WT-1基因在肾胚瘤中的表达及其对G401细胞系肿瘤逆转的研究

【作者】 胡兰；

【导师】 任常山；

【作者基本信息】 中国医科大学 ， 肿瘤学， 2002， 博士

【摘要】 前言 肾胚瘤是一种较常见的儿科实体肿瘤，诊断年龄平均为15个月，其中1-5岁者占75％，而90％见于7岁以前。该肿瘤的遗传学基础比较复杂，现已发现有多个染色体位点与其发生有关，其一是定位于染色体11p13的抑癌基因-WT-1，据统计约5-25％的肾胚瘤发生WT-1基因的改变，现在认为WT-1蛋白在细胞增殖、分化及凋亡等方面均有一定作用。现在对于肾胚瘤的治疗主要为放疗、化疗或肾脏切除，但由于肾胚瘤的发病年龄早，放化疗所造成的方法副作用比成年人更大，所以有待寻找一条更为安全有效的治疗途径。 目的 本实验拟检测WT-1基因在肾胚瘤各细胞中的表达情况，然后根据对WT-1和EGR-Ⅰ基因在肾胚瘤中表达情况的统计分析，探讨其在肿瘤发生方面的作用；并且通过RT-PCR扩增产生³²P标记的cDNA探针进行原位杂交，从cDNA文库中筛选含有WT-1基因的克隆，进一步将WT-1基因连到表达载体导人肾胚瘤细胞系G401中表达，分子水平研究G401细胞的生长、凋亡及肿瘤逆转情况，从而为研究WT₁基因的功能提供参考，为基因水平治疗肾胚瘤提供重要的依据。 方法 （1）采用免疫组化法检测61例肾胚瘤石蜡标本上皮细胞、胚基细胞、间质细胞wT一1和ECR一I基因的表达情况，然后通过单人进行半定量估计WT一1和EGR一I在特定位置的阳性细胞数，采用SPSSro.0统计软件包确定wT一1和EGR一I的表达与病理阶段之间的关系，通过犷检验及Kendall色相关分析研究Wl，一1和ECR一I表达的相关性。 （2）提纯胎肾mRNA进行RT一PCR扩增，将Wl，一1 cDNA文库转人感受态大肠杆菌，采用RT一PCR扩增的32P标记的CDNA作为探针进行原位杂交，筛选所需克隆。 （3）利用质粒提取试剂盒提取原位杂交呈阳性的克隆的重组质粒DNA，再经限制性内切酶酶切及纯化，将其与表达载体连接，利用脂质体介导法将WT一1基因导人肾胚瘤以01细胞系，用流氏细胞仪和电泳方法检测细胞凋亡情况，用Westem blot法检测Wl，一l蛋白的表达情况。结果 （l）免疫组化结果表明WT一1蛋白定位于细胞核内，正常肾脏肾小球内皮细胞核染色较深，而肾小管核染色较浅。61例肾胚瘤样品中有36（59%）例的胚基细胞呈阳性;而EGR一I蛋白定位于细胞质，正常肾脏的近曲小管和远曲小管EGR一I免疫组化胞质染色均较深，而肾小球染色较浅。肾胚瘤的胚基、上皮、间质成分均可发生EGR一I免疫反应。61例肾胚瘤样品中有56%样品胚基发生EGR一I免疫反应，87%样品的上皮细胞发生免疫反应，但间质细胞EGR一I表达较少。与wT一1基因相比，EGR一I基因表达更广泛，而且染色更深，胚基表达EGR一I蛋白水平与病理阶段相关，统计分析表明胚基中WT一1和EGR一I的表达明显相关。 （2）提取胎肾mRNA经RT一PcR扩增产生一32P标记的289bp的扩增产物，此序列为WT一1基因C一末端锌指区的一段。我们将购买的人胎肾cDNA文库转化人感受态大肠杆菌JM109，经氨节青霉素及X一列和IPTG筛选可见有转化成功的白色菌落，出现白斑比率为85%，利用RT，-- PCR扩增产物作为WT一1基因cDNA探针对阳性菌落进行原位杂交，结果在4个菌落中均出现放射自显影阳性结果。 （3）提纯原位杂交筛选出的阳性菌落重组质粒DNA，并进行Ec0RI和Nod酶切，电泳结果显示载体上有两个插人片段，其一约0.skb，其二约1.skb。我们回收了大片段，将其连接到表达载体，然后导人肾胚瘤G401细胞系，经药物筛选得到阳性克隆。应用流式细胞仪分析细胞凋亡情况，结果表明导人Wl，一1基因的以01细胞和未导人WT一l基因的G401细胞的凋亡细胞比例分别为9 .8%和6.1%，Westem杂交结果显示导人WT一l基因后细胞中有Wl，一1基因表达，而在未导人此基因的细胞中没有WT一1基因的表达。讨论 （l）本研究结果表明，在肾胚瘤的胚基细胞和上皮细胞中均有较高水平的WT一1和EGR一I的表达，而在间质成分中表达水平较低。这些结果表明肾胚瘤发生的分子基础有一定区别，间质成分为主的肿瘤可能VZF一1基因的表达完全缺失。这说明WT一1和EGR一I只在特定的细胞中或在特定的分化阶段表达;肿瘤内W下T一l的表达水平可能和肿瘤·内存在的某种细胞的相对数量有关;以胚基和上皮细胞为主的肿瘤比以间质细胞为主的肿瘤的Wl，一1和EGR一I表达水平高。最近有人通过RNA印迹杂交分析，也表明肾胚瘤中EGR一I高表达。当然，在不同肿瘤中的EGR一I所起的作用可能完全相反，这可能归因于细胞内环境的影响或特定细胞内其它蛋白质的存在等原因。肾胚瘤中Wl，一1基因的表达和EGR一I基因的表达存在一定的相关性，特别是在胚基细胞中呈负相关关系。最近十年，国外已有学者在mRNA水平、临床表现、组织学及分子特性等方面研究了Wl，一1和EGR一I的相互作用，有人认为WT一l和EGR一I均连于相同的DNA反应元件中，这表明可通过二者竞争结合于DNA靶分子来刺激肾胚瘤的生长。尽管这种作用机制还不完全清楚，但现在公认wT一1和EGR-I在肾脏中可通过一种或更多条途径刺激肿瘤的发生。 （2）通过流式细胞仪和电泳分析，结果均表明将wT一l基因导人后，凋亡细胞数目有所增加，另外通过用裸鼠研究以01的致瘤?更多还原

