【作者】 张晓波；

【导师】 齐琳；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 前列腺癌在许多西方国家是男性最常见的恶性肿瘤,占男性癌症死因的第二位。前列腺癌在我国临床住院病人中所占比率逐渐升高,成为威胁老年男性健康的一个重要因素。由于前列腺癌细胞的生长具有依赖于雄激素的生物学特性,导致几乎所有初始对内分泌治疗敏感的前列腺癌患者最终都将产生雄激素抵抗,由雄激素依赖型前列腺癌发展成高度恶化且易广泛转移,并最终死于激素非依赖性前列腺癌。因此,激素非依赖性前列腺癌的发病机制已经成为泌尿肿瘤学者遇到的最常见和最棘手的难题之一,是当前前列腺癌研究的热点。深入研究其分子机制,寻求有效的干预手段阻断,对于提高前列腺癌的整体治疗水平具有重要的理论和现实意义。第一章非依赖性前列腺癌差异表达蛋白的筛选研究目的：筛选HRPC的发生发展的重要蛋白分子靶点。方法：通过提取雄激素依赖型前列腺癌和雄激素非依赖型前列腺癌组织总蛋白,用双向电泳技术分离转膜,正常人血清封闭,分别用两组患者血清作为一抗行Western blot检测作为引导,从正常、雄激素依赖和激素难治性三个视角去显示差异表达的蛋白。结果：筛选获得差异表达的蛋白点：自吞噬蛋白1(Beclinl),谷胱苷肽S转移酶P(GSTP1-1), ZBTB7、Dihydrodiol dehydrogenase 2(DDH),葡萄糖依赖性胰岛素释放肽受体(GIPR)、热休克因子1(heat shock factor 1,HSF1)、α烯醇酶(ENO1)、锰,超氧化物歧化酶(MnSOD)、磷酸甘油酸变位酶1(PGAM1)、肽基脯氨基顺反异构酶(PPIA)、磷脂酰乙醇胺结合蛋白(PEBP)等。结论：本研究改进的血清免疫印迹技术引导的蛋白质组学方法,筛选差异表达蛋白具有可行性。为激素非依赖性前列腺癌研究提供了一种新的方法和候选分子。目的：探讨热休克因子1在前列腺癌发生发展中起重要的作用。方法：采用免疫组化和Western blot方法检测了HSF1在前列腺癌组织中的表达中,并分析其表达水平与前列腺癌临床病理指标的相关性。结果：(1)HSF1在前列腺癌组织中的表达高于良性前列腺增生组织(3.01±0.09 vs 1.25±0.07),差异有统计学意义(P<0.05)；HSF1在激素依赖前列腺癌组织中表达低于激素非依赖型前列腺癌组织(1.25±0.07 vs 3.12±0.09),差异有统计学意义(P<0.05),HSP27在前列腺癌组织中的表达低于良性前列腺增生组织(1.47±0.06vs3.12±0.01),差异有统计学意义(P<0.05)；HSP27在激素依赖前列腺癌组织中表达高于激素非依赖型前列腺癌组织(2.37±0.02vs1.12±0.04),差异有统计学意义(P<0.05)。HSF1和HSP27表达呈负相关(r=-8.753,P<0.05)。HSP27在激素依赖性前列腺癌细胞株LNCaP中的表达高于激素非依赖性前列腺癌细胞株PC-3细胞中的表达(3.02±0.06 vs 1.35±0.05),差异有统计学意义(P<0.05),HSF1和HSP27表达呈负相关(r=-8.695,P<0.05)。(2)HSF1的表达与PSA、临床分期的具有显著性相关性。HSF1表达与Gleason评分呈正相关(r=0.66,P<0.001)。HSF1表达与PSA水平随PSA升高而升高,呈正相关(r=0.76,P<0.001)结论-HSF1蛋白在激素非依赖性前列腺癌发生发展中起重要的调控作用。第三章HSF1在PC,3细胞中的作用与机制研究目的：HSF1在前列腺癌中发生发展中的作用及机制。方法：通过构建逆转录RNA干扰HSF1质粒,转染激素非依赖性前列腺癌细胞株PC-3细胞,体外采用划痕愈合实验、Transwell实验、流式细胞术等方法,体内通过裸鼠皮下成瘤实验,从体外和体内两个层面进行HSF1的表达调节作用机制研究。结果：(1)体外实验中抑制HSF1的表达可以抑制PC-3细胞的侵袭迁移。采用划痕实验比较了PC-3HSF1RNAi+和PC-3HSF1RNAi-的迁移能力,结果显示PC-3HSF1RNAi+较PC-3HSF1RNAi-细胞迁移能力明显下降,24小时划痕愈合率分别为62%vs77%,差异具有显著性意义(P<0.05)。采用Transwell侵袭小室测定法比较PC-3HSF1RNAi+和PC-3HSF1RNAi-的侵袭能力,结果显示PC-3HSF1RNAi+较PC-3HSF1RNAi,细胞侵袭能力明显下降,24小时培养后穿过Transwell的每低倍视野细胞数分别为63±11 vs 129±13,差异具有显著性意义(P<0.05)。(2)体外HSF1表达沉默的可以促进PC-3细胞的凋亡。pSUPERHSF1RNAi+转染细胞48h后出现大量的凋亡细胞,较空白对照组明显增加,差异有统计学意义(31.3%±2.8%vs3.9%±0.7%,P<0.05),而pSUPER HSF1RNAi-组和空白对照组细胞凋亡率差异无统计学意义(P>0.05)。(3)体内抑制HSF1的表达可以抑制PC-3细胞的成瘤能力。比较了MiceHSF1RNAi+和MiceHSF1RNAi-两组裸鼠模型中PC-3细胞原发瘤的大小,分别为3.01±0.22和3.45±0.23,差异具有显著性意义(P<0.05),这提示体内抑制HSF1的表达能够抑制PC-3细胞的生长。(4)HSF1表达沉默的PC-3细胞后蛋白的表达.应激在上调HSF1表达的同时下调了HSP27表达。且Caspase-3蛋白(1.13±0.03 vs 2.13±0.08)、Fas蛋白表达上调(1.31±0.09 vs2.69±0.03),Cyclin E蛋白表达下调(1.98±0.02 vs 1.06±0.05),差异具有统计学意义(P<0.05),Mdr1蛋白表达无变化(2.44±0.05vs2.53±0.07),差异具无统计学意义(P>0.05)。结论：HSF1表达上调在激素非依赖性前列腺癌细胞侵袭转移中起重要的作用。HSF1通过上调Caspase-3蛋白、Fas蛋白表达,下调HSP27蛋白、Cyclin E蛋白表达在激素非依赖性前列腺癌细胞凋亡中起重要的作用机制。更多还原

