人视网膜母细胞瘤肿瘤干细胞的分离鉴定及相关表面标志的初步研究

发布时间：2018-03-01 01:03

本文关键词： 视网膜母细胞瘤肿瘤干细胞细胞系致瘤性 ABCG2 CD133 CD44　出处：《复旦大学》2011年博士论文　论文类型：学位论文

【摘要】：视网膜母细胞瘤(retinoblastoma, RB)是婴幼儿最常见的眼内恶性肿瘤,目前国际上针对RB开展了以化疗为主结合局部治疗的综合疗法,极大改善了患儿的生命预后,然而化疗存在最大的问题就是肿瘤的复发和转移。肿瘤化疗失败后复发的主要原因是肿瘤细胞对化疗药物产生耐药性,而耐药性的产生机制相当复杂,主要包括多耐药性蛋白的功能表达、静息期细胞对化疗不敏感以及肿瘤细胞凋亡耐受等,虽然在这些方面的研取得了一定的进展,但在阐释耐药性机制及指导临床应用上并未取得根本性突破。肿瘤干细胞(Cancer stem cells, CSCs)是一类极少的具有自我更新、不定潜能性并促使肿瘤形成的细胞,对肿瘤的生长、形成及转移起到关键作用’,更为重要的是这部分肿瘤起始性细胞对化疗药物的敏感性较其它非致瘤性肿瘤细胞明显降低。目前,临床上化疗很大程度上是针对大的非致瘤性肿瘤细胞组成的肿瘤块,而不能有效杀灭或抑制肿瘤干细胞,导致了化疗后肿瘤的复发和转移。虽然不是所有恶性肿瘤的生物学特点都符合肿瘤干细胞理论,但CSCs假说为理解RB化疗耐药性提出一种更为合理的细胞水平的理论解释。通过研究肿瘤干细胞,将有利于进一步理解RB发生、发展及转移的机制,可能为减少RB化疗后复发和转移提供新的治疗策略。目前在多种实体瘤中均已证实有肿瘤干细胞存在,包括乳腺癌、脑肿瘤、胰腺癌、结肠癌、黑色素瘤、前列腺癌和卵巢癌等。然而和其他肿瘤研究相比,作为眼内肿瘤的RB由于新鲜标本获取的困难性以及样本的变异性使得其肿瘤干细胞的研究相对滞后。本实验通过对人RB肿瘤细胞体外采用无血清限定培养基长期培养,分离得到了以肿瘤球方式生长的细胞系,并可以在连续传代的同时保持了其自我更新、增殖及分化能力,同时通过化疗耐药性试验及小鼠致瘤试验评价其对化疗药物的反应性及异体致瘤能力。同时文章第三部分还测定了表面标志物ABCG2(ATP-binding cassette superfamily G member 2, ATP结合膜转运蛋白2)、CD133和CD44在该群细胞中的表达情况,为进一步研究视网膜母细胞瘤肿瘤干细胞(retinoblastoma cancer stem-like cell, RCSC)及其鉴定所需的表面标志奠定了基础。
[Abstract]:Retinoblastoma (RBB) is the most common intraocular malignant tumor in infants and young children. At present, a combination of chemotherapy and local therapy has been developed for RB in the world, which has greatly improved the life prognosis of children with retinoblastoma.The retinoblastoma (RBB) is one of the most common intraocular malignancies in infants. However, the biggest problem in chemotherapy is tumor recurrence and metastasis. The main reason for tumor recurrence after chemotherapy failure is that tumor cells develop resistance to chemotherapeutic drugs, and the mechanism of drug resistance is quite complex. These include the functional expression of multidrug resistance proteins, the insensitivity of resting cells to chemotherapy and the tolerance of tumor cells to apoptosis, although some progress has been made in these areas. However, no fundamental breakthrough has been made in explaining the mechanism of drug resistance and guiding clinical application. Tumor Stem Cell Cancer stem cells (CSCs) are a group of very few cells that have self-renewal, indeterminate potential, and promote tumorigenesis, the growth of tumors. Formation and metastasis play a key role. More importantly, the sensitivity of these tumor initiation cells to chemotherapeutic drugs is significantly lower than that of other non-tumorigenic tumor cells. Chemotherapeutic therapy is largely targeted at tumor blocks made up of large, non-tumorous tumor cells, rather than effectively killing or inhibiting tumor stem cells. Which leads to tumor recurrence and metastasis after chemotherapy. Although not all malignant tumors have biological characteristics consistent with cancer stem cell theory, However, the CSCs hypothesis provides a more reasonable theoretical explanation for understanding the chemoresistance of RB at cellular level. The study of tumor stem cells will be helpful to further understand the mechanism of RB occurrence, development and metastasis. It may provide new treatment strategies to reduce the recurrence and metastasis of RB after chemotherapy. Cancer stem cells have been confirmed in a variety of solid tumors, including breast cancer, brain tumor, pancreatic cancer, colon cancer, melanoma. However, compared with other tumor studies, RB, as an intraocular tumor, lags behind in the study of tumor stem cells due to the difficulty of obtaining fresh samples and the variability of samples. In this experiment, human RB tumor cells were cultured in serum-free medium for a long period of time, and the cell lines which grew in the form of tumor balls were isolated, and their ability of self-renewal, proliferation and differentiation were maintained at the same time of continuous passage. The reactivity to chemotherapeutic drugs and the ability of allogeneic tumorigenesis were evaluated by chemotherapeutic drug resistance test and mouse tumorigenicity test. The surface markers ABCG2(ATP-binding cassette superfamily G member 2 and ATP binding membrane transporter 2hmd CD133 were also determined in the third part of the paper. And the expression of CD44 in this group of cells, The results provide a basis for the further study of retinoblastoma cancer stem-like cells (RCSCs) and the surface markers needed for identification of retinoblastoma.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R739.7

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