【Abstract】 Wilms tumor is one of the most common solid tumors in children. The Wilms’ tumor 1 gene ( WT -1) has been identified as a tumor suppressor gene involved in the etiology of Wilms’ tunior. Approximately 5-25% of all Wilms’ tumors carry mutations in the WT, gene. Alterations in the WTj gene have also been observed in other tumor types, such as leukemia, mesothelioma and desmoplastic small round cell tumor. Dependent on the tumor type, WT - 1 proteins might either function as tumor suppressor proteins or as survival factors. The Wilms tumor suppressor gene WT, encodes a zinc finger protein, expressed as different splicing variants, that has all the hallmarks of a transcription factor. The - KTS form of WT, displays a homogeneous localization within the nucleus and has been shown to activate or repress the activity of various target genes. In contrast, the WT, ( + KTS) variant demonstrates a speckled pattern of expression within the nucleus. This and its association with factors of the splicing machinery has led to the hypothesis that WTt ( + KTS) might play a role in post -transcriptional processes.Mutations in the WT -1 gene can also result in congenital abnormalities as observed in Denys - Drash and Frasier syndrome patients. Mouse models have proven the critical importance of WT - 1 expression for the development of several organs, including the kidneys, the gonads and the spleen. The WT -1 proteins seem to perform two main functions. They regulate the transcription of a variety of target genes and may be involved in post - transcriptional processing of RNA. These isoforms have partially distinct biological functions and effects, which in many cases are also specific for the model system in which WT - 1 is studied. This discusses the molecular mechanisms by which the various WT, isoforms exert their functions in normal development and how alterations in WT - 1 may lead to developmental abnormalities and tumor growth. , Nephroblastomas ( Wilms’ tumors) are curable with survival rates about 80%. Wilms’ yumor pa-tients can now be achieved using combination therapy with chemotherapy, surgery, and in some cases radiotherapy. But these methods can produce more damages to children than to adults. So we hope a new approaches to therapy Wilms’ tumor patients. In addition to there is little research about the biology of nephroblastoma in our country, so many work need to do.PURPOSES( 1) Our purposes is to study the expression of both genes WT - 1 and EGR - I in Wilms’ tumor using an immunohistochemical approach. We analysis the expression of both genes in normal kidney tissues , in the blastema! , epithelial component of Wilms’ tumor and stromal tissue . We hope to detect the association between the expression of WT - 1 and EGR -1 in the three cells of Wilms’ tumor and the clinical progression. morever,We study the relationship between WT - 1 and EGR -1 expression in clinical nephroblastomas. (2) We want to get 32P labled cDNA probe by Reverse Transcription - PCR , and then find WT - 1 gene through in situ blot with the the colon of cDNA libarary of embryomic kidney tissue. (3) To determine whether Wilms’ tumor cell lines are suppressed by WT - Igene, we performed chromosome transfer experiments : transfect WT -1 gene into the pediatric kidney - derived cell line G401. we want to Detecting the effects on the population doubling times of the cell line, proliferative capacity , the expression of WT - 1 gene in G401 cell line,and the apoptosis of G401.METHODS(1) Immunohistochemistry: The following primary antibodies were used: a mouse monoclonal antibody against WT - 1; and rabbit polyclonal antibody a-gainst, The peroxidase - antiperoxidase technique was used. Serial sections from all samples which were incubated overnight, in a 60 incubator. After dewa-xing in fresh xylene for 10 min and rehydration in 100% methanol for 10 min, the sections were rinsed in methanol containing 3% hydrogen peroxide for 20min to block endogenous peroxidase activity. Slides were incubated in PBS ?5% BSA for 15 min a更多还原