【作者】 李建华；

【导师】 王禾；

【作者基本信息】 第四军医大学 ， 外科学， 2009， 博士

【摘要】 前列腺癌为老年男性常见疾病。全球范围居男性癌症发病率第3位,病死率第6位,在美国则分别居男性癌症发病率第1位,病死率第2位。我国前列腺癌发病率虽远低于欧美国家,但近年随着前列腺癌检出率的提高、人口老龄化及生活习惯的改变,发病率呈明显增加趋势,目前已经成为泌尿系统除膀胱癌和肾癌以外的第3大恶性肿瘤,并有逐渐接近膀胱癌和肾癌的趋势。有关前列腺癌诊断和治疗的研究已经成为泌尿外科的重要研究课题。目前普遍的观点认为从正常前列腺组织发展到前列腺癌经历了前列腺上皮细胞内瘤、局部前列腺癌、侵袭性前列腺癌和转移性前列腺癌等几个阶段。对于晚期的前列腺癌一般采用内分泌治疗,大多数患者起初都对内分泌治疗有效,但经过中位时间14~30个月后,几乎所有患者病变都将逐渐发展为雄激素非依赖性前列腺癌。目前,临床上尚无有效的方法来治疗,病死率高。只有从肿瘤的发生发展的分子机制方面来治疗恶性肿瘤,才是彻底根治的唯一途径。因此基因治疗目前成为前列腺癌治疗的选择之一。本论文以针对前列腺癌的基因治疗方法和前列腺癌发生、发展以及预后评价相关新分子的研究为切入点,对两方面的内容进行了较为深入的实验研究。第一部分是关于受体型酪氨酸激酶TrkB参与前列腺癌发生与发展进程分子机制的研究;第二部分是关于我课题组前期发现的一个凋亡诱导分子—Survivin显性负突变分子Sur-D71A诱导前列腺癌细胞凋亡分子机制的研究。TrkB是一种受体型酪氨酸激酶,属于Trk神经营养因子受体家族,其基因表达产物通过与其配体脑衍生营养因子(BDNF)相互作用诱导神经系统细胞的持续性增殖、活化及抵抗外源凋亡刺激因素引起的细胞死亡。近年来发现TrkB及其配体BDNF在多种肿瘤中的表达存在异常,其过量表达高度提示与肿瘤细胞的增殖和转移密切相关,但在前列腺癌方面的研究却为数不多。本部分实验观察了TrkB在前列腺癌中的表达情况,并利用分子生物学方法调节前列腺癌细胞内源TrkB的表达水平,分析TrkB对前列腺癌细胞的生物学行为影响,更进一步的分析了前列腺癌细胞内源TrkB表达水平的改变所导致的胞内信号转导通路变化。目的:①分析TrkB在前列腺癌中的表达是否与疾病进程有关;②观察TrkB对前列腺癌细胞生物学行为的影响;③评价TrkB是否可能成为前列腺癌治疗的分子靶点;④观察TrkB对前列腺癌细胞信号转导通路的影响。方法:①利用免疫组化的方法研究TrkB在前列腺良性增生性病变组织与前列腺癌组织中的表达,分析TrkB的表达与肿瘤的病理分级之间的关系和TrkB的组织定位与前列腺癌进程之间的关系;②构建pSilencer-TrkB干涉表达载体,利用稳定转染pSilener-TrkB干涉表达载体的方法观察TrkB表达水平改变对前列腺癌细胞生物学行为的影响。通过细胞计数实验观察TrkB对前列腺癌细胞增殖能力的影响,通过软琼脂克隆形成实验观察TrkB对前列腺癌细胞恶性表型的影响。③通过观察前列腺癌肿瘤细胞中MAPK信号通路和PI3K/Akt信号通路的变化,分析TrkB分子对前列腺肿瘤细胞增殖活化相关信号转导通路的影响。结果:①TrkB在前列腺癌细胞中高表达,其高表达趋势与前列腺癌的病理分级水平成正相关。TrkB在前列腺癌细胞上主要分布在细胞浆和细胞膜,但是在部分病例中发现TrkB具有非常特异和明确的核定位现象,但由于检测病例标本例数尚不足以进行统计学分析,还无法判定TrkB的核定位与疾病进程之间的关系。②构建的pSilencer-TrkB干涉载体能显著抑制前列腺癌细胞中TrkB的表达。TrkB有促进前列腺癌细胞增殖和恶性转化的能力。③TrkB介导的胞内活化信号通路主要是通过PI3K/Akt通路实现的。结论:①TrkB可能参与了前列腺癌的发生与发展的进程;②TrkB在前列腺癌细胞的增殖和活化中发挥着重要的作用;③TrkB对前列腺癌细胞增殖和活化的影响主要是通过PI3K/Akt信号通路的活化实现的;④以TrkB为靶点的治疗策略有可能成为前列腺癌肿瘤治疗的理想靶点。肿瘤组织中Survivin的高表达可促进肿瘤细胞和肿瘤血管内皮细胞的增殖以及抗凋亡能力。以Survivin为靶点的治疗策略通过抑制其表达或破坏其功能而诱发肿瘤细胞凋亡已经成为共识。我实验室张剑博士在前期研究中通过对以往关于Survivin晶体结构相关文献的分析提出Survivin分子第71位天冬氨酸的突变体可能是一个尚未被实验证实的Survivin显性负突变分子,他通过复制缺陷性腺病毒介导的基因导入方式证明Sur-D71A氨基酸点突变分子具有诱导前列腺癌细胞凋亡的生物学效应。在其基础上,我们通过观察凋亡相关分子Bcl-2家族成员Bcl-2、Bax和Bcl-xl的表达变化,以及Sur-D71A与野生型Survivin分子形成同源或异源二聚体能力的差异分析Sur-D71A对前列腺癌细胞诱导凋亡作用的可能机制。目的:分析Sur-D71A显性负突变分子诱导肿瘤细胞凋亡的可能分子机制。①检测Sur-D71A导入前列腺癌肿瘤细胞对线粒体相关凋亡分子Bcl-2家族成员表达的影响;②观察野生型Survivin分子与Sur-D71A显性负突变分子形成同源二聚体或异源二聚体结合力的差别。方法:①包装、扩增并纯化表达Sur-D71A的复制缺陷性腺病毒,感染前列腺癌细胞后检测Sur-D71A的表达;②利用免疫印迹实验观察Sur-D71A对肿瘤细胞内Bcl-2家族抗凋亡和抑凋亡分子表达的影响;③利用分子生物学方法构建融合标签(flag,HA)的野生型Survivin和Sur-D71A真核表达载体和融合GST的野生型Survivin和Sur-D71A原核表达载体,并在此基础上进行蛋白诱导表达、蛋白纯化和免疫印迹实验检测构建分子的表达状况;④利用免疫共沉淀(co-immunoprecipitation,Co-IP)和GST-pulldown实验检测野生型Survivin分子和Sur-D71A形成同源或异源二聚体结合能力的差别。结果:①成功获得了高滴度表达Sur-D71A的复制缺陷型腺病毒,免疫印迹实验证明Sur-D71A分子可在前列腺癌细胞中正确表达;②免疫印迹实验结果显示Sur-D71A可导致前列腺癌细胞中Bcl-2家族抗凋亡分子Bcl-2和Bcl-xl表达水平的下降和促凋亡分析Bax表达水平的上升;③成功构建融合标签(flag,HA)的野生型Survivin和Sur-D71A真核表达载体和融合GST的野生型Survivin和Sur-D71A原核表达载体,蛋白表达检测方法证明各种分子的表达正确;④利用Co-IP和GST-pulldown实验证明野生型Survivin和Sur-D71A分子在体内和体外均具有类似的二聚体结合能力。结论:①Sur-D71A显性负突变分子通过影响线粒体相关凋亡分子实现诱导肿瘤细胞凋亡的生物学活性;②外源表达的Sur-D71A通过竞争结合的方式抑制了野生型Survivin分子形成同源二聚体从而破坏了野生型Survivin分子抵抗细胞凋亡的生物学活性。更多还原

