【作者】 贾万健；

【导师】 张元芳；

【作者基本信息】 复旦大学 ， 外科学， 2004， 博士

【摘要】 背景 同源框基因( Homeobox Genes)最早在果蝇中发现,存在于酵母到人类几乎所有的真核生物细胞中。其含有称为同源框(Homeobox)的 183bp 高度保守的核苷酸序列,是真核生物转录因子。同源框基因对胚胎发育有重要的调节作用,近年的研究发现一些肿瘤的发生、发展与调控细胞分化的同源框基因家族的异常表达密切相关。同源框基因 Nkx3.1 定位于小鼠 14 号染色体的中间区,与 NK 家族的 NK3 有最高同源性。人类 Nkx3.1 写作 NKX3.1, cDNA 全长 3266bp,开放阅读框(ORFs)705bp,定位于 8p21,正好是前列腺癌发病时频繁发生丢失的“热斑区”。研究表明,NKX3.1 几乎只局限于前列腺组织表达,特异度超过前列腺特异抗原 PSA;其 mRNA 转录水平受雄激素调节;对前列腺器官的发生、分化、发育及成熟器官的功能维持有重要作用。另外,有实验提示 NKX3.1 表达缺失与激素抵抗性前列腺癌和晚期前列腺癌关系密切;Nkx3.1 可抑制其表达缺失株前列腺癌细胞体外生长,接种裸鼠后其肿瘤生长能力明显降低; Nkx3.1 失功能的Nkx3.1 突变基因工程化鼠增龄饲养,发现有前列腺上皮内瘤(PIN)产生,类似于人类组织发现;实验中 Nkx3.1 与抑癌基因 PTEN 同时丧失功能的小鼠发生了前列腺癌,并证实淋巴结转移,提示 Nkx3.1 功能丧失是前列腺癌发生过程中的严重事件――因而,Nkx3.1 可能是新的前列腺组织特异的候选抑癌基因。NKX3.1有正常变异但未发现影响其功能,前列腺癌病理组织中亦未发现其编码区存在变异,因而其失活方式可能是表达缺失而非变异。PTEN 是近年来发现的“广谱”抑癌基因,定位于 10q23,此区在进展期前列腺癌频繁发生缺失,因而 PTEN 被认为在前列腺癌发病中是一个中央调节因子。 本课题拟采用免疫组化技术,检测一组前列腺恶性肿瘤中前列腺特异性同源框基因 NKX3.1 与 PTEN 基因的表达情况,并分析两者间可能存在的关联。并采用目前分子生物学实验室较为成熟与常用的脂质体介导的真核基因转移技术,利用携带有 NKX3.1 基因 cDNA(OFRs)全长序列的真核基因表达载体 pcDNA3.1(+)-NKX3.1,稳定转染 NKX3.1 表达缺失的人雄激素非依赖型前列腺癌细胞株 PC3,获得稳定表达 NKX3.1 蛋白的 PC3-NKX3.1 细胞株,观察 NKX3.1基因转染对 PC3 细胞的生物学效应。进一步进行裸鼠肿瘤接种,观察 NKX3.1基因转染后 PC3 裸鼠实验动物致瘤能力的变化,探讨 NKX3.1 基因的生物学功能与意义,以及为未来其可能的作为前列腺癌肿瘤标志物与基因治疗靶点的潜在价值提供实验依据。第一部分 前列腺特异性同源框基因 NKX3.1 与抑癌基因 PTEN 在前列腺癌组织 中表达的免疫组化研究 目的 免疫组化方法检测前列腺恶性肿瘤与良性前列腺增生组织标本中前列腺同源框基因 NKX3.1 与抑癌基因 PTEN 的表达情况,并分析二者间的相关性。 材料与方法 前列腺恶性肿瘤标本 31 例,其中腺癌 30 例,肉瘤 1 例;良性前列腺增生标本 10 例。 所有标本均病理检验证实诊断,两步法分别行 NKX3.1 与 PTEN 抗体的免疫组化染色,HRP 标记的二抗加 DAB 显色;光镜下观察,阳性反应为预期的细 1<WP=5>胞定位处出现棕黄色染色,高倍镜下阳性细胞数>50%时为强阳性(++)。SPSS11.0 统计软件进行统计分析,行χ2检验并分别计算 Kappa 系数与 Gamma系数。 结果 NKX3.1 与 PTEN 在前列腺恶性肿瘤标本中阳性率总分别为 48.39%与 29.03%,良性前列腺增生为 70%与 50%,与文献报道中数据相近。Kappa 系数与Gamma 系数均提示 NKX3.1 与 PTEN 基因在本组中的表达未发现明确相关性。 结论 本研究采用免疫组化方法检测的前列腺恶性组织标本中前列腺特异性同源框基因 NKX3.1 与抑癌基因 PTEN 的表达与文献报道相近,但未发现二者的表达之间存在明确的相关性。更多还原

