【作者】 戚士芹；

【导师】 肖现民； 李凯； 陈莲； 郑继翠；

【作者基本信息】 复旦大学 ， 儿科学， 2010， 博士

【摘要】 研究背景神经母细胞瘤(neuroblastoma, NB)是小儿最常见的恶性实体肿瘤之一,占儿童期肿瘤的8-10%,其恶性程度高,多数患儿最终愈后不良,故深入研究NB的发生、发展机理至关重要。我们课题组在对NB的发生、发展机理进行研究时发现：雌激素(estrogen, E)和环境雌激素双酚A(bisphenol A, BPA)均能促进神经母细胞瘤SK-N-SH细胞株的增殖、侵袭和转移,即E和BPA均可促进NB的发展。在此基础上,我们试图探寻E和BPA对NB起始环节的作用,而肿瘤干细胞(cancer stem cell, CSC)即属于肿瘤的起源细胞,故我们选择研究“E和BPA对神经母细胞瘤SK-N-SH细胞株的CSC的影响”来探寻“E和BPA对NB起始环节有无作用”。研究目的SP(side population, SP)分析法从神经母细胞瘤SK-N-SH细胞株中分离并鉴定出CSC,明确E和BPA干预对SK-N-SH细胞株的CSC有无影响,最终了解E和BPA对NB的起始环节有无作用。材料与方法一、SK-N-SH细胞株侧群细胞的检测及检测条件的优化：不同的Hoechst孵育时间、不同的维拉帕米浓度检测SK-N-SH细胞株的SP细胞(侧群细胞)比例,根据SP细胞的检测效果选择各自的最佳条件；不同浓度Hoechst染色后的表型观察、CCK-8活细胞数评定了解Hoechst对SK-N-SH细胞株的毒性；不同浓度Hoechst染色后的流式细胞仪细胞周期分析、Annexin V-FITC和PI双标法流式细胞仪凋亡及死亡分析、再培养后SP细胞比例的检测,探讨Hoechst对SK-N-SH细胞株毒性的机理；最后对比不同浓度Hoechst对SK-N-SH细胞株的SP细胞检测效果,选择出能有效检测SP细胞并最低毒性的Hoechst浓度。二、SK-N-SH细胞株侧群细胞的分选及其干细胞特性的鉴定：流式细胞仪于SK-N-SH细胞株中分选出SP细胞和非SP细胞(NSP细胞)；SP和NSP细胞均分别接种于正常生长培养基和干细胞培养基中培养并传代,观察细胞表型的变化；电镜观察SP和NSP细胞的超微结构；免疫荧光和免疫印迹法了解SP和NSP细胞的干细胞和分化细胞标志物的表达；CCK-8分析了解SP和NSP细胞的增殖能力；连续培养的SP和NSP细胞的表型观察、蛋白标志物表达及流式细胞仪SP细胞比例再检测了解各细胞亚型的分化潜能；平板克隆实验、Transwell侵袭实验、裸鼠成瘤实验明确SP和NSP细胞的恶性程度。三、雌激素和双酚A对SK-N-SH细胞株侧群细胞作用的研究：E2和BPA干预后流式细胞仪检测SK-N-SH细胞株的SP细胞比例；E2和BPA干预后免疫印迹法检测干细胞标志物c-kit和ABCG2蛋白的表达；CCK-8分析和流式细胞周期分析确认E2和BPA对SK-N-SH细胞株的促增殖作用；CCK-8分析了解E2和BPA对SK-N-SH细胞株的SP和NSP细胞各亚型的促增殖作用。结果一、SK-N-SH细胞株侧群细胞的检测及检测条件的优化：60min的Hoechst孵育时间、50μmol/L的维拉帕米浓度可有效于SK-N-SH细胞株检测出SP细胞；SK-N-SH细胞株特异地对低浓度的Hoechst毒性反应敏感,其机理与Hoechst干扰细胞周期、诱导细胞死亡和凋亡有关；对比不同浓度Hoechst的检测效果和毒性,最终选择5μM的浓度用于SK-N-SH细胞株的SP细胞分选；以上述的检测条件检测出SK-N-SH细胞株的SP细胞比例为3.6±0.2%。二、SK-N-SH细胞株侧群细胞的分选及其干细胞特性的鉴定：成功于SK-N-SH细胞株中分选出SP和NSP细胞；SP细胞胞体小、有神经丝样突起,贴壁能力弱、集聚性生长,核质比高、胞浆细胞器少,高表达干细胞标志物、低表达分化细胞标志物,增殖速度快,具有快速分化能力,克隆形成能力强、侵袭性高、裸鼠成瘤率高；SK-N-SH细胞株的NSP细胞胞体大、无神经丝样突起,贴壁能力强、单层生长,核质比低、胞浆细胞器多,低表达干细胞标志物、高表达分化细胞标志物,增殖速度慢,具有缓慢的分化能力,克隆形成能力消失、侵袭性弱、无裸鼠成瘤能力。三、雌激素和双酚A对SK-N-SH细胞株侧群细胞作用的研究：E2和BPA均能使SK-N-SH细胞株的SP细胞比例下降直至检测不出、均能下调干细胞标志物c-kit和ABCG2蛋白的表达；E2和BPA均对SK-N-SH母细胞有显著的体外促增殖作用,但对其NSP细胞亚型的促殖作用较对其SP细胞亚型更显著。结论1.SP细胞研究时要警惕Hoechst的毒性,对任一将行SP细胞研究的新细胞株,SP细胞检测、分选的系列条件均应予优化以寻及最佳检测条件。2.SK-N-SH细胞株的SP细胞有CSC的特征。3.E2和BPA对SK-N-SH细胞株中具有干细胞特性的SP细胞的作用较对其NSP细胞小,故E2和BPA对NB的发生相对作用不大。更多还原

