【作者】 刘冉录；

【导师】 徐勇； 张志宏；

【作者基本信息】 天津医科大学 ， 外科学， 2007， 博士

【摘要】 前列腺癌是欧美国家男性人群最常见的恶性肿瘤。在我国，随着人口老龄化的发展，饮食结构等因素的变化，前列腺癌发病率及死亡率呈明显上升趋势。前列腺癌已经成为一个重要的公共健康问题，前列腺癌对放疗、化疗均不敏感，激素剥夺疗法容易变为难治性前列腺癌，特别是对于局部及全身转移的进展期患者，基因治疗是一种有希望的治疗方法。前列腺癌基因治疗，从引起前列腺癌的致病基因入手，意在从根本上治疗肿瘤，已经成为研究的热点，各国科学家也在不断的探索新的靶基因。核干因子(nucleostemin，NS)是新近发现的一个p53结合蛋白，研究认为NS基因表达于干细胞和肿瘤细胞，在已分化的成体组织中不表达，NS基因在维持干细胞和多种癌细胞的增殖和自我更新中发挥重要作用。NS则可能是干细胞和癌细胞中重要的共同调节基因，已经证明在前列腺癌中存在肿瘤干细胞(tumorstem cells，TSCs)，TSC与前列腺癌的发生发展密切相关，因此对NS基因功能及其调节途径的研究对前列腺癌的诊断与治疗有重要意义。然而，NS基因在前列腺癌中是否也存在表达，其在前列腺癌发生、增殖过程中的作用及作用机制如何，尚不清楚。因此，本研究将检测NS基因在前列腺癌中的表达，并利用RNA干扰及表达谱基因芯片技术初步探讨其作用机制。第一部分前列腺癌组织及细胞系中Nucleostemin基因表达的检测目的揭示NS基因在前列腺癌组织及细胞系中的表达水平，探讨NS基因水平与前列腺癌患者临床指标的关系，并作为后续研究的基础。方法利用RT-PCR、蛋白印迹及免疫组化等方法对前列腺癌组织及PC-3、LNCap及DU145细胞中NS基因的表达进行检测，并探讨NS基因表达水平与前列腺癌患者临床指标的关系。结果前列腺癌标本及PC-3、LNCap及DU145细胞中都表达NS基因，NS基因在前列腺癌中的表达水平显著高于前列腺增生中的水平，NS基因表达水平与前列腺癌的分化程度呈负相关，细胞分化程度越差，NS基因表达水平越高。结论前列腺癌中高表达NS基因，NS基因在前列腺癌的不良分化和恶性增殖中可能起着重要作用。第二部分Nucleostemin基因表达水平下调对PC-3细胞增殖能力的影响目的探讨NS基因与前列腺癌恶性增殖的关系，为前列腺癌的基因治疗探索新的靶基因。方法通过shRNA质粒表达载体沉默PC-3细胞中NS基因的表达，研究NS基因水平下调后PC-3细胞形态、周期、增殖能力及凋亡的变化。结果NS基因在PC-3细胞中的表达水平下调后，细胞胞膜边缘突起增多，更趋向于分化；S期细胞的百分率降低，G1期的百分率升高；细胞的体外增殖速率明显降低，在裸鼠体内致瘤能力、肿瘤的生长速度及肿瘤的最终体积和重量也明显降低，细胞凋亡增多。结论NS基因可能是前列腺癌PC-3细胞周期中G1／S检查点的一个重要的调节因子，在PC-3细胞恶性增殖中发挥重要作用，NS可能是前列腺癌基因治疗的理想靶基因之一。第三部分PC-3细胞Nucleostemin基因下调后全基因组表达谱的变化分析目的筛选与NS相互作用的基因及可能的信号通路，探讨NS基因在前列腺癌中的作用机制。方法利用表达谱基因芯片技术分析PC-3细胞NS基因下调后全基因组表达谱的变化，筛选与NS相互作用的差异基因及信号通路，并利用实时荧光定量PCR对重要差异基因进行验证。结果筛选出了219个差异表达的基因，这些基因涉及到细胞周期、细胞增殖、信号转导、细胞凋亡及细胞分化等多个方面，重要差异基因被实时荧光定量PCR进一步证实。NS基因下调后主要引起INK4家族基因(P15，P16，P18)的上调和cyclin D1及HDAC1基因的下调，主要作用点位于CDK4／6-Cyclin D和pRb-E2F1复合体。结论在基因水平上证实了NS基因是细胞周期G1／S检查点的重要调控因子，在含突变型p53的前列腺癌中，NS可能主要是通过抑制p15、p16、p18等INK4家族的肿瘤抑制基因促进肿瘤的发生发展。发现了许多NS所涉及的肿瘤相关基因，但目前的工作仍不能完全揭示NS的信号通路，仍需进一步的实验去发现和证实。更多还原

