【作者】 包世新；

【导师】 叶章群； 杨为民；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 前列腺癌(Prostate Cancer, PCa)是男性最常见的恶性肿瘤之一,在不同的国家和地区,PCa的发病率和死亡率差别很大。随着我国老龄化人口比例逐渐增高,前列腺癌发病率有逐年上升的趋势。近来研究表明,细胞因子白细胞介素-6(interleukin-6,IL-6)在PCa发生发展的过程中发挥了重要作用,其启动子区的单核苷酸多态性与PCa的发生发展之间存在着相关性。本研究检测了IL-6基因及蛋白在前列腺癌及其细胞系中的表达；构建携带基因IL-6-shRNA特异性腺病毒表达载体并将其感染人前列腺癌PC-3细胞系,观察IL-6-shRNA对PC-3细胞生长及凋亡的影响；对湖北地区汉族人群中前列腺癌患者IL-6基因启动子区的单核苷酸多态性(single nucleotide polymorphisms, SNPs)进行分型,以探讨IL-6基因SNP与湖北地区汉族人群前列腺癌的关系。分四个部分简述如下。第一部分IL-6蛋白及基因在前列腺癌中的表达及意义目的：探讨细胞因子白细胞介素-6(IL-6)在前列腺癌发生发展中的作用及其临床意义。方法：①采用免疫组化SABC法和逆转录-聚合酶链反应(RT-PCR)法对前列腺癌组织及其相应的癌旁前列腺组织和前列腺癌细胞系PC-3及LNCaP中的IL-6表达进行检测；②采用酶联免疫吸附试验(ELISA)检测前列腺癌患者及随机正常人群外周血中IL-6的浓度和前列腺癌细胞系PC-3及LNCaP培养上清液中的IL-6浓度。结果：①前列腺癌组织中IL-6表达明显强于相应的癌旁前列腺组织,且与肿瘤的分期分级相关；PC-3细胞中IL-6呈强阳性表达,而LNCaP细胞中IL-6呈弱阳性表达。②前列腺癌患者外周血中IL-6浓度显著高于正常人群组；而PC-3细胞组培养上清液中IL-6浓度也明显高于LNCaP细胞组。结论：IL-6基因可能在前列腺癌的发生发展中起重要作用,有可能是前列腺癌从激素依赖性转化为激素非依赖性的因素之一。第二部分携带基因IL-6-shRNA特异性腺病毒表达载体的构建与鉴定目的：构建并鉴定含有基因IL-6-shRNA的腺病毒表达载体,感染前列腺癌细胞PC-3,并检测重组质粒对IL-6的沉默效应,为后期实验提供靶细胞。方法：分别设计三条以基因IL-6mRNA为靶序列的shRNA和阴性对照序列(HK),应用基因重组技术将其克隆到真核表达载体pGensil-1中,构建的3条重组shRNA真核表达质粒分别命名为pGensil-1_IL-6-shRNA1、_IL-6-shRNA2和_IL-6-shRNA3。将重组质粒转化到大肠杆菌DH5α中,经筛选后对提取质粒进行酶切和测序鉴定。测序正确后,利用腺病毒载体系统Adeno-X构建重组腺病毒质粒rAd-IL-6-shRNA1-3,酶切、PCR鉴定重组腺病毒质粒,鉴定正确后,将rAd-IL-6-shRNA1-3转染至HEK293细胞,包装成复制缺陷型腺病毒质粒rAd-IL-6-shRNA1-3。大量扩增rAd-IL-6-shRNA1-3,测病毒滴度。将构建的重组质粒感染到PC-3细胞中,感染72小时后,应用RT-PCR及Western-blot鉴定IL-6mRNA和蛋白的表达变化。观察各组质粒对IL-6mRNA和蛋白的抑制效率,筛选最有效的干扰序列。结果：酶切分析、测序鉴定表明rAd-IL-6-shRNA1-3构建成功；转染PC-3细胞后,RT-PCR及Westen blot结果显示,pGensil-1-IL-6-shRNA3干扰效果最强。结论：携带有IL-6-shRNA基因的重组腺病毒质粒构建成功,并筛选出pGensil-1-IL-6-shRNA3为沉默效应最强的质粒,它能有效抑制IL-6基因表达；建立了rAd-IL-6-shRNA感染人前列腺癌细胞PC-3的长期稳定细胞系,为进一步探讨IL-6在前列腺癌生物学行为中的作用奠定了基础。第三部分IL-6-shRNA对前列腺癌细胞PC-3生物学行为的影响目的：观察shRNA沉默IL-6基因对前列腺癌细胞PC-3增殖和凋亡的影响,初步探讨IL-6基因在前列腺癌发生和进展过程中的作用。方法：实验分为三组：实验组(PC-3/IL-6-shRNA组,感染rAd-IL-6-shRNA)、空白对照组(PC-3组,未感染腺病毒质粒)和阴性对照组(PC-3／HK组,感染rAd-HK)。利用MTT法细胞计数并绘制细胞生长曲线；Hoechst33258染色、流式细胞仪检测细胞凋亡情况；细胞迁移实验观察感染后细胞侵袭能力的变化。结果：MTT法观察显示,感染rAd-IL-6-shRNA后细胞生长增殖速度较对照组明显减慢；Hoechst33258染色显示部分细胞呈现凋亡形态学改变；流式细胞仪定量检测表明感染IL-6-shRNA基因后,PC-3细胞出现较为明显的凋亡峰；细胞迁移力显著低于对照组。结论：通过RNA干扰沉默IL-6基因的表达能有效地抑制PC-3细胞的生长增殖和迁移,并诱导细胞的凋亡。IL-6基因可能在前列腺癌发生和进展过程中扮演重要角色,抑制IL-6基因的表达可能成为前列腺癌基因治疗的可行策略。第四部分白细胞介素-6基因单核苷酸多态性与前列腺癌的关系目的：探讨白细胞介素-6基因启动子区-174G／C、-634C／G位点的单核苷酸多态性与湖北地区汉族人群前列腺癌的关系。方法：采用TaqMan探针荧光定量PCR技术对IL-6基因启动子区-174G／C、-634C／G位点的单核苷酸多态性进行基因分型,观察前列腺癌组和正常对照组的基因型频率、等位基因频率及其患病风险。结果：①所有实验对象-174G／C位点的基因型均为GG型,无CG型及CC型存在；②前列腺癌组与正常对照组-634C／G位点的GG、CG和CC基因型频率及其G和C等位基因频率差异显著(P<0.05)；且GG+CG基因型频率随临床分期及病理分级的增加而增加。结论：IL-6基因启动子区-174G／C位点在湖北地区汉族人群中可能不存在单核苷酸多态性；而-634C／G位点的单核苷酸多态性与前列腺癌的发生发展具有一定程度的相关性,具有GG+CG基因型的人患前列腺癌的危险性相对较高。更多还原

【Abstract】 Prostate cancer (PCa) is one of the commonest male malignancies worldwide and its mortality and morbidity vary greatly according to countries and regions. With increase of aging population, the incidence of PCa has been on the rise over years. Recent studies showed that interleukin-6 (IL-6) plays a critical role in the development and progression of PCa and the polymorphisms of the single nucleotide at its promoter region are intimately correlated with the development and progression of PCa. This study examined the expressions of IL-6 gene and protein in PCa cancer and their