【作者】 陈亮；

【导师】 唐文渊；

【作者基本信息】 重庆医科大学 ， 外科学， 2010， 硕士

【摘要】 目的胶质瘤是严重危害人类健康的疾病之一,是一种最常见的颅内原发恶性肿瘤。近年来,胶质瘤的发病率和死亡率在国内外均有明显增加,其恶性程度高者预后十分差。胶质瘤细胞本身超乎寻常的局部侵袭、转移能力是疾病治疗过程中所面临的巨大挑战。研究表明,通过对肿瘤转移抑制因子的检测可预判患者预后的情况,甚至可以为胶质瘤的基因治疗提供潜在靶点的理论依据。已有研究表明CD82/KAI1、CD9/MRP-1在众多实体性恶性肿瘤转移和增殖中发挥作用,为进一步研究CD82/KAI1、CD9/MRP-1在胶质瘤细胞发生迁移、增殖中的作用,本文以两种恶性程度存在差异的胶质瘤细胞株(CHG-5、U251)为研究对象,采用细胞培养、免疫细胞化学、PT-PCR等方法从mRNA和蛋白水平初步检测CD82/KAI1、CD9/MRP-1的表达水平,探索其与胶质瘤细胞迁移力和增殖力的关系。方法1.免疫细胞化学技术分别观察CHG-5、U251中CD82/KAI1、CD9/MRP-1的表达情况;2. RT-PCR分别观察CHG-5、U251中CD82/KAI1mRNA、CD9/MRP-1mRNA的表达情况;3.分别设立1:100、1:200CD82/KAI1抗体干预组和空白对照组,通过划痕试验于24h、48h观察CD82/KAI1对细胞迁移能力的影响并进行统计分析;4.分别设立1:100、1:200、1:400 CD9/MRP-1抗体干预组和空白对照组,通过流式细胞术于24h、48h观察CD9/MRP-1对细胞周期的影响并进行统计分析。结果1. U251细胞组中CD82/ KAI1及CD9/MRP-1的蛋白水平表达均高于CHG-5细胞组,差异有显著性(P<0.01);2. U251细胞组中CD82/KAI1mRNA及CD9/MRP-1mRNA表达均高于CHG-5细胞组,差异有显著性(P<0.01);3. U251细胞CD82/KAI1抗体干预组迁移细胞数量高于对照组(P<0.05),且迁移细胞数量随抗体浓度升高而增加(P<0.05);4. CHG-5细胞CD9/MRP-1抗体干预组中细胞周期阻滞于G0/G1期,高于对照组(24h:P<0.05;48h:P<0.01),且比例随抗体浓度升高而增加(P<0.05);5. U251细胞CD9/MRP-1抗体干预组中细胞周期阻滞于S期,高于对照组(24h:P<0.01;48h:P<0.01),且比例随抗体浓度升高而呈现上升趋势(P<0.05)。结论CD82/KAI、CD9/MRP-1均在恶性胶质瘤细胞株中表达,且两者在U251细胞中表达较CHG-5细胞都高;CD82/KAI1可抑制胶质瘤U251细胞的迁移能力,CD9/MRP-1可抑制胶质瘤CHG-5细胞和U251细胞的增殖能力;推测CD82/KAI、CD9/MRP-1的检测能为胶质瘤临床治疗方案的选择和预后的评估提供一定依据。更多还原

【Abstract】 Objective: Glioma is one of the serious diseases which threat human beings, and one of the most common primary intracranial tumors. In recent years, morbidity and mortality rates of gliomas were significantly increased at home and abroad, their highly malignant and the prognosis are very poor. Glioma cells them exceptional local invasion and metastasis are enormous challenges of the disease course of treatment. Studies have shown that inhibitors of tumor metastasis by testing can predict prognosis of the situation, even for gene therapy of gliomas theoretical basis for potential targets. Studies have also shown that CD82/KAI1, CD9/MRP-1 play important roles in the many entities of tumor metastasis and proliferation. To further study the CD82/KAI1, CD9/MRP-1 in glioma cell migration and proliferation of the role, this paper differ in two malignant glioma cell lines (CHG-5, U251) for the study, using cell culture, immunocytochemistry, PT-PCR methods such as mRNA and protein levels from the initial detection of CD82/KAI1, CD9/ MRP-1 expression, in order to explore the relationship between the ability of tumor cell migration and proliferation and its expression. Methods: 1. Immunocytochemistry was observed CHG-5, U251 in CD82/KAI1, CD9/MRP-1 expression; 2. RT-PCR was observed CHG-5, U251 in CD82/KAI1mRNA, CD9/MRP-1mRNA expression; 3. 1:100, 1:200 CD82/KAI1 antibody were set up in the intervention group and control group, by wound healing in 24h, 48h observation CD82/KAI1 ability of cell migration and statistical analysis; 4. 1:100, 1:200, 1:400 CD9/MRP-1 antibody were set up in the intervention group and control group, by flow cytometry at 24h, 48h CD9/MRP-1 observed on the cell cycle and statistical analysis.Results: 1. The expression level of CD82 / KAI1 protein and CD9/MRP-1 protein in U251 cell group was higher than that in CHG-5 cells, the difference was significant (P < 0.01); 2.The expression level of CD82/KAI1mRNA and CD9/MRP-1 mRNA in U251 cell group was higher than that in CHG-5 cells, the difference was significant (P<0.01); 3.The number of migrated cells in U251 cell group which be intervened by CD82/KAI1 antibody were higher than that in their control group (P<0.05), and the number of migrated cells with the antibody concentration increased (P< 0.05); 4. Cell cycle arrest in G0/G1 phase in CHG-5 cell group which be intervened by CD9/MRP-1 antibody, difference compared with the control group(24h:P<0.05;48h:P<0.01), and the proportion increased with antibody concentration(P<0.05); 5. Cell cycle arrest at S phase in U251 cell group which be intervened by CD9/MRP-1 antibody, difference compared with the control group(24h:P<0.01;48h:P<0.01), and the proportion increased with antibody concentration(P<0.05).Conclusions : CD82/KAI, CD9/MRP-1 expressed in malignant glioma cell lines, and both in U251 cells compared with CHG-5 cells are high;CD82/KAI1 inhibits U251 cell migration, CD9 / