抑制Polo样激酶1选择性阻断RAS突变膀胱肿瘤的生长及患者源性膀胱肿瘤动物模型的初步建立

发布时间：2018-03-28 20:09

本文选题：膀胱癌　切入点：RAS　出处：《华东师范大学》2014年硕士论文

【摘要】：1.抑制Polo样激酶1选择性阻断RAS突变膀胱肿瘤的生长膀胱癌是泌尿系统最常见的恶性肿瘤,以移行上皮细胞癌最为多见。随着膀胱癌的发生、发展分子机制的不断阐明,膀胱肿瘤分子靶向治疗受到人们越来越多的关注。HRAS是原癌基因RAS家族成员之一,它的突变被证实在膀胱肿瘤发生及发展中有重要作用。正常的Ras蛋白参与调节细胞的生长、增殖和分化等功能,突变后会使其下游信号通路持续激活导致细胞发生癌变。目前直接靶向Ras蛋白及其下游信号通路的抑制剂并未取得良好疗效,这使得RAS突变肿瘤的治疗策略成为研究难点。最新的研究结果表明,PLK1基因与RAS基因存在“协同致死”关系,提示Plk1可能是RAS突变肿瘤的潜在治疗新靶点。Polo样激酶1(Plk1)是一种蛋白激酶,在细胞周期中发挥重要调控作用。本论文假设为抑制Plkl可选择性阻断RAS突变膀胱肿瘤的生长。在多种膀胱癌细胞上,利用siRNA技术或用小分子抑制剂(BI-2536和BI-6727)干预Plk1,结果显示Plk1的功能缺失可以选择性抑制RAS突变的细胞株的存活(P0.01)。当Plk1激酶活性被抑制后,与RAS非突变细胞株BIU-87相比,RAS突变细胞株5637细胞周期被阻断在G2/M期,且发生明显凋亡。分子机制研究中,结果显示在RAS突变细胞株5637中,Plk1功能被抑制导致Ras下游p-AKT和p-ERK的水平降低。在体内动物实验中,BI-2536(20mg/kg,每周两次,连续给药22天)能明显抑制膀胱癌RAS突变5637肿瘤的体积及重量,但不影响裸鼠的正常体征。肿瘤组织免疫组化结果分析发现,与对照组相比,给药组裸鼠肿瘤组织中Ki-67表达明显减少,p-AKT和p-ERK的表达降低。综上所述,本文研究证明抑制Plk1通过下调Ras下游关键蛋白从而选择性抑制RAS突变膀胱肿瘤,研究成果为RAS突变膀胱癌的治疗提供线索。2患者源性膀胱肿瘤动物模型的初步建立动物模型是肿瘤分子机理研究和靶向新药研发的的重要载体。目前膀胱癌研究动物模型主要是细胞系皮下荷瘤和细胞系原位荷瘤动物模型,然而该两类模型并不能有效模拟临床膀胱癌肿瘤组织的微环境和异质性。近年,患者源性肿瘤模型应运而生,越来越受到推崇,被认为是最贴近临床且最具有应用价值的动物模型。为了进一步解析膀胱癌转移分子机制以及发现抑制膀胱癌的有效药物,本文旨在建立患者源性膀胱肿瘤动物模型。本课题组通过与长海医院泌尿外科建立科学合作关系,经患者同意,获得新鲜膀胱癌肿瘤组织,建立患者源性膀胱肿瘤动物模型。具体研究方法如下：使用套管针将新鲜膀胱肿瘤小块移植于裸鼠皮下,经过为期四个月的观察测量,定义肿瘤体积≥500mm3则认为病人原代组织成瘤,进一步对肿瘤进行分离传代和冻存。目前本研究已成功建立一例膀胱肿瘤动物模型,肿瘤组织已传至第三代。经分子突变鉴定,初步发现该模型肿瘤组织RAS基因热点突变区域不存在突变,深入分子的鉴定工作有待进一步开展。该模型的成功建立为膀胱癌发生发展分子机制研究以及抗膀胱癌肿瘤新药筛选和研发提供重要研究系统。
[Abstract]:1. inhibition of Polo like kinase 1 selectively blocking the growth of bladder cancer RAS mutation in bladder cancer is the most common malignant tumor of urinary system in transitional cell carcinoma is the most common. With the occurrence of bladder cancer, we clarify the molecular mechanism of the development, to the treatment of bladder tumor targeting molecules has attracted more and more attention is one of the.HRAS RAS gene family, the mutation was confirmed in bladder cancer occurrence and development has an important role. The normal Ras protein involved in the regulation of cell growth, proliferation and differentiation of function mutation will make its continued activation of downstream signaling pathways lead to cancer. At present, directly targeted inhibitors of Ras protein and its downstream the signal pathway has not achieved good effect, which makes the strategy for the treatment of tumors with RAS mutations has become difficult. The latest research results show that the existence of "PLK1 gene and RAS gene co lethal "Relationship, suggesting that Plk1 may be RAS mutated tumors a new potential target for therapy of.Polo like kinase 1 (Plk1) is a protein kinase that plays an important role in the cell cycle. The hypothesis for the inhibition of Plkl can selectively block RAS mutation in bladder tumor growth. In a variety of bladder cancer cells, using siRNA technology or with small molecule inhibitors (BI-2536 and BI-6727) Plk1 intervention, the results showed that functional loss of Plk1 can survive the selective inhibition of RAS mutant cell line (P0.01). When Plk1 kinase activity