cell lines. A specific adenovirus expression vector with IL-6-shRNA and was constructed and introduced into the PC-3 cell line of human PCa in order to observe the effect of IL-6-shRNA on the growth and apoptosis of PC-3 cells. The polymorphisms of single nucleotide located at the promoter region of IL-6 gene in the Han People with PCa living in Hubei region were analyzed with an attempt to examine the relationship between the single nucleotide polymorphisms (SNPs) and development of PCa in Han People in Hubei region. The study falls into four parts.PartⅠ. The expression of interleukin-6 in prostate cancer and its clinical significanceObjective:To study clinical significance and the relationship between the interleukin-6(IL-6) and disease progression in prostate cancer.Methods:①Immunohistochemistry and RT-PCR were used to detect the expression of IL-6 protein and mRNA in frozen prostatic adenocarcinoma,adjacent benign prostatic tissue and prostate cancer cell lines such as PC-3 and LNCaP.②The serum levels of IL-6 in patients with prostate cancer and healthy control and the supernatants of prostate cancer cell cultures were measured by using ELISA.Results:①The IL-6 protein levels in prostate cancer tissue and PC-3 cell were significantly higher than those in adjacent benign prostatic tissue and LNCaP cell.②The serum IL-6 levels in the patients with prostate cancer were markedly higher than those in the healthy control.The IL-6 levels in supernatants in PC-3 cell were notably higher than those in the LNCaP cell.Conclusions:The IL-6 gene may act as an important regulator in prostate cancer progression and may be one of the causes of prostate carcer conversion from an initially androgen-dependent state into an androgen-independent state.PartⅡ. The construction and identification of Adenovirus expression vector targeting IL-6-shRNA geneObjective:To construct and identify adenovirus vector carrying shRNA targeting IL-6(rAd-IL-6-shRNA) and to establish a monoclone PC-3 cell line transfected with plasmid IL-6-shRNA, and detect its influence on expression of IL-6.Methods:To design 3 pairs of shRNA sequence targeting IL-6 and a pair of shRNA sequence making negative control(HK) respectively, and clone them into pGensil-1 vector by using technology of gene recombination. The recombinants were named pGensil-1_IL-6-shRNA1-、_ IL-6-shRNA2 and_ IL-6-shRNA3. They were transfected into Bacterium coli DH5a, and extracted with nucleic acid purification Kits. The sequence of recombinant plasmids was evaluated by electrophoresis and DNA sequencing. The adenovirus plasmid rAd-IL-6-shRNAl-3 was constructed by adeno-X expression system, and the control adenovirus plasmid rAd-HK was constructed by the same system. After verification by enzymolysis and PCR, the rAd-IL-6-shRNA1-3 vector was cotransfected into 293 cells where they were packed as the replication-deficient adenovirus rAd-IL-6-shRNAl-3. rAd-IL-6-shRNA1-3 was abundantly amplified and then virus titer was evaluated. RT-PCR and Western Blot were used to detect the IL-6 mRNA and protein expression in PC-3 cell lines.Results:rAd-IL-6-shRNA was successfully constructed and verified by enzymolysis and sequencing.RT-PCR and W-B analysis showed that