干性基因Nanog在膀胱癌干细胞干性维持中的作用研究

发布时间：2019-05-09 09:55

【摘要】：膀胱癌是我国男性泌尿生殖系统发病率最高的恶性肿瘤，具有对放化疗耐受以及治疗后易复发等特点，因此，我们需要寻找膀胱肿瘤新的治疗策略。肿瘤干细胞在多种肿瘤中的相继发现为我们研究膀胱肿瘤发生、发展以及治疗提供了崭新的研究视角。研究表明，肿瘤干细胞具有自我更新和多向分化潜能，其中肿瘤干细胞可以通过对称分裂和不对称分裂，形成与母细胞性质和功能完全一致的子代细胞，维持其自我更新；肿瘤干细胞中存在的突变可通过自我更新在细胞中代代相传，保持肿瘤细干胞的致瘤性。这说明自我更新是肿瘤干细胞致瘤的重要原因之一。干细胞的自我更新受到多种途径调节，干性基因在其自我更新中起重要作用，包括Oct4，Sox2和Nanog等转录因子，通过激活某些关键基因的转录，从而调控细胞增殖与分化的平衡，维持干细胞的未分化状态。其中Nanog是最近鉴定的维持未分化胚胎细胞多能性和自我更新的关键转录因子，在多种肿瘤中异常表达，其高表达可以增强肿瘤细胞的增殖能力，且常伴随不良预后。这提示Nanog可通过维持肿瘤干细胞的自我更新能力导致肿瘤发生，促进肿瘤发展。在膀胱癌干细胞的研究中发现，膀胱癌干细胞同样具有较强自我更新能力，并且伴随Nanog的表达升高，提示我们Nanog可能参与了膀胱癌干细胞的干性维持。目前关于Nanog在膀胱癌发生、发展中的作用尚不明确，为了研究Nanog在膀胱癌干细胞干性维持中的作用，我们首先采用Western Blot和免疫组化方法检测Nanog蛋白在膀胱癌干细胞和46例膀胱癌组织中的表达，发现Nanog在膀胱癌干细胞和组织中均为高表达，且其表达与组织的病理分级呈正相关；为深入探讨Nanog在膀胱癌细胞中的作用，我们利用慢病毒载体，构建了稳定高表达外源Nanog的膀胱癌T24和5637细胞株；进一步通过平板克隆形成实验观察Nanog对膀胱癌细胞增殖的作用，采用悬浮成球技术检测Nanog对膀胱癌细胞自我更新能力的影响，选取化疗药物顺铂，利用克隆形成实验和MTT法检测Nanog对膀胱癌细胞耐药性的影响，运用裸鼠移植瘤模型分析Nanog在膀胱癌细胞致瘤性中所起作用，结果表明，，与对照组相比，高表达Nanog的T24和5637细胞克隆形成率上升，悬浮成球能力增强，对顺铂的敏感性降低，以及体内成瘤能力增强。本研究首次报道了Nanog在膀胱癌发生、发展中的表达变化，并从肿瘤干细胞特性角度探讨Nanog与膀胱癌干细胞的干性维持的关系，发现Nanog通过影响膀胱癌细胞的增殖，自我更新，耐药性和致瘤性，促进膀胱癌干细胞的干性维持。为阐明Nanog基因与膀胱癌发生、发展关系以及膀胱癌复发、耐药的机制奠定了基础，也提示Nanog可作为膀胱癌干细胞的新型标志物，在临床中，可作为膀胱癌的早期诊断分子以及个性化治疗新靶标，为膀胱癌的诊断，治疗提供新思路。
[Abstract]:Bladder cancer is a malignant tumor with the highest incidence in male genitourinary system in China, which has the characteristics of tolerance to radiotherapy and chemotherapy and easy recurrence after treatment. Therefore, we need to find a new treatment strategy for bladder cancer. The successive discovery of tumor stem cells in many kinds of tumors provides a new research perspective for us to study the genesis, development and treatment of bladder tumors. Studies have shown that tumor stem cells have the potential of self-renewal and multi-directional differentiation, in which tumor stem cells can form progeny cells which are identical to the nature and function of mother cells by symmetrical division and asymmetric division, and maintain their self-renewal. Mutations in tumor stem cells can be transferred from generation to generation through self-renewal to maintain the tumorigenicity of tumor stem cells. This suggests that self-renewal is one of the important reasons why tumor stem cells are tumorigenic. Stem cell self-renewal is regulated by many ways, and stem genes play an important role in its self-renewal, including transcription factors such as Oct4,Sox2 and Nanog, which regulate the balance of cell proliferation and differentiation by activating transcription of some key genes. Maintain the undifferentiated state of stem cells. Among them, Nanog is recently identified as a key transcription factor for maintaining pluripotency and self-renewal of undifferentiated embryonic cells. Its high expression in a variety of tumors can enhance the proliferation of tumor cells, and often accompanied by poor prognosis. These results suggest that Nanog can promote tumor development by maintaining the self-renewal ability of tumor stem cells. In the study of bladder cancer stem cells, bladder cancer stem cells also have a strong ability of self-renewal, and with the increase of Nanog expression, it is suggested that Nanog may be involved in the dry maintenance of bladder cancer stem cells. At present, the role of Nanog in the carcinogenesis and development of bladder cancer remains unclear. In order to study the role of Nanog in the dry maintenance of bladder cancer stem cells, We first detected the expression of Nanog protein in bladder cancer stem cells and 46 cases of bladder cancer by Western Blot and immunohistochemistry. We found that Nanog was highly expressed in stem cells and tissues of bladder cancer, and its expression was positively correlated with the pathological grade of bladder cancer. In order to investigate the role of Nanog in bladder cancer cells, we constructed bladder cancer T24 and 5637 cell lines with stable and high expression of exogenous Nanog using lentiviral vector. Furthermore, the effect of Nanog on the proliferation of bladder cancer cells was observed by plate cloning assay, and the effect of Nanog on the self-renewal ability of bladder cancer cells was detected by suspension globulation technique. The chemotherapeutic drug cisplatin was selected. Clone formation test and MTT method were used to detect the effect of Nanog on the drug resistance of bladder cancer cells, and the role of Nanog in the tumorigenicity of bladder cancer cells was analyzed by using nude mice transplantation tumor model. The clone formation rate of T24 and 5637 cells with high expression of Nanog increased, the ability of suspension spheroidization increased, the sensitivity to cisplatin decreased, and the tumorigenicity of T24 and 5637 cells increased in vivo. In this study, we first reported the expression of Nanog in the carcinogenesis and development of bladder cancer, and discussed the relationship between Nanog and dry maintenance of bladder cancer stem cells from the point of view of the characteristics of tumor stem cells. It was found that Nanog could affect the proliferation and self-renewal of bladder cancer cells. Drug resistance and tumorigenicity promote the dry maintenance of bladder cancer stem cells. It lays a foundation for elucidating the relationship between Nanog gene and carcinogenesis and development of bladder cancer, as well as the mechanism of recurrence and drug resistance of bladder cancer. It is also suggested that Nanog can be used as a new marker of bladder cancer stem cells in clinical practice. It can be used as a new target for early diagnosis and individualized treatment of bladder cancer, and provide a new idea for the diagnosis and treatment of bladder cancer.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14

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