肿瘤的1/3,2005年前列腺癌相关死亡30,350例,死亡率居所有肿瘤第二位。我国前列腺癌发病率也逐年升高,目前已位居男性泌尿系统恶性肿瘤第三位。雄激素阻断治疗是前列腺癌的治疗中不可替代的一种治疗方法,包括药物或外科手术阻断雄激素疗法(ADT),用于外科手术及放疗后的辅助治疗以及转移性前列腺癌的治疗,然而,这些治疗方法却伴随着许多副作用,例如脂质代谢紊乱和心血管疾病。ADT通常只能使肿瘤暂时缓解,最终将进展为雄激素非依赖性前列腺癌(AIPC)。近年来研究表明,高脂肪/胆固醇等“西方饮食”同前列腺癌的发病和进展密切相关。高血清胆固醇水平能够促进前列腺癌的发展并且刺激雄激素上调表达,通过改变饮食及药物降低血清胆固醇水平将有助于减缓前列腺癌的进展。一系列的病理和临床前观察已经证实,通过改变饮食及药物降低胆固醇水平有助于减缓前列腺癌的进展。尽管其具体作用机制尚未阐明,但是数据清楚表明这不是通过雄激素介导的机制。许多研究表明,普通前列腺增生和前列腺中都聚集了不同寻常的高水平胆固醇。目前尚不清楚为什么高胆固醇可变信号分子和导致肿瘤细胞增生,有证据表明肿瘤细胞细胞膜上的胆固醇积聚直接改变,甚至导致异常的信号转导。为什么改变细胞膜胆固醇水平影响肿瘤细胞的信号转导?我们的体外实验研究证实在低胆固醇水平条件下前列腺癌细胞LNCAP-AD, LNCAP-AI及PC-3增殖均受到抑制,分泌至细胞外的Hedgehog蛋白下调,而细胞内的Hedgehog蛋白表达上调。Hedgehog基因是一种分节极性基因,因突变的果蝇胚胎呈多毛团状,酷似受惊刺猬而得名。哺乳动物中存在三个Hedgehog的同源基因：Sonic Hedgehog(SHH、Indian Hedgehog(IHH)和Desert Hedgehog(DHH),分别编码Shh、Ihh和Dhh蛋白。Hedgehog在细胞分化、胚胎发育、器官形成、损伤修复和肿瘤发生中都有重要生理和病理意义。胆固醇促进前列腺癌细胞增殖的具体分子机制目前尚未完全阐明。胆固醇是否是Hedgehog信号通路的重要影响因子,以及在低胆固醇条件下是否是通过Hedgehog下调作用抑制前列腺癌细胞增殖目前尚无相关报道。本研究通过MTT (CCK-8)实验检测分别培养于普通培养基(NM)、胆固醇耗竭培养液(CDM),高胆固醇培养基(HCM)以及加入辛伐他汀后各组中的前列腺癌PC-3细胞、LNCaP细胞以及LNCaP-AI细胞的增殖情况,进一步采用半定量逆转录聚合酶链式反应(RT-PCR)、Real-time PCR检测Hedgehog的mRNA水平变化,同时检测Hedgehog下游信号通路蛋白Ptc, Smo和Gli的mRNA表达变化;ELISA方法检测分泌至胞外的Hedgehog蛋白水平变化；并进一步采用激光共聚焦方法检测Hedgehog蛋白的亚细胞定位情况；确定在低胆固醇和降血脂(辛伐他汀)条件下,是否是抑制Hedgehog信号通路激活从而抑制细胞增殖,从而论证在前列腺癌细胞中胆固醇是Hedgehog信号通路的重要影响因子。初步揭示高胆固醇条件下通过上调Hedgehog胞外分泌表达而促进前列腺癌发展的分子机制,为治疗前列腺癌提供新的思路。一、胆固醇规律前列腺癌细胞中Hedgehog的表达及意义目的：研究比较分别培养于NM、CDM、HCM各组中人前列腺癌PC-3细胞、LNCaP细胞以及LNCaP-AI细胞的增殖凋亡情况以及Hedgehog信号通路的表达差异,分析胆固醇对Hedgehog信号通路的影响与前列腺癌进展的关系。方法：以人前列腺癌PC-3细胞、LNCaP细胞以及LNCaP-AI细胞系为研究对象,分别培养于NM、CDM和HCM中。MTT方法检测细胞增殖凋亡情况,采用半定量逆转录聚合酶链式反应(RT-PCR)、Real-time PCR、ELISA方法检测Hedgehog的mRNA和蛋白水平变化；同时检测Hedgehog下游信号通路蛋白Ptc, Smo和Gli1的mRNA表达变化；并以激光共聚焦观察不同胆固醇浓度条件下Hedgehog在细胞内定位情况。结果：1、培养于CDM的前列腺癌细胞增殖曲线明显低于培养于NM和HCM的细胞。2、培养于CDM的前列腺癌细胞分泌Hedgehog的蛋白水平明显低于培养于NM和HCM的细胞表达,两者有显著差异。3、培养于CDM的前列腺癌细胞Hedgehog, Smo和Gli表达增加,而Ptc表达下降。4、激光共聚焦观察发现CDM组前列腺癌细胞的Hedgehog蛋白在细胞内表达明显高于NM和HCM组。结论：1、低胆固醇抑制前列腺癌PC-3细胞、LNCaP细胞以及LNCaP-AI细胞体外增殖。2、低胆固醇抑制前列腺癌PC-3细胞、LNCaP细胞以及LNCaP-AI细胞中的Hedgehog蛋白胞外分泌过程。3、低胆固醇抑制前列腺癌细胞增殖与Hedgehog的胞外分泌下调有关。二、辛伐他汀规律前列腺癌细胞中Hedgehog的表达及意义目的：研究比较分别培养于不同浓度辛伐他汀的NM、HCM各组中人前列腺癌PC-3细胞、LNCaP细胞以及LNCaP-AI细胞的增殖凋亡情况以及Hedgehog信号通路的表达差异,分析辛伐他汀对Hedgehog信号通路的影响与前列腺癌进展的关系。方法：以人前列腺癌PC-3细胞、LNCaP细胞以及LNCaP-AI细胞系为研究对象,分别培养于NM和HCM中,各组中分加不同浓度辛伐他汀。MTT方法检测细胞增殖凋亡情况,采用半定量逆转录聚合酶链式反应(RT-PCR)、Real-time PCR、ELISA方法检测Hedgehog的mRNA和蛋白水平变化；同时检测Hedgehog下游信号通路蛋白Ptc, Smo和Gli的mRNA表达变化；并以激光共聚焦观察不同辛伐他汀浓度条件下Hedgehog在细胞内定位情况。结果：1、培养于2.4μM辛伐他汀的NM和HCM前列腺癌细胞增殖曲线明显低于培养于未加辛伐他汀的NM和HCM培养基的细胞,呈浓度依赖关系。2、培养于2.4μM辛伐他汀的NM和HCM中的前列腺癌细胞上清液中Hedgehog的蛋白水平明显低于未加辛伐他汀的NM和HCM的细胞,两者有显著差异。3、培养于2.4μM辛伐他汀的NM和HCM中的前列腺癌细胞Hedgehog, Smo和Gli表达增加,而Ptc表达下降。4、激光共聚焦观察发现2.4μM辛伐他汀的NM和HCM组前列腺癌细胞的Hedgehog蛋白在细胞内表达明显高于未加辛伐他汀的NM和HCM组。结论：1、高浓度辛伐他汀抑制前列腺癌PC-3细胞、LNCaP细胞以及LNCaP-AI细胞体外增殖。2、高浓度辛伐他汀抑制前列腺癌PC-3细胞、LNCaP细胞以及LNCaP-AI细胞中的Hedgehog蛋白胞外分泌过程。3、高浓度辛伐他汀抑制前列腺癌细胞增殖与Hedgehog的胞外分泌下调有关。4、前列腺癌患者中应用降血脂药物可以减缓前列腺癌进展。更多还原

【Abstract】 Prostate cancer is one of the most prevalent malignant tumors threaten male health. It is the most common form of non-cutaneous cancer and second most lethal cancer in American men with an incidence of 232,090 new cases, and 30,350 deaths in 2005 alone. The incidence rate of prostate cancer in China is going up recently, and reported cases already up to the third in urinary malignant tumor. Although in many cases this cancer is treatable through hormone therapy and/or surgery, these cures are associated with a host of medical problem(e.g. lipid metabolic