the silencing effect of pGensil-1-IL-6-shRNA3 was more evident than other recombinant vectors.Conclusions:Successful construction of the recombinant adenovirus vector containing the IL-6-shRNA gene was achieved, pGensil-1_IL-6-shRNA3 was selected for the further study as the most effective plasmid in silencing expression of IL-6. A PC-3 cell line transfected with rAd-IL-6-shRNA was obtained.PartⅢ. The Effects of IL-6-shRNA Gene on the Biological Behaviors of Prostate Cancer Cell (PC-3) in VitroObjective:To investigate whether the expression of gene IL-6-shRNA can effect the growth, proliferation and apoptosis of prostate cancer PC-3 cells in vitro. and research their inter-relation between IL-6 and tumorigenesis of prostate.Methods:The whole experiment was divided into 3 groups. The rAd-IL-6-shRNA was used to infect the human prostate cancer PC-3 cells. For control experiments, the vector rAd-HK was also transfected into PC-3 cells and nontransfected PC-3 cells. The effect of IL-6-shRNA on the cellular proliferation capacity of PC-3 cells was assayed by the growth curve. The cell apoptosis was detected by Hoechst33258 staining, and flow cytometry analysis. The cell migration assay was used to detect the difference of invasion and metastases between transfected and non-transfected cells.Results:MTT showed the cell proliferation was markedly inhibited compared with the control cells. Partial cancer cells presented morphological changes of apoptosis by Hoechst33258 staining and flow cytometry analysis. Contrast to control cells and rAd-HK transfected PC-3, the invasion ability of rAd-IL-6-shRNA transfected cells decreased obviously.Conclusion:IL-6-shRNA gene can suppress PC-3 cell growth and proliferation, in addition to acceleration of its apoptosis in vitro. IL-6 gene might play a crucial role in the development and progression of prostatic cancer and suppression of IL-6 gene expression might be a promising strategy for the treatment of human prostate cancer.PartⅣ. Relationship between Single Nucleotide Polymorphisms in Interleukin-6 and Prostate CancerObjective:To examine the association between the single nucleotide polymorphisms (SNPs) in-174G/C and-634C/G of interleukin-6(IL-6) promoter region and prostate cancer in the population of Han People in Hubei region.Methods:TaqMan PCR was employed for the gene-typing of-174G/C and-634C/G in promoter region of IL-6 gene to compare the prostate cancer patients and normal controls in terms of genetype frequency, allele frequency and risk of prostate cancer.Results:In all the subjects, the genetype of genetic locus-174G/C was found to be GG and no CG and CC were found. There were a significant difference in gene frequency of GG, CG and CC of-634C/G and allele frequency of G and C between prostate cancer patients and normal controls (P<0.05) and the gene frequency of GG+CG increased with the clinical stages and pathological grades of prostate cancer.Conclusion:No SNP in-174G/C IL-6 promoter region was found in the population of Han People in Hubei region. The SNP in-634C/G was, to some extent, associated with the development and progression of prostatic cancer. The population with GG+CG genetype have higher risk of prostate cancer.更多还原