【Abstract】 Prostate cancer is the most common cancer in male, and the second most common cause of cancer-related death in Western countries,. Prostate cancer has gradually increased in proportion in China, becoming the threat to the health of male elders.Since the growth of prostate cancer depends on the biological characteristics of androgen, almost all the initial treatment of the endocrine-sensitive prostate cancer patients will eventually become androgen resistance and change from androgen-dependent prostate cancer into hormone refractory prostate cancer(HRPC) which is highly deteriorated, and eventually died of hormone refractory prostate cancer. Thus, the pathogenesis of HRPC has become the most common and one of the most difficult point in the current focus of prostate cancer research. Study of the molecular mechanism of HRPC and find effective interventions to block or even reverse the genesis of HRPC,will be helpful for the treatment of prostate cancer and extend overall level of great theoretical and practical significance.Discovering important protein molecular target for the occurrence and development of HRPC is a scientific problem to be solved.Objective:Screening of the important molecular target protein in tumorigenesis and development of HRPC.Methods:Extracting the total protein of androgen-dependent prostate cancer and androgen-independent prostate cancer tissues, and using two-dimensional electrophoresis to separate transfer membrane, blocked with three groups of serums from normal male, androgen-dependent prostate cancer patients and androgen-independent prostate cancer patients as the primary antibody, screening the differentially expressed proteins with Western blot. Results: We have screened differentially expressed protein as follow:swallowing protein 1 (Beclinl),glutathione S transferase P (GSTP1-1),ZBTB7, Dihydrodiol dehydrogenase 2 (DDH), glucose-dependent insulin-releasing peptide receptor(GIPR), heat shock factor 1 (HSF1),a enolase (ENO1),Mn-superoxide dismutase (MnSOD), phosphoglycerate mutase 1 (PGAM1),peptide-based breast amino-cis-trans isomerase (PPIA), phosphatidylethanolamine binding protein (PEBP) and so on. Conclusion: The modified serum immunoblotting proteomics technique is feasible to screen differentially expressed proteins. We screened differentially expressed proteins of Claudin-4, Beclin 1,HSF1 which may play an important role in development of hormone-independent prostate cancer.Objective:To investigate the role of heat shock factor 1 in the development of prostate cancer. Methods:Immunohistochemistry and Western blot methods were used to detect the expression levels of HSF1 in prostate cancer tissues and the correlation between the expression levels and prostate cancer clinical pathological characters was analyzed. Results:(1)HSP27 was lower in prostate cancer tissues than benign prostatic hyperplasia tissues(1.47±0.06 vs 3.12±0.01),with significant difference (P<0.05);HSP27 in hormone refractory prostate cancer tissues was significantly higher than hormone-independent prostate cancer (2.37±0.02 vs 1.12±0.04, P<0.05).The negative correlation was found between HSF1 and HSP27 expression (r=-8.753, P<0.05).HSP27 in hormone-dependent prostate cancer cell line LNCaP was higher than in hormone-independent prostate cancer cell line PC-3 (3.02±0.06 vs 1.35±0.05),the difference was significant (P<0.05),HSF1 and HSP27 expression was negatively correlated (r=-8.695,P<0.05).