disturbance and heart diseases). And often provide only temporary relief, as prostate cancer reoccurs in part of cases as a far less treatable and much more malignant disease-androgen independent prostate cancer(AIPC).Recent studies showed high fat/high cholesterol’Western-type diets’have been linked to PCa incidence and progression. High levels of serum cholesterol contribute to prostate cancer progression and upon stimulation with androgen. Modification of cholesterol levels through diet, exercise and the use of pharmaceuticals may contribute to slowing prostate cancer Progression.In our studies we demonstrate that Hedgehog protein expression decreased after treatment in cholesterol depletion media (CDM) and the tumor cells apoptosis ratio increased. Hedgehog gene is a sub-segment polarity genes, it is named because that in Drosophila embryos showed hairball-like which resembles a frightened Hedgehog to follow gene mutations. There are three Hedgehog homologous genes in mammalian: Sonic Hedgehog (SHH), Indian Hedgehog (IHH) and Desert Hedgehog (DHH), encoding Shh, Ihh, and Dhh proteins respectively. Hedgehog have important physiological and pathological significance in cell differentiation, embryonic development, organ formation, injury repairs and tumorigenesis, but the specific molecular mechanism has not been fully clarified about cholesterol promoting the apoptosis of prostate cancer. Whether or not Hedgehog is the major cholesterol-sensitive factors which affect the prostate cancer cell proliferation, as well as low-cholesterol conditions, whether it is inhibited by Hedgehog downward effect on the proliferation of prostate cancer there were no relevant reports. Our study research the prostate cancer PC-3 cells, LNCaP cells and LNCaP-AI cell proliferation, apoptosis by MTT experiments, and the prostate cancer cells were cultured in normal medium (NM), cholesterol depletion of culture medium (CDM), high cholesterol medium (HCM) and the addition of simvastatin in each group, then we use semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Real-time PCR, ELISA method to detect the mRNA and protein levels of Hedgehog change; the same time Detection of Hedgehog signaling pathway downstream of protein Ptc, Smo and Gli expression of the mRNA; and last we use confocal laser scanning detection of sub-cellular localization of Hedgehog; to determine the low cholesterol and lipid-lowering (simvastatin) conditions, whether inhibited Hedgehog promotes cell apoptosis and inhibit prostate cancer cell from hormonal-dependent to-independent transformation, thus demonstrated that the Hedgehog is a cholesterol-sensitive cytokine hypothesis in prostate cancer cells. Revealed the fact that low-cholesterol can down-regulating the expression of Hedgehog, then inhibits the development of prostate cancer, provide a new way of thinking for the treatment of prostate cancer.Objective:To comparative study the proliferation, apoptosis as well as differences in the expression of Hedgehog signaling pathway of human prostate cancer PC-3 cells, LNCaP cells and LNCaP-AI cell which were cultured in NM, CDM, HCM groups, To analyze the impact of cholesterol on Hedgehog signaling pathway and prostate cancer progression.Method:Human prostate cancer PC-3 cells, LNCaP cells and LNCaP-AI cells were cultured in NM, CDM, and in HCM. MTT method to detect cell proliferation and