【Abstract】 BACKGROUND: Homeobox genes were firstly found in Drosophila Bagpipe, exist broadly in thecreatures from yeast to human being. Containing a 183bp ribonucleotide fragmentnamed Homeobox, all homeobox genes play a role of eukaryotic transcriptional factor.Homeobox genes are important regulators in embryogenesis, recent findings suggestthat they are closely related to some malignant neoplasm’s genesis, development, asregulator of cell differentiation. Homeobox genes Nkx3.1 maps to mid-region ofmouse chromosome 14, has topmost homogeneity with homeobox gene NK family.Human’s Nkx3.1 has been written as NKX3.1,with 3266bp full cDNA length, 705bpopen reading frame(ORFs),maps to chromosome 8p21,just the “hot spot”superposition site that frequently lost in prostate carcinoma. Researches suggest thatNKX3.1 nearly only expresses in prostate tissue, has higher specificity than prostatespecific antigen(PSA);its mRNA transcriptional level is regulated by androgen; playsa important role in the genesis, differentiation, development and function maintenanceof prostate organ. In addition, some experimental results indicate that depletion ofNKX3.1 expression is strongly related with hormone-resistance prostate carcinomaand late phase prostate cancer; in vitro Nkx3.1 can suppresses the growth of Nkx3.1loss-of-expression prostate carcinoma cell, tumor growth ability decreases afterinoculation into nude mouse; in those enhanced-age Nkx3.1 null mutant mice inducedby genetic engineering method, prostatic intraepithelial neoplasia(PIN) has beenfound, and resemble the findings in human tissue; in experiments, the mice ofcompound loss-of-function of Nkx3.1 and PTEN genes developed invasive prostaticcarcinoma, and lymph node metastasis were confirmed; these demonstrate thatNkx3.1 loss-of-function is a critical event in prostate cancerinitiation.— herein,Nkx3.1 may be a new prostate tissue specific candidatetumor-suppressing gene.NKX3.1 normal mutation doesn’t influence its function, itscoding region hasn’t been found mutation in prostate cancer pathological tissue, so itsloss-of-function manner may be expression depletion but not mutation. PTEN is abroad spectrum tumor-suppressing gene found in recent years, maps to 10q23,afrequently lost in progressing-phase prostatic cancer, thus PTEN has been regarded asa central regulator in prostatic carcinogenesis. This study plans to use immunohistochemical technique, to examine expression ofprostate specific homeobox gene NKX3.1 and tumor-suppressing gene PTEN in aserial specimen of prostate malignant tumors, and probe into the relationship betweenthese two genes; by mature liposome-induced gene transferring technique currentlybroadly used in molecular biology labs, using a eukaryotic gene expression vectorpcDNA3.1(+)-NKX3.1 which carrying NKX3.1 cDNA(ORFs) full length sequences,we plans to transfect NKX3.1 loss-of-expression human androgen-independentprostatic cancer cell line PC3, obtain the cell line PC3-NKX3.1 which stablyexpresses NKX3.1 protein, and investigate the biologic effects that derived fromNKX3.1 transfection. Moreover, we will inoculate nude mice with these cells, to 5<WP=9>explore the change of tumor-growth ability in nude mice, and discuss biologicfunction and significance of NKX3.1 gene, and to provide experimental evidence forits possible utilities as a tumor marker and gene-therapy target site in the future.Part I Immunohistochemical Research on the Expression of Prostate Specific Homeobox NKX3.1 & Tumor Suppressing Gene PTEN in Prostatic Carcinoma TissuesOBJECTIVE To investigate the expression of prostatic specific homeobox gene NKX3.1 andtumor suppression gene PTEN in prostate malignant tumors and benign prostatehyperplasia tissue specimen by immunohistochemical assay method, and analyse therelationship between the two genes.MATERIAL AND METHODS Prostate maligna更多还原