(2) Expression of HSF1 was significantly correlated with PSA level, Gleason score, clinical stage in prostate cancer. Expression of HSF1 was observed positive correlation with Gleason score(r=0.66,P<0.001)and with PSA level (r=0.76, P<0.001).Conclusion:HSF1 plays an important role in the development of hormone refractory prostate cancer.Objective:To investigate the mechanism of HSF1 on development of prostate cancer. Methods:RNA interference plasmid of HSF1 was constructed and was transfected into hormone-independent prostate cancer cell line PC-3 cells by reverse transcription. Scratch healing, Transwell experiments, flow cytometry and other methods as in vitro experiments and subcutaneous tumor formation experiment in vivo were used to explain the regulation mechanism. Results:(1)Inhibition of the expression of HSF1 lead to decrease of the invasion ablitiy of PC-3 cell migration In vitro. The results of scratch healing showed that migration ability of PC-3 HSF1RNAi+ Was significantly impaired,24-hour scratch healing rates of PC-3 HSF1RNAi+ and PC-3 HSF1RNAi- cells were 62% vs 77%,the difference was significant (P<0.05).Different invasive ability were found in PC-3 HSF1RNAi+ and PC-3 HSF1RNAi- by Transwell assay. After 24 hours cell culture, cell count of permeate through Transwell (lowpower field) were 63±11 vs 129±13,difference was significant (P<0.05).These results suggest that in vitro inhibition of HSF1 expression in PC-3 cells can inhibit the invasion and metastasis of prostate cancer. (2) In vitro, the silence of HSF1 expression can promote the apoptosis of PC-3 cells.48h after transfection, pSUPER HSF1RNAi+ cells increased more apoptotic cells number than control group, significantly (31.3%±2.8% vs 3.9%±0.7%, P<0.05),but the apoptosis rate of pSUPER HSF1RNAi- group and control group showed no significant difference (P>0.05).(3)The silence of HSF1 expression in PC-3 cells can inhibit the ability tumor formation in vivo.The size of primary tumor of PC-3 cells in nude Mice HSF1RNAi+ and nude Mice HSF1RNAi-,were 3.01±0.22 and 3.45±0.23,significant difference (P<0.05),suggesting that the silence of HSF1 expression in vivo can inhibit the growth of PC-3 cells. (4) Thermal stimulation up-regulated the expression of HSF1-silenced in PC-3 cells and down-regulated expression of HSP27.Caspase-3 protein (1.13±0.03 vs 2.13±0.08), Fas protein expression(1.31±0.09 vs 2.69±0.03),Cyclin E protein expression were decreased(1.98±0.02 vs 1.06±0.05),with statistically significant difference (P<0.05).No statistically significant difference was found in Mdrl protein expression (2.44±0.05 vs 2.53±0.07,P>0.05).Conclusion:Over-expression of HSF1 and down-regulation of HSP27 play an important role in the invasion and metastasis of hormone resistant prostate cancer.更多还原