apoptosis, using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Real-time PCR, ELISA method to detect the change of Hedgehog mRNA and protein levels; Simultaneous detect Hedgehog signaling pathway protein Ptc, Smo and Glil; and observe the cell location of Hedgehog under the conditions of different concentrations of cholesterol by confocal laser.Result: 1, the growth curves of prostate cancer cells cultured in CDM were significantly lower than cultured in NM and HCM cells.2, the Hedgehog protein levels of prostate cancer cells cultured in CDM were significantly lower than that in NM and HCM, it is a significant difference.3, Hedgehog signaling pathway-related protein of prostate cancer cells cultured in CDM, the expression of Hedgehog, Smo and Gli increased and Ptc expression reduced.4, we also observed that the CDM group of prostate cancer cells express Hedgehog protein in cells was significantly higher than NM and HCM groups by laser confocal.Conclusions:1, low-cholesterol inhibits prostate cancer PC-3 cells, LNCaP cells and LNCaP-AI cells in vitro.2, low-cholesterol inhibits Hedgehog protein in the process of extracellular secretion of prostate cancer PC-3 cells, LNCaP cells and LNCaP-AI cells.3, low-cholesterol inhibit prostate cancer cell proliferation correlate with the fall of extracellular secretion of Hedgehog.Objective:To comparative study the proliferation, apoptosis as well as differences in the expression of Hedgehog signaling pathway of human prostate cancer PC-3 cells, LNCaP cells and LNCaP-AI cell which were cultured in NM, HCM with different concentration of Simvastatin, To analyze the impact of Simvastatin on Hedgehog signaling pathway and prostate cancer progression.Method:Human prostate cancer PC-3 cells, LNCaP cells and LNCaP-AI cells were cultured in NM, and in HCM, each group plus different concentrations of simvastatin. MTT method to detect cell proliferation and apoptosis, using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Real-time PCR, ELISA method to detect the change of Hedgehog mRNA and protein levels; Simultaneous detect Hedgehog signaling pathway protein Ptc, Smo and Gli1; and observe the cell location of Hedgehog under the conditions of different concentrations of simvastatin by confocal laser.Result:1, the growth curves of prostate cancer cells cultured in 2AμM simvastatin NM and HCM were significantly lower than cultured in NM and HCM cells.2, the Hedgehog protein levels of prostate cancer cells cultured in 2.4μM simvastatin NM and HCM were significantly lower than that in NM and HCM, it is a significant difference between the two.3, Hedgehog signaling pathway-related protein of prostate cancer cells cultured in 2.4μM simvastatin NM and HCM, the expression Hedgehog, Smo and the Gli increased and Ptc expression decreased.4, we also observed that the 2.4μM simvastatin NM and HCM group of prostate cancer cells express Hedgehog protein in cells was significantly higher than NM and HCM groups by laser confocalConclusions:1, simvastatin inhibit prostate cancer PC-3 cells, LNCaP cells and LNCaP-AI cells in vitro.2, simvastatin inhibit Hedgehog protein in the process of extracellular secretion of prostate cancer PC-3 cells, LNCaP cells and LNCaP-AI cells.3, simvastatin inhibit prostate cancer cell proliferation correlate with the fall of extracellular secretion of Hedgehog.4, prostate cancer patients apply the lipid-lowering drugs can slow the progress of prostate cancer.更多还原