【作者】 陶志华；

【导师】 尹一兵；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2010， 博士

【摘要】 前列腺癌是欧美国家男性最常见的恶性肿瘤,其死亡率仅次于肺癌,居男性肿瘤的第二位。美国癌症学会估计, 2009年美国大约有19.228万名男性被诊断为前列腺癌,约有2.736万前列腺癌患者死亡[1]。目前,我国前列腺癌发病率虽远低于西方国家,但近年来随着人们生活方式的改变、寿命的延长及医疗保健和诊断水平的提高,我国前列腺癌发病率呈显著增长趋势,而且与西方国家前列腺癌发生有所不同,我国前列腺癌患者在初诊时往往已为进展期,丧失了手术良机,预后较差,生存期也较短。雄激素去势治疗是除外科手术与放射治疗外的标准前列腺癌治疗方法,然而经过中位数为18~24个月的缓解期后,雄激素依赖性前列腺癌(Androgen-Dependent Prostate Cancer,ADPC)转变为雄激素非依赖性前列腺癌(Androgen-Independent Prostate Cancer,AIPC),表现为前列腺癌细胞在缓解期过后又开始增殖,PSA进行性升高,极易发生骨转移,转变后的治疗非常棘手,死亡率极高,前列腺癌已成为危害老年男性人群健康的重大疾病。对AIPC的转变机制研究,是目前国内外前列腺癌研究领域的热点,主要涉及:雄激素受体(androgen receptor,AR)基因的扩增;雄激素受体突变;凋亡调控基因的异常,由于前列腺癌的生物学行为极其复杂,以上理论并不能合理地阐述AIPC的发生机制,雄激素非依赖性前列腺癌发病仍需进一步深入研究。建立一株能模拟临床上前列腺癌由雄激素依赖转变为雄激素非依赖过程的细胞模型,能模拟人前列腺癌发生、发展、结局的生理病理全过程,能够体现出人前列腺癌各阶段的特点,能模拟人前列腺癌基因的改变和前列腺癌对治疗的反应。采用现代细胞生物学、分子生物学以及蛋白组学和代谢组学的研究手段,进一步揭示雄激素依赖性前列腺癌向雄激素非依赖性前列腺癌转化过程中发生机制,有望为雄激素非依赖性前列腺癌的治疗提供新的理论依据。主要研究内容:1.在去雄激素的培养环境和雄激素阻断剂氟他胺作用下,诱导前列腺癌LNCaP细胞向雄激素非依赖性转化,建立雄激素非依赖性前列腺癌细胞系-LNCaP-AI细胞系和LNCaP-AI+F细胞系,鉴定雄激素非依赖性前列腺癌细胞系生物学特征变化。2.研究雄激素非依赖性前列腺癌细胞系-LNCaP-AI细胞系和LNCaP-AI+F细胞系的AR受体通路变化,包括目前AR受体突变分析、AR受体表达变化(mRNA和蛋白质水平);AR受体对雄激素反应性以及PSAmRNA和PSA蛋白质水平变化;细胞信号通路主要信号蛋白变化。3.采用PCR、Western Blot和免疫组织化学染色方法检测了RON基因表达情况,研究受体酪氨酸激酶RON在雄激素非依赖性前列腺癌细胞向雄激素非依赖性前列腺癌细胞转化过程中作用。4.利用Solexa高通量测序技术分别对LNCaP和LNCaP-AI+F细胞系的小分子RNA进行测序,利用生物信息学技术对所得到的大规模序列进行分析,筛选出与雄激素非依赖性前列腺癌相关的miRNA,同时可能发现一些新的miRNA,最后对感兴趣的miRNA的靶基因进行预测。主要结果:1. LNCaP-AI和LNCaP-AI+F细胞系细胞形态学发生变化:雄激素依赖性前列腺癌LNCaP细胞转化为雄激素非依赖性前列腺癌细胞- LNCaP-AI和LNCaP-AI+F细胞系,其形态学发生了变化,表现为:细胞胞体变小,呈扁平状,少见圆形或梭形,多见单个细胞独立生长,同时出现聚集生长现象;而LNCaP细胞形态呈三角形或长梭型,小部分为圆形,分布均匀。2. LNCaP-AI和LNCaP-AI+F细胞系细胞生长特性发生了变化:2.1在去雄激素环境下,LNCaP-AI和LNCaP-AI+F细胞系生长速度显著高于LNCaP细胞;氟他胺对生长抑制作用明显减弱.2.2 LNCaP-AI细胞系对雄激素的生长反应性明显下降,仅高浓度雄激素才刺激细胞增殖生长,而低浓度雄激素却无增殖反应性,但不同浓度雄激素对LNCaP-AI+F细胞生长均无刺激作用,说明LNCaP-AI+F细胞和LNCaP-AI对雄激素反应性也存在差异。2.3 LNCaP-AI和LNCaP-AI+F细胞系细胞侵袭能力和细胞迁移活性高于LNCaP细胞,特别是LNCaP-AI+F细胞系细胞迁移活性明显高于LNCaP细胞。3. LNCaP-AI和LNCaP-AI+F细胞系细胞AR信号通路变化:3.1在雄激素依赖性前列腺癌细胞向雄激素非依赖性前列腺癌细胞转化过程中,LNCaP-AI和LNCaP-AI+F细胞系细胞AR并没有发生突变,但LNCaP-AI细胞AR表达上调,而LNCaP-AI+F细胞AR表达下调。3.2 PSA表达在LNCaP-AI和LNCaP细胞之间无差异(P>0.05),但在LNCaP-AI+F细胞中表达明显下调(P<0.01);氟他胺、双氢睾酮和十一酸睾酮均能刺激LNCaP、LNCaP-AI和LNCaP-AI+F细胞表达和分泌PSA,但LNCaP-AI和LNCaP-AI+F细胞对双氢睾酮和十一酸睾酮反应性下降,特别是LNCaP-AI+F细胞对双氢睾酮和十一酸睾酮刺激存在PSA转录和分泌反应性和对药物浓度敏感性下降。4. LNCaP-AI和LNCaP-AI+F细胞受体酪氨酸激酶表达:结果发现,LNCaP-AI和LNCaP-AI+F细胞的RONmRNA、RON蛋白质均为阴性,说明RON基因在前列腺癌从雄激素依赖性向雄激素非依赖性转变过程中不起作用。5. LNCaP-AI+F细胞的miRNA表达谱变化:5.1实验组LNCaP-AI+F细胞共检测出376个miRNA,而对照组LNCaP细胞共检测出357个miRNA。存在表达差异miRNAs有94个,其中48个表达上调,46个表达下调。5.2预测新miRNA共有15个,其中LNCaP-AI+F细胞有9个新miRNA,而LNCaP细胞有6个新miRNA。主要结论:1.建立了雄激素非依赖性前列腺癌细胞系-LNCaP-AI和LNcaP-AI+F细胞系,其细胞生物学生长特性和对雄激素反应性与LNCaP细胞之间存在明显差异,为雄激素非依赖性前列腺癌发病机制的研究提供了良好的细胞学模型。2.在LNCaP细胞系转变为LNCaP-AI和LNCaP-AI+F细胞系过程中,AR均没有发生突变,但LNCaP-AI细胞的AR表达上调,而LNCaP-AI+F细胞的AR表达呈下降趋势;AR信号通路并没有被阻断,但对雄激素反应性下降。提示在长期的去雄激素环境和氟他胺抑制作用,LNCaP-AI和LNCaP-AI+F细胞的AR通路存在差异,虽然AR信号通路还是有效地,但对雄激素反应性下降。3.在LNCaP-AI和LNCaP-AI+F细胞系中RON基因均没有表达,提示:RON基因在LNCaP-AI和LNCaP-AI+F细胞系从ADPC转变为AIPC过程中可能不起作用。4.在激素依赖性前列腺癌LNCaP细胞和雄激素非依赖性前列腺癌LNCaP细胞之间存在差异表达的miRNAs,有可能在前列腺癌雄激素非依赖性转变过程中起重要的作用,为进一步研究前列腺癌的发生、发展机制提供了前期数据和基础。更多还原