【Abstract】 Prostate cancer is the most common primary malignant tumor among malepopulation in the European and American countries. In China, the incidence andmortality rate have increased significantly due to population aging, changes in lifestyles and other causes. Prostate cancer has become a world-wide health problemwhich is not so sensitive to radiotherapy and chemotherapy, and many cases willbecome hormone-refractory prostate cancer when treated with hormone deprivation.Therefore, the gene therapy is a promising method, especially for the patients withlocal or systemic metastasis. The gene therapy for the treatment of prostate cancer,which targets the virulence genes of prostate cancer to cure this diseasefundamentally, has become the focus of research and many Scientists are nowexploring the new target genes.Recently, a p53-binding protein, named nucleostemin (NS) exclusively expressedin the stem cells and cancer cells but not in differentiated adult tissues and cells, hasbeen shown to be essential for the proliferation and self-renewal of stem and cancercells. NS may be the common controlling gene of stem and cancer cells. Tumor stemcells (TSCs) which are thought to be related to the carcinogenesis of prostate cancerhave been identified in prostate cancer. Therefore, studies on the function andpathway of NS are of great importance to the diagnosis and treatment of prostatecancer. However, it is currently unknown whether NS is expressed in prostate cancerand how this newly identified molecule involved in prostate cancer pathogenesis. Thepresent study will detect the expression level of NS in prostate cancer andinvestigate its mechanism by RNA interference and DNA microarray technology. PartⅠDetection of NS expression in prostate cancer tissuesand cell linesObjective To reveal the NS expression level in prostate cancer tissues and celllines and investigate the correlation between NS gene level and clinical variables ofprostate cancer, taking this part as a base for later study. Methods RT-PCR,western blot and immunohistochemistry were used to detect the NS mRNA andprotein level in prostate cancer tissues and PC-3, LNCap and DU 145 cells, analyzingthe correlation between NS protein level and clinical variables of prostate cancer.Results NS gene was expressed in all the detected samples and cell lines, the NSmRNA and protein level in prostate cancer tissues were significantly higher thanthose in BPH tissues. NS protein level was negatively correlated with the degree ofcell differentiation in prostate cancer tissues, the worse the differentiation, the higherthe NS protein level. Conclusion NS gene is highly expressed in prostate cancerand may be of great importance to the adverse differentiation and malignantproliferation of prostate cancer.PartⅡThe effect on the proliferation of PC-3 cells afterdown-regulating the NS gene levelObjective To investigate the relationship between the NS gene and themalignant proliferation of prostate cancer and explore the new target gene for prostatecancer gene therapy. Methods NS-specific short-hairpin RNA (shRNA) expressionplasmid was used to transfect PC-3 cells. The changes of cell morphology, cell cycle,proliferation ability and apoptosis were studied after down-regulating the NS genelevel. Results The cells became larger and showed more pseudopodia, having atendency to differentiate, the detection of cell cycle showed a decrease of S stage andan increase of G1 stare, cell proliferation ability in vitro and tumorigenesis ability in nude mice were discounted, the final tumor volume and weight were also decreasedafter knocking down NS gene. Conclusion NS may act as an important G1/Sregulator to regulate the malignant proliferation of prostate cancer PC-3 cells. NSmay serve as an ideal therapeutic gene target for prostate cancer.PartⅢGene profiling after knocking-down the expression of NSgene in PC-3 cellsObjective To screen the genes and possible signal transduction pathways withwhich NS interact and explore the mechanism of NS in pro.state cancer. MethodsOligonucleotide DNA microarray was used to screen the genome changes afterknocking down expression of NS in PC-3 cells and real-time quantitative PCR wasused to further confirm the important differentially expressed genes. Results 219differentially expressed genes were found and theses genes were involved in cellcycle, cell proliferation, signal transduction, cell apoptosis and cell differentiation.Some important differentially expressed genes were further Certified using real-timequantitative PCR. INK4 family genes (P15, P16, P18) were up-regulated and cyclinD1, HDAC1 were down-regulated, the main action points were CDK4/6-Cyclin Dand pRb-E2F1 complexes. Conclusion NS is an important G1/S checkpointregulator and its regulatory activity is certified at the gene level. Many tumor-relatedgenes are found involving in the NS-regulating pathways. However, the present studystill can not completely reveal the NS signal pathways and more studies are needed tofurther discover and confirm other mechanism.更多还原