下咽癌FADU细胞系中CD133阳性细胞的基础研究

发布时间：2018-10-22 07:07

【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,P0.01。 [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。
[Abstract]:[Background and Purpose of Study] Hypopharyngeal carcinoma is a head and neck malignant tumor, which is one of the most malignant tumors of upper gastrointestinal tract and upper respiratory tract. It is not clear that the main causes of poor prognosis include local recurrence, easy lymph node metastasis, distant metastasis. Due to poor clinical consequences, the patient's life and quality of life seriously affect the patient's life and quality of life. It is the current Otolaryngology-Head and Neck Surgery. Tumor stem cells are a new theoretical point in the development of tumorigenesis in recent years. The new viewpoint holds that only a very small number of tumor cells in tumor tissue have stem cell-like function, they are a group of small numbers but have unlimited self-renewal potential and can A cell that promotes the formation of a tumor in a tumor. It plays a decisive role in long term. These cells have strong resistance to radiation therapy and chemotherapy, and may be associated with various tumors such as immune escape, abnormal transmission of growth signals, and so on. The tumor stem cells are extracted from various tumor tissues such as brain tumor, leukemia, breast cancer, laryngeal cancer, lung cancer and the like, From the current research results, the biological characteristics of tumor stem cells in different tumors have commonalities, such as infinite self-renewal potential, differentiation ability, etc., but the surface of tumor stem cells Markers are different, which suggests that we need to study different tumors to learn about tumors in different tumors The marker features of stem cells. In recent years, many studies have demonstrated that CD133 It is one of the important markers of tumor stem cells. CD133 is a 120kDa molecular weight with 5 transmembrane domains. Glycoprotein of the region belonging to an early antigen. The 7th International Human Leukocyte Differentiation Antigen, held in England in 2000 CD133, a normal hematopoietic stem cell surface marker, is characterized by a rapid down-regulation of CD133 expression with the differentiation of cells, selectively in bone marrow and peripheral blood hematopoietic stem cells and endothelial progenitor cells Da, but not expressed on mature endothelial cells, is an important separation and Identification of molecular markers of hematopoietic stem or progenitor cells. The invasive ability is one of the characteristics of malignant tumors. At present, it is an important direction to study the characteristics of malignant tumor behavior. In vitro, Transwell chamber is used to study swelling in vitro. In this study, CD133 positive cells were selected from human FADU cell lines by CD133 as a sorting marker, and the number and morphology of CD133 positive cells were studied in human FADU cell lines. To investigate the invasion ability of CD133 positive cells in FADU cell lines, and to improve CD133 positive cells in the future. intracellular The cytological feature study provides a corresponding cytological laboratory basis. CD1 in the first hypopharyngeal carcinoma FADU cell line[Objective] To study the expression of CD133 in FADU cell line of hypopharyngeal carcinoma. The expression of CD133 in the FADU cell line of hypopharyngeal carcinoma was studied by immunocytochemistry and flow cytometry. The cells accounted for only a small number of cells, whereas after study using a flow cytometer, FADU was found Cells in C The proportion of D133 positive cells was only 2. 01%.[Conclusion] The number of CD133 positive cells is a few in FADU cell lines. Sorting of CD133-positive cells in hypopharyngeal carcinoma FADU cell line[Objective] To apply a reliable sorting device from hypopharyngeal carcinoma In FADU cell line, CD133 positive cells were sorted and identified.[Methods] FADU cells of hypopharyngeal carcinoma were cultured and passaged, and immunomagnetic beads were applied. Separation of CD133 positive cells by sorting technique.[Results] After using immunomagnetic beads sorting technique, it can be very good. to be selected from the ground CD133 positive cells, the purity of CD133 positive cells after sorting can reach 1. 2%.[Conclusion] Immunomagnetic bead sorting technique can be well applied to hypopharynx Isolation of CD133-positive cells in carcinoma FADU cell lines. Study on invasion ability of CD133-positive cells in the third hypopharyngeal carcinoma FADU cell line[Objective] To study CD13 in the hypopharyngeal carcinoma FADU cell line 3. The invasive ability of positive cells.[Methods] Applied immune magnetism the CD133 positive cells and the negative cells after sorting by the bead sorting technique are respectively used as Tr. An analysis of invasive ability in an answell cell, the number of cells transferred to the outer surface of the upper chamber substrate film by the inversion phase contrast microscope, and the number of cells that can pass through the substrate film indicates the cell's invasion ability. Each sample counts 5 fields of view (Table 2 00) Number of cells to be compared.[Results] Negative with CD133
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.63

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