【Abstract】 The incidence and mortality of adenocarcinoma of the prostate continue to rise and will continue to pose a major public health problem.Prostate cancer is the second most common cause of cancer deaths on men.Despite progress in diagnosis and local therapy,fundmental questions remain with regard to the etiology, prevention,and treatment of prostate cancer.Our knowledge of prostate adenocarcinoma remains significantly less than of most other neoplasm.Finding a more sensitivity marker or oncogenic potential gene will provide us a useful tool to diagnose and cure prostate carcinoma. In our study, we focus our research on the prostate cancer gene therapeutics and the biological function of a novel identified tumor-associated molecule in the program of prostate cancer. The first part of our study is about the molecular mechanism of TrkB, a receptor-like tyrosine kinase, in the program of prostate carcinogenesis. And the second part is about the further mechanistic explanation of how dominant negative form of Survivin—Sur-D71A does induce apoptosis in prostate cancer cells.TrkB, together with TrkA and TrkC are neurotrophin receptors regulating the proliferation and differentiation of neuronal cells. Upon binding to its ligand brain-derived neurotrophic factor (BDNF), TrkB will dimerise, auto- phosphorylate and initiate multiple signal transduction pathways that are involved in neurite growth, morphological plasticity and the synthesis of proteins for differentiated function of neurons and synapses. Recent reports showed that TrkB may also have important fuhnction outside of the central neural system. Though the investigations are limited and the underlying mechanism remains unclear, yet it strongly indicates that TrkB may be involved in the tumorigenesis. In the study of prostate cancer, the relationship between TrkB and carcinogenesis has not been established. In this study, we investigated the expression pattern of TrkB in prostate cancer tissues with different pathological degrees. We performed RNAi technology to knockdown TrkB expression in prostate cancer cell lines and detected the malignant phenotype of the cells. To further analysis the cellular signal pathway mediated by TrkB,we used immunostaining to determine the changes of MAPK and PI3K/Akt signal pathwayhs.Objective:To determine the expression of TrkB in the prostate cancer tissues and to estimate its clinical significance. To investigate the biological function of TrkB in the malignant behavior of prostate cancer cell lines. To prove if TrkB can be a proming target in the prostate cancer gene therapeutics. To detect the change of cellular signal pathway mediated by TrkB in prostate cancer cells. Method:Immunohistochemistry was performed to detect TrkB expression in malignant tumor of prostate and the tissue of benign neoplasia was used as the control. Vector-based siRNA was constructed and the stable transfectants were established. Cell counting assay was used to detect the proliferation rate of prostate cancer cells with a low level of TrkB expresion. And soft agar assay was performed to investigate the transformation capacity of prostate cancer cells with a low level of TrkB expresion. Wester blot was used to detect the change of cellular signal pathway involved in TrkB-mediated tumor growth.Result:TrkB expressed with a much higher level in prostate cancer tissues with comparison to that in the benign neoplasia. And its expression trend was correlated with the malignant degree of the carcinoma. TrkB was mainly expressed on the cellular membrane and the cytoplasma, however, in some samples, TrkB was obviously localized in the nucleus of cancer cells. Its clinical significance was not clear since there is no sufficient tumor tissues to be detected. We successfully constructed the plasmid expression siRNA targeting TrkB. It can effectively downregulate TrkB expression in cancer