【Abstract】 BackgroundsNeuroblastoma (NB) is a common malignant pediatric tumor and accounts for 8-10% of childhood cancers. Long-term survival in high-risk cases is still less than 50% in current treatment strategies, thus research on the genesis and progress of NB is urgently needed to improve treatment. Our results of research on the genesis and progress of NB showed that estrogen (E) and bisphenol A (BPA) which is the most common environmental estrogen not only promoted the growth but also improved the invasion and metastasis for human NB SK-N-SH cells, i.e. E and BPA promote the progress of NB. On the basis of our research results for NB, we presume that E and BPA maybe contributes to the genesis of NB. It is well known that cancer stem cells (CSC) are the origin cells of tumors, thus we investigate the effects of E and BPA on the CSCs of SK-N-SH cells in order to identify if E and BPA have a tumorigenic ability for NB.PurposeThe aim of this study was to sort the side population (SP) cells from SK-N-SH cell line and identify if the sorted SP cells have the cancer stem cell-like properties. At last, the effects of E and BPA on the SP cells were investigated to understand if E and BPA promote the genesis of NB.Materials and Methods1. The detection of SP cells in SK-N-SH cell line and optimization of the detecting procedure:The comparison of detecting effect for SP cells by different incubating time, different concentrations of verapamil was performed to select the suitable incubating time and verapamil concentration. Morphology observation and cell counting kit-8 (CCK-8) assay for re-cultured cells following different concentrations of Hoechst exposure were carried out to confirm the toxicity of Hoechst to SK-N-SH cells. Flow cytometry analysis for cell cycle, Annexin V-FITC and propidium iodide staining flow cytometry, and detection of the SP cells for re-cultured cells following different concentrations of Hoechst exposure were used to investigate the toxicity mechanism of Hoechst to SK-N-SH cells. The effective and relatively nontoxic concentration of Hoechst for SP analysis in SK-N-SH cells was selected by comparing the SP cells detecting effect and cytotoxicity of different concentrations of Hoechst.2. The sorting of SK-N-SH SP cells and identification for their cancer stem cell-like characteristics:SP and non-SP (NSP) cells were separated from the SK-N-SH cell line by flow cytometry and cultured in normal growth medium or serum-free medium containing some growth factors, and then their characteristics were analyzed by light and electron microscopy for morphology; we also used western blotting to analyze marker proteins, CCK-8 assay for proliferative ability, series differentiation assays for differentiation properties; single cell clone experiment, Matrigel invasion assays and tumorigenicity assays were performed to analyze their malignant potential.3. The research of the effects of E and BPA on the SK-N-SH SP cells: Following by E2 or BPA exposure, the detection of SP cells was performed by flow cytometry, and the expression of stem cell marker proteins c-kit and ABCG2 was analyzed by western bloting. The growth-promoting effect of E2 or BPA on SK-N-SH parent cells was confirmed by the CCK-8 assay and flow cytometry cell cycle analysis. The SK-N-SH SP and NSP sub-types were treated by E2 or BPA, respectively, and then the number of viable cells for these two sub-types was evaluated by CCK-8 assay.Results1. The detection of SP cells in SK-N-SH cells and optimization of the detecting procedure:The incubation time of 60 minute, the verapamil concentration of 50μmol/L are suitable to detect the SP cells in SK-N-SH cell line. SK-N-SH cells had strong toxic reaction to Hoechst even at lower concentrations. The mechanism of the strong Hoechst sensitivity of SK-N-SH cells relates to the perturbation of cell cycle, the acute cell death and apoptosis induced by Hoechst. We ultimately selected the Hoechst concentration of 5μM to carry out the SP analysis for SK-N-SH cell line after we compared the SP cells detection effect and cytotoxicity of different Hoechst concentrations.2. The sorting of SK-N-SH SP cells and identification for their cancer stem cell-like characteristics:We successfully sorted the SP and NSP cells from SK-N-SH cell line. SP cells are small, have neurites, grow as clumps of cells with weakly substrate-adherent, and have few cytoplasmic organelles and high nuclear-cytoplasmic ratio; SP cells express high levels of the stem cell marker protein c-kit, have high proliferative and malignant ability, and have a capacity for fast differentiation. NSP cells are large and flat, do not have neurites, grow as a contact-inhibited monolayer with tightly substrate-adherent, and have many cytoplasmic organelles and low nuclear-cytoplasmic ratio; NSP cells express the differentiated cell marker proteins peripherin and S100 at high levels, have slow differentiate ability and have low proliferative and malignant ability.3. The research of the effects of E and BPA on the SK-N-SH SP cells:Both E2 and BPA gradually decreases the percentage of SP cells in SK-N-SH cell line to the level that can not be detected by flow cytometry and depresses the expression of the stem cell marker proteins c-kit and ABCG2. Both E2 and BPA significantly promotes growth not only for SK-N-SH parent eells but also for its SP and NSP sub-types, but the growth-promoting effect of E2 and BPA for NSP cells is significant higher than for SP cells.Conclusions1. It is essential to minimize or, at the very least, be aware of Hoechst toxicity in SP analysis. The detecting procedure of SP cells needs to be carefully optimized for each new cell line studied.2. SK-N-SH SP cells have cancer stem cell-like properties.3. E2 and BPA have lower effects on the SP cells which are enriched for the origin cells of tumor, thus E2 and BPA maybe don’t have the capacity to promote the genesis of NB.更多还原