【作者】 梁辉；

【导师】 潘新良；

【作者基本信息】 山东大学 ， 耳鼻咽喉科学， 2010， 博士

【摘要】 [研究背景与目的]下咽癌是一种头颈部恶性肿瘤,是上消化道及上呼吸道恶性程度最高的肿瘤之一,其发生的机制并不清楚。预后差的主要原因包括局部复发、易发生淋巴结转移及远处转移。由于临床后果差,对患者生命及生活质量影响严重,是当前耳鼻咽喉-头颈外科的研究重点。肿瘤干细胞是近年来对肿瘤发生发展过程的一种新的理论观点,新观点认为肿瘤组织中只有极少量的肿瘤细胞有干细胞样作用,它们是一群数量较少但具有无限自我更新潜能并能促使肿瘤形成的细胞,在肿瘤生长中起着决定性作用。这类细胞有较强的抗拒放疗及化疗的作用,并可能与免疫逃逸,生长信号异常传递等多种肿瘤的重要生物学特点有关。目前已从脑肿瘤,白血病,乳腺癌,喉癌,肺癌等多种肿瘤组织中提取到了肿瘤干细胞,并进行了相关细胞学及分子生物学研究。从当前的研究结果来看,不同的肿瘤中,尽量其肿瘤干细胞生物学特性有共同点,如无限自我更新潜能,分化能力等,但肿瘤干细胞其表面标志物是不尽相同的,这提示我们需要对不同的肿瘤进行相应研究,了解不同肿瘤中肿瘤干细胞的标志物特点。近年来,许多研究证实,CD133是肿瘤干细胞的重要标志物之一。CD133是一种分子量为120kDa,具有5个跨膜区的糖蛋白,属于一种早期抗原。在2000年英国举行的第7届国际人类白细胞分化抗原研讨会上被正式命名为CD133。它是一种正常的造血干细胞表面标志物,它的一个显著特点就是：CD133的表达随着细胞的分化迅速下调,选择性地在骨髓和外周血造血干细胞及内皮祖细胞表达,但在成熟内皮细胞上不表达,是一个重要的分离和鉴定造血干或祖细胞的分子标志。侵袭能力是恶性肿瘤的标志性行为特点之一,是当前研究恶性肿瘤行为特点的一个重要方向。目前利用Transwell小室是体外研究肿瘤细胞侵袭和转移行为的简便且快速的方法。本研究拟以CD133为分选标志物,从人类FADU细胞系中分选出CD133阳性细胞,研究其数量分布特点,形态学特点,并拟通过应用Transwell小室的研究方法,研究在FADU细胞系中CD133阳性细胞的侵袭能力,从而为未来完善对CD133阳性细胞拥有的细胞学特点研究提供相应的细胞学实验室基础。第一部分下咽癌FADU细胞系中CD133表达情况研究[目的]研究下咽癌FADU细胞系中CD133表达情况。[方法]对下咽癌FADU细胞进行培养和传代,分别应用免疫细胞化学和流式细胞仪的方法对下咽癌FADU细胞系中CD133表达情况进行研究。[结果]免疫细胞化学的方法发现,在下咽癌FADU细胞中CD133阳性细胞仅占少数,而在利用流式细胞仪的方法研究后发现,FADU细胞中CD133阳性细胞比例仅为2.01%。[结论]CD133阳性细胞数量在FADU细胞系中占少数。第二部分下咽癌FADU细胞系中CD133阳性细胞的分选[目的]应用可靠的分选手段,从下咽癌FADU细胞系中分选CD133阳性细胞并鉴定。[方法]对下咽癌FADU细胞进行培养和传代,应用免疫磁珠分选技术进行CD133阳性细胞的分选。[结果]应用免疫磁珠分选技术后,可很好地分选出CD133阳性细胞,其分选后CD133阳性细胞纯度可达91.2%。[结论]免疫磁珠分选技术可很好地应用于下咽癌FADU细胞系中CD133阳性细胞的分选。第三部分下咽癌FADU细胞系中CD133阳性细胞的侵袭能力研究[目的]研究下咽癌FADU细胞系中CD133阳性细胞的侵袭能力。[方法]对应用免疫磁珠分选技术进行分选后的CD133阳性细胞、阴性细胞分别应用Transwell小室进行侵袭能力研究,倒置相差显微镜下照相,计数迁移到上室基质膜外表面的细胞数目,以能穿过基质膜的细胞数目多少表示细胞的侵袭能力强弱。每个样本计数5个视野(×200)的细胞数,取其平均值进行比较。[结果]与CD133阴性细胞相比,CD133阳性细胞透膜数量较阴性细胞明显高,其穿过基质膜的细胞数分别为54.0±2.3,17.0±1.1,其差异具有统计学意义,P<0.01。[结论]在下咽癌FADU细胞系中CD133阳性细胞比阴性细胞侵袭能力强。更多还原

【Abstract】 Hypopharyngeal cancer is one of head and neck malignant tumor, which is also one of the highest level of malignant tumor in upper digestive tract and upper respiratory tract. The mechanism isn’t clear.The reasons of poor prognosis include: local recurrence, lymphatic metastasis and metastasis.Because of poor prognosis,and may affect the quality of patient’s life severely, hypopharyngeal cancer is one of the research priorities in otorhinolaryngology.Cancer stem cells is a new theoretical perspective, the new view is that only a very small amount of tumor cells have stem cell-like role,.they have potential for unlimited self-renewal and promote tumor formation, The cells play a decisive role in tumor growth. Such cells have a strong resistance to radiation therapy and chemotherapy, and may be related to immune escape, the growth of abnormal signal transmission and many other important feature of the tumor. At present, cancer stem cells have been identified in brain neoplasms, leukemia, breast cancers laryngeal cancer and lung cancer through an experimental strategy. From the current research results, the different tumors, as some biological characteristics have in common, such as the unlimited self-renewal potential and differentiation capacity, but the cancer stem cell markers on its surface is not the same, which prompt us to the need for accordingly different tumor research, find the different tumor markers of cancer stem cells in different tumors.In recent years, many studies have confirmed that, CD 133 is one of the important markers for cancer stem cells,. CD133 is an early antigen whose molecular weight is 120 kDa, with five transmembrane glycoprotein. In 2000, the antigen was officially named CD133 in the United Kingdom at the 7th session of the International seminar on human leukocyte differentiation.CD133 is a normal surface markers for hematopoietic stem cell.one of its important features is:CD133 expresses with cell differentiation and down rapidly.It selectively expressed in stem cells in the bone marrow and peripheral blood and endothelial progenitor cells, but never in mature endothelial cells.CD133 is an important separation and identification marker for hematopoietic stem or progenitor cell.Invasion capacity is a sign of behavior characteristics of malignant tumors.It is one of the important direction in current study of the characteristics of malignant tumors. Transwell chamber is currently using in vitro for the study of tumor cell invasion and metastasis behavior.This study is intended to CD133 as a sorting marker of cell lines from human FADU cell line.CD133-positive cells were selected to study the distribution of their number, morphological characteristics, and intends to study the FADU cell lines CD133-positive cells in the invasion capacity through Transwell chamber. PartⅠStudy of the expressions of CD133 in FADU cell lineObjective:Study of the expressions of CD 133 in FADU cell line.Methods:FADU cells are cultured and passaged, the expression of CD133 was studied by immunocytochemistry and flow cytometry methods.Results:CD133-positive cells accounted for only a small number of the FADU cells by mmunocytochemistry method, while the ratio of CD133-positive cell in FADU cell line is only 2.01% in the use of flow cytometry method.Conclusion:The number of CD133-positive cells accounted for a small number of cell lines FADU. PartⅡCells sorting of CD133-positive in Hypopharyngeal FADU cell lineObjective:Applied with the magnetic cell sorting(MACS) technology, we purified CD133-positive cells from a FADU cell line and identified.Methods:FADU cells are cultured and passaged, we purified CD133-positive cells from a FADU cell line by magnetic cell sorting(MACS) technology and identified.Results:Application of magnetic cell sorting(MACS) technology, may be a good choice for purifying of CD133-positive cells, purity of the CD133-positive cells after sorting is 91.2%.Conclusion:Application of magnetic cell sorting(MACS) technology, may be a good choice for purifying of CD133-positive cells PartⅢInvasion of the CD133 positive cells in FADU cell lineObjective:To investigate the Invasion of the CD 133 positive cells in FADU cell line.Methods:In our study, Immunomagnetic beads were applied to purify CD133-positive cells in FADU cell line. Invasion of CD133-positive, CD133-negative cells were was measured by Transwell experiment.Results:The cell number that infiltrated Transwell membrane in CD133-positive group (54±2.3) was significantly higher than that in CD133-negative group (17±1.1).Conclusion:CD133-positive cells in FADU cell line show higher Invasion.更多还原