cells. The proliferation rate and transformation capacity of prostate cancer cells decreased with the downregulation of TrkB by RNAi technology. Downregulation of TrkB decreased the phosphorylation level of Akt kinase while had slight effect on MAPK signal pathway.Conclusion:Receptor-like tyrosine kinase TrkB involved in the program of prostate carcinogenesis. It accelerated the proliferation rate of prostate cancer cells and played an important role in the process of transformation. TrkB-mediated cell survival, proliferation and transformation were mediated by PI3K/Akt signal pathway. And TrkB targeting therapeutics may be a promising strategy for cancer therapy of prostate cancer.Survivn is over expressed in many types of cancers. It plays important roles in cell division and antiapoptosis. Survivin-targeting therapeutics is a proming strategy for the cancer therapy. Our previous study demonstrated that adenovirus-mediated a novel dominant negative form of Survivin, Sur-D71A,increased the apoptosis rate of prostate cancer cells. But its molecular mechanism has not been elucidated. In our study, we investigated the expression pattern of Bcl-2 family members and detected its capacity to form homodimer or heterodimer with itself or wild-type Survivin protein.Objective: To investigated the expression pattern of Bcl-2 family members in prostate cancer cells after infected with the adenovirus expressing Sur-D71A or GFP control. To detect the capacity of Sur-D71A to form homodimer or heterodimer with itself or wild-type Survivin protein. Method: To package, amplify and purify the adenovirus expressing Sur-D71A. Western blot was used to detect the expression of Sur-D71A in prostate cancer cells after infection with adenovirus expressing Sur-D71A. We used western blot to detect the expression of Bcl-2 family members after transduction of Ad-Sur-D71A. We fused Flag-tag or Myc-tag to the terminal of Survivin or Sur-D71A. and also inserted the open reading frame of survivin or sur-D71A into the prokaryotic expression vector pGEX-4T-1.We used coimmunoprecipitation and GST-pull down assay to detect the capacity of Sur-D71A to form homodimer or heterodimer with itself or wild-type Survivin protein.Result: Adenovirus mediated expression of Sur-D71A induced apoptosis of prostate cancer cells by robustly changing the expression patterns of mitochandria-associated Bcl-2 family members. Antiapoptotic molecule Bcl-2 and Bcl-xl were downregulated and proapoptotic molecules Bax was upregulated in prostate cancer cells transfected with Ad-Sur-D71A in comparison with Ad-GFP-infected control or intact cells. Coimmunoprecipitation and GST-pull down assay showed that Sur-D71A had the similar binding affinity to form homodimer with Sur-D71A or heterodimer with wild-type Survivin.Conclusion: Adenovirus mediated expression of Sur-D71A induced obvious apoptosis of prostate cancer cells by disrupting the balance of antiapoptosis maintained by the homodimer of wild-type Survivin in a Sur-D71A dependent competitive binding manner to form heterodimer of wild-type Survivin and Sur-D71A, instead of the functional homodimer of wild type Survivin. And on the other hand, downregulation of Bcl-2 and Bcl-xl and upregulation of Bax also involved in the apoptosis of prostate cancer cells induced by Sur-D71A. This study provided a molecular mechanistic explaination for the Sur-D71A induced apoptosis in cancer cells.更多还原