下咽癌FADU細(xì)胞系中CD133陽性細(xì)胞的基礎(chǔ)研究
發(fā)布時間:2018-10-22 07:07
【摘要】: [研究背景與目的] 下咽癌是一種頭頸部惡性腫瘤,是上消化道及上呼吸道惡性程度最高的腫瘤之一,其發(fā)生的機(jī)制并不清楚。預(yù)后差的主要原因包括局部復(fù)發(fā)、易發(fā)生淋巴結(jié)轉(zhuǎn)移及遠(yuǎn)處轉(zhuǎn)移。由于臨床后果差,對患者生命及生活質(zhì)量影響嚴(yán)重,是當(dāng)前耳鼻咽喉-頭頸外科的研究重點(diǎn)。 腫瘤干細(xì)胞是近年來對腫瘤發(fā)生發(fā)展過程的一種新的理論觀點(diǎn),新觀點(diǎn)認(rèn)為腫瘤組織中只有極少量的腫瘤細(xì)胞有干細(xì)胞樣作用,它們是一群數(shù)量較少但具有無限自我更新潛能并能促使腫瘤形成的細(xì)胞,在腫瘤生長中起著決定性作用。這類細(xì)胞有較強(qiáng)的抗拒放療及化療的作用,并可能與免疫逃逸,生長信號異常傳遞等多種腫瘤的重要生物學(xué)特點(diǎn)有關(guān)。目前已從腦腫瘤,白血病,乳腺癌,喉癌,肺癌等多種腫瘤組織中提取到了腫瘤干細(xì)胞,并進(jìn)行了相關(guān)細(xì)胞學(xué)及分子生物學(xué)研究。從當(dāng)前的研究結(jié)果來看,不同的腫瘤中,盡量其腫瘤干細(xì)胞生物學(xué)特性有共同點(diǎn),如無限自我更新潛能,分化能力等,但腫瘤干細(xì)胞其表面標(biāo)志物是不盡相同的,這提示我們需要對不同的腫瘤進(jìn)行相應(yīng)研究,了解不同腫瘤中腫瘤干細(xì)胞的標(biāo)志物特點(diǎn)。 近年來,許多研究證實(shí),CD133是腫瘤干細(xì)胞的重要標(biāo)志物之一。CD133是一種分子量為120kDa,具有5個跨膜區(qū)的糖蛋白,屬于一種早期抗原。在2000年英國舉行的第7屆國際人類白細(xì)胞分化抗原研討會上被正式命名為CD133。它是一種正常的造血干細(xì)胞表面標(biāo)志物,它的一個顯著特點(diǎn)就是:CD133的表達(dá)隨著細(xì)胞的分化迅速下調(diào),選擇性地在骨髓和外周血造血干細(xì)胞及內(nèi)皮祖細(xì)胞表達(dá),但在成熟內(nèi)皮細(xì)胞上不表達(dá),是一個重要的分離和鑒定造血干或祖細(xì)胞的分子標(biāo)志。 侵襲能力是惡性腫瘤的標(biāo)志性行為特點(diǎn)之一,是當(dāng)前研究惡性腫瘤行為特點(diǎn)的一個重要方向。目前利用Transwell小室是體外研究腫瘤細(xì)胞侵襲和轉(zhuǎn)移行為的簡便且快速的方法。 本研究擬以CD133為分選標(biāo)志物,從人類FADU細(xì)胞系中分選出CD133陽性細(xì)胞,研究其數(shù)量分布特點(diǎn),形態(tài)學(xué)特點(diǎn),并擬通過應(yīng)用Transwell小室的研究方法,研究在FADU細(xì)胞系中CD133陽性細(xì)胞的侵襲能力,從而為未來完善對CD133陽性細(xì)胞擁有的細(xì)胞學(xué)特點(diǎn)研究提供相應(yīng)的細(xì)胞學(xué)實(shí)驗(yàn)室基礎(chǔ)。 第一部分 下咽癌FADU細(xì)胞系中CD133表達(dá)情況研究 [目的]研究下咽癌FADU細(xì)胞系中CD133表達(dá)情況。 [方法]對下咽癌FADU細(xì)胞進(jìn)行培養(yǎng)和傳代,分別應(yīng)用免疫細(xì)胞化學(xué)和流式細(xì)胞儀的方法對下咽癌FADU細(xì)胞系中CD133表達(dá)情況進(jìn)行研究。 [結(jié)果]免疫細(xì)胞化學(xué)的方法發(fā)現(xiàn),在下咽癌FADU細(xì)胞中CD133陽性細(xì)胞僅占少數(shù),而在利用流式細(xì)胞儀的方法研究后發(fā)現(xiàn),FADU細(xì)胞中CD133陽性細(xì)胞比例僅為2.01%。 [結(jié)論]CD133陽性細(xì)胞數(shù)量在FADU細(xì)胞系中占少數(shù)。 第二部分 下咽癌FADU細(xì)胞系中CD133陽性細(xì)胞的分選 [目的]應(yīng)用可靠的分選手段,從下咽癌FADU細(xì)胞系中分選CD133陽性細(xì)胞并鑒定。 [方法]對下咽癌FADU細(xì)胞進(jìn)行培養(yǎng)和傳代,應(yīng)用免疫磁珠分選技術(shù)進(jìn)行CD133陽性細(xì)胞的分選。 [結(jié)果]應(yīng)用免疫磁珠分選技術(shù)后,可很好地分選出CD133陽性細(xì)胞,其分選后CD133陽性細(xì)胞純度可達(dá)91.2%。 [結(jié)論]免疫磁珠分選技術(shù)可很好地應(yīng)用于下咽癌FADU細(xì)胞系中CD133陽性細(xì)胞的分選。 第三部分 下咽癌FADU細(xì)胞系中CD133陽性細(xì)胞的侵襲能力研究 [目的]研究下咽癌FADU細(xì)胞系中CD133陽性細(xì)胞的侵襲能力。 [方法]對應(yīng)用免疫磁珠分選技術(shù)進(jìn)行分選后的CD133陽性細(xì)胞、陰性細(xì)胞分別應(yīng)用Transwell小室進(jìn)行侵襲能力研究,倒置相差顯微鏡下照相,計(jì)數(shù)遷移到上室基質(zhì)膜外表面的細(xì)胞數(shù)目,以能穿過基質(zhì)膜的細(xì)胞數(shù)目多少表示細(xì)胞的侵襲能力強(qiáng)弱。每個樣本計(jì)數(shù)5個視野(×200)的細(xì)胞數(shù),取其平均值進(jìn)行比較。 [結(jié)果]與CD133陰性細(xì)胞相比,CD133陽性細(xì)胞透膜數(shù)量較陰性細(xì)胞明顯高,其穿過基質(zhì)膜的細(xì)胞數(shù)分別為54.0±2.3,17.0±1.1,其差異具有統(tǒng)計(jì)學(xué)意義,P0.01。 [結(jié)論]在下咽癌FADU細(xì)胞系中CD133陽性細(xì)胞比陰性細(xì)胞侵襲能力強(qiáng)。
[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
【學(xué)位授予單位】:山東大學(xué)
【學(xué)位級別】:博士
【學(xué)位授予年份】:2010
【分類號】:R739.63
本文編號:2286455
[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
【學(xué)位授予單位】:山東大學(xué)
【學(xué)位級別】:博士
【學(xué)位授予年份】:2010
【分類號】:R739.63
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1 王春梅,趙育楨,高凌寒,劉芳莉,李鈺,李璞;應(yīng)用重組基底膜侵襲技術(shù)對人肺腺癌細(xì)胞侵襲能力的研究[J];哈爾濱醫(yī)科大學(xué)學(xué)報;2000年05期
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