【作者】 朱海斌；

【导师】 谢立平；

【作者基本信息】 浙江大学 ， 外科学， 2011， 博士

【摘要】 野生型p53(wild-type p53,wtp53)是重要的抑癌基因,可诱导细胞周期阻滞、凋亡、分化、静息、DNA损伤、血管生成和转移抑制。多种恶性肿瘤伴有wtp53的错义突变,以及大量突变型p53(mutant p53,mp53)蛋白的聚集。研究表明,wtp53丧失抑癌基因的功能后,一些mp53蛋白将获得新的功能,比如参与肿瘤细胞的增殖,提高肿瘤的化疗耐药性。这些新的变化被称作mp53的获得性功能(mutant p53gain-of-function,mp53GOF).mp53作为肿瘤治疗的一个有效靶点,成为目前肿瘤研究的重要方向之一。研究mp53GOF的重要方法是siRNA干扰试验,siRNA能有效沉默p53基因,排除显性负效应的干扰,为检测与研究mp53GOF提供了有力的技术支持。目前很少有研究探索抑制mp53在泌尿系统肿瘤治疗中的作用。本研究选择泌尿系统中的膀胱癌与前列腺癌作为研究对象。前者是我国发病率最高的泌尿系统恶性肿瘤,后者去势抵抗前列腺癌的治疗为临床难点。针对前列腺癌细胞株DU145、膀胱癌细胞株T24、5637,我们使用siRNA干扰技术,研究靶向mp53的siRNA对细胞生长、凋亡、以及化疗耐药性的影响,评估其治疗应用的可行性。主要研究内容及成果如下：1.mp53干扰后,体外细胞培养及RT-PCR检测发现：前列腺癌DU145、膀胱癌T24、5637细胞的mp53表达明显降低,p53-siRNA能有效抑制肿瘤细胞的生长和活力。MTT实验发现抑制作用呈时间一剂量依赖性。2.流式细胞术以及蛋白印记实验结果表明：干扰mp53后,DU145细胞阻滞在G1和G2期,而膀胱癌T24、5637表现为G2期细胞周期阻滞。同时,p53-siRNA具有较强的凋亡诱导能力,其诱导的凋亡可能与Bcl-2家族蛋白相关。3.mp53沉默后PI3K/Akt抗凋亡信号通路受到了抑制,可能是引起细胞凋亡和周期阻滞的作用机制之一。4.MTT法和流式细胞术结果提示p53-siRNA与cisplatin联用较两者单用更能明显抑制膀胱癌5637细胞活性,两者对抑制细胞存活率具有协同作用。总之,siRNA靶向抑制mp53或许能成为治疗泌尿系统肿瘤的一种有效手段,对于去势抵抗前列腺癌以及耐药性膀胱癌具有一定的治疗效果和应用前景。该技术也可以应用于其它具有mp53的恶性肿瘤研究与治疗中。但是,在临床应用之前,还需要进行进一步探索和验证,包括机理研究与动物实验等。更多还原

【Abstract】 Wild-type p53(wtp53) is a major tumor suppressor whose function is critical for protection against cancer. It has been reported that wtp53can induce cell cycle arrest、DNA damage、angiogenesis and metastasis inhibition. Many human tumors carry missense mutations in the p53gene and the affected tumor cells accumulate excessive amounts of the mutant p53(mp53) protein. Various lines of evidence indicate that, in addition to abrogating the tumor suppressor functions of wtp53, the common types of cancer-associated p53mutations also endow the mutant protein with new activities that can contribute actively to various stages of tumor progression and to increased resistance to anticancer treatments. Collectively, these activities are referred to as mp53gain-of-function(mp53GOF). At present, the research on mp53becomes increasingly important as a valid target for inactivation by prospective anticancer therapies. An effective approach to explore mp53GOF, which has become feasible by the advent of siRNA technology, relies on knocking down endogenous mp53in tumor derived cells. It is suitable for monitoring the changes in cell phenotype while excluding dominant-negative effects over wtp53. However, the researches on its distinctive roles in the urogenital cancers are rare.This paper focused on the mp53GOF in prostatic cancer and bladder cancer. Chemotherapy was thought to have minimal clinical efficacy in men with metastatic, hormone-refractory prostate cancer and drug resistant bladder cancer. Therefore, novel therapeutic strategies for the treatment are urgently required. In this study, we knocked down the endogenous mp53in DU145human androgen-independent prostate cancer cell line and T24、5637bladder cancer cells with the siRNA technology, analyzed the outcomes of cells growth, viability, apoptosis and drug resistance, evaluates its therapeutic potential.The main investigations and results are as follows:1. Expressions of mp53mRNA and protein were down-regulated significantly in cell lines DU145、T24and5637after siRNA transfections which were detected by real-time RT-PCR and Western blotting. Knockdown of mp53inhibits cells growth and viability and this phenomenon was concentration and time-dependent demonstrated by MTT assay.2. The impact of silencing mutant p53on cell cycle and apoptosis was examined by flow-cytometric analysis of cells labeled by PI and annexin V, and the related proteins of cell cycle and apoptosis were detected by Western blotting. p53-siRNA induced the arrest of cell proliferation at the both G1and G2phase in DU145cells while induced G2-phase cell cycle arrest in5637and T24cells. The p53-siRNA could also induce remarkable apoptosis in these cell lines and it maybe occurs with the involvement of Bcl-2family pathway.3. Suppression of p53mutants by siRNA resulted in attenuation of PI3K/Akt pathway., which might be one of the mechanisms related to the effects of p53-siRNA.4. Silence of mp53by siRNA increased responsiveness to chemotherapeutic cisplatin in5637cells by MTT assay and flow-cytometric analysis. They exhibited a characteristic of synergic action to reduce the cells viability.In conclution, suppression of p53mutants by siRNA could be an effective and important technology in the treatment of urogenital cancers, especially for treating the hormone-refractory prostate cancer and the drug resistant bladder cancer. Meanwhile, our study also provided evidence and basis for its future clinical application in the therapeutics of other cancer with mutant p53. However, there are still a lot of problems need to be investigated and improved, including the exact mechanisms and in vivo therapeutic experiments.更多还原