【Abstract】 Prostate cancer is the most frequently diagnosed cancer and the second-leading cause of cancer death in men in the United States and Europe. American Cancer Society estimated that in 2009 about 192,280 American men are diagnosed with prostate cancer, about 27,360 died of prostate cancer.At present, the incidence of prostate cancer, although far lower than western countries, but showed a significant growth trend in china with changing lifestyles, longer life expectancy and the improvement of diagnosis. our patients with newly diagnosed prostate cancer often have advanced, loss of opportunity for surgery and poor prognosis with compared to the West national prostate cancer. Androgen deprivation therapy is the standard methA for prostate cancer besides surgery and radiation therapy. However, after a median of 18 to 24 months, androgen dependent prostate cancer (ADPC) turned into androgen independent prostate cancer (AIPC), extremely high mortality rate of AIPC has become a major health hazard for older men.The studies for mechanism of AIPC pathogenesis is a hot research field, mainly: androgen receptor (AR) gene amplification; androgen receptor mutation; apoptosis gene abnormalities and so on. But the above theory can not reasonably explain the pathogenesis of AIPC because the biological behavior of prostate cancer is extremely complex.AIPC still need further study. To establish the cell mAel of prostate cancer from androgen dependent into androgen-independent can simulate gene changes and response to treatment in human prostate cancer. Mechanism of AIPC pathogenesis expected to be revealed by mAern cell biology, molecular biology and proteomics and metabolomics research tools and provide a new theory for AIPC treatment.Main contents:1. In the environment of free androgen hormones and androgen antagonist flutamide, prostate cancer LNCaP cells were transformed into androgen-independent cell line-LNCaP-AI cell line and LNCaP- AI + F cell line and their biological characteristics were identified.2. The changes of androgen receptor(AR) pathway were studied in LNCaP-AI and LNCaP-AI+F, including the AR receptor mutation, AR receptor expression (mRNA and protein level); AR receptor esponsiveness to androgen and PSA protein and PSAmRNA levels; the changes of the main protein in cell signaling pathway.3. The changes of the receptor tyrosine kinase RON in LNCaP-AI and LNCaP-AI+F. the RON gene expression was analysed by the methAs of PCR, Western Blot and immunohistochemical staining.4. Using high throughput Solexa sequencing, the profile of small RNA molecules was analysed in LNCaP-AI+F and LNCaP respectively. The miRNAs related to androgen-independent prostate cancer were screened and new miRNAs may be found.The main results:1. The morphological changes of LNCaP-AI and LNCaP-AI+F cell lines: its morphological changes as follows: the cell bAy became smaller, flat and round or spindle rare, simultaneous growth of aggregation; But LNCaP cells appeared in triangle or a long shuttle-type, the circular uniform distribution.2. Growth characteristics of LNCaP-AI and LNCaP-AI + F cell lines:2.1 In free androgen hormones environment, the growth rate of LNCaP-AI and LNCaP-AI+F cell lines were significantly higher than that of LNCaP cells; Growth inhibition of Flutamide on the LNCaP-AI and LNCaP-AI+F cell lines