is inhibited, and the mutation of BIU-87 cells compared to RAS, RAS mutant cell lines of 5637 cell cycle was blocked in G2/M phase. And the occurrence of apoptosis. The molecular mechanism in the study, the results showed that in RAS mutant cell lines 5637, Plk1 function was inhibited leading to Ras downstream of the p-AKT and p-ERK levels decreased. In vivo animal experiment, BI-2536 (20mg /kg, two times a week. Continuous administration for 22 days) volume and weight can obviously inhibit the bladder cancer RAS mutations in 5637 tumors, but does not affect the normal signs of tumor tissue in nude mice. Immunohistochemistry results showed that compared with the control group, the tumor tissue of mice to Ki-67 medicine group significantly decreased the expression, decreased expression of p-AKT and p-ERK. To sum up, this paper the research indicated that inhibition of Plk1 by downregulation of Ras downstream key protein to selective inhibition of RAS mutation in bladder cancer, provide clues to patients with.2 bladder tumor animal model for research of RAS mutation in bladder cancer preliminary establish the animal model of tumor molecular mechanism and targeted drug development important carrier. The animal model of primary bladder cancer is cell line subcutaneous tumor cell lines and in situ tumor animal model, however, the two models can not effectively simulate clinical bladder cancer tissue microenvironment and heterogeneity . in recent years, as patients with tumors of the model, more and more respected, is considered to be the most close to the animal model and clinical application value. In order to further analyze the molecular mechanism of bladder cancer metastasis and find effective drugs that inhibit bladder cancer, this paper aims to establish the source of patients with bladder tumor animal model. The relationship between scientific cooperation by the research group and the Department of Urology, Changhai Hospital, the patients agreed to get fresh bladder cancer patients, establish bladder tumor animal model. Specific research methods are as follows: using trocar small fresh bladder tumor transplanted subcutaneously in nude mice, through the observation period of four months, the definition of tumor volume more than 500mm3 that patients primary tumor tissue, further separation and subculture and cryopreservation on tumor. At present, this study has successfully established an animal model of bladder tumor, tumor The organization has been passaged for third generations. The molecular identification of mutations, initially found that the model of RAS gene hotspot mutations of tumor tissue region no mutations, identifying the molecular depth need to be further carried out. The model successfully established for bladder cancer molecular mechanism of bladder cancer development and anti tumor drug screening and development provides an important research system.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14

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