in vitro significantly reduced.2.2 The growth role of androgen responsiveness decreased in the LNCaP-AI cell lines, only the high concentration of androgen to stimulate cell growth, but in the LNCaP-AI+F cell line, different concentrations of androgen can not stimulate the cell growth. It indicated that LNCaP-AI+F might be different from LNCaP-AI cells in the cellcharacteristics.2.3 Cell invasion and migration activities of the LNCaP-AI and LNCaP-AI+F cell line were higher than the LNCaP cells, in particular, the activities were significantly higher in the LNCaP-AI+F cell line.3. The changes of AR signaling pathway in the LNCaP-AI and LNCaP-AI+F cell lines:3.1 AR mutation was not detected in the LNCaP-AI and LNCaP-AI+F cells, but AR expression was upregulated in LNCaP-AI cells, while downregulated in the LNCaP-AI+F cells in the transformation process of androgen dependent prostate cancer to androgen-independent prostate cancer.3.2 PSA expression was not difference between LNCaP-AI and LNCaP cells(p>0.05), but significantly reduced in the LNCaP-AI+F cells(p<0.01); flutamide, DHT and testosterone undecanoate can stimulat PSA expression and secretion in the LNCaP, LNCaP-AI and LNCa P-AI+F cells, but responsiveness of PSA expression to dihydrotestosterone and testosterone undecanoate in the LNCaP-AI and significantly decreased in the LNCaP-AI+F cells; the reactivity and sensitivity to the drug concentration decreased in the LNCaP-AI and LNCaP-AI+F cells. It indicated that AR signaling pathway is effective, but decreased in the responsiveness to androgen hormnones in the transformation process of androgen dependent to androgen independent.4. Expression of receptor tyrosine kinase RON in the LNCaP-AI and LNCaP-AI+F cells: The results showed that RONmRNA and RON protein were not detected in the LNCaP-AI and LNCaP-AI+F cells. It indicated that RON gene did not play role in the transformation process.5. The changes of miRNA expression profile in the LNCaP-AI+F cells:5.1 376 miRNAs were detected in the LNCaP-AI-F cells, while 357 miRNAs in the LNCaP cells. 94 miRNAs including 48 upregulated, 46 downregulated were differentially expressed in the LNCaP-AI-F cells compared to LNCaP cells.5.2 15 new miRNA were predicted, including 9 new miRNAs in the LNCaP-AI+F cells and 6 new miRNAs in the LNCaP cells.The main conclusions:1. Androgen-independent prostate cancer cell lines-LNCaP-AI and LNcaP-AI+F cell line were successfully established, their androgen responsiveness and growth characteristics significantly different from the LNCaP cells. It is a goA cellular mAel for studying the mechanisms for androgen-independent prostate cancer.2. In the transformation process of LNCaP cell line to LNCaP-AI and LNCaP-AI+F cell line, AR mutation was not detected and AR expression was decreasing; AR signaling pathway was not blocked, but reduced in the responsiveness to androgen.3. RON gene did not play role in the transformation process of LNCaP cell line to LNCaP-AI and LNCaP-AI+F cell line4. miRNAs might play important role in the transformation process of LNCaP cell line to LNCaP-AI and LNCaP-AI+F cell line, provided the foundation for further study of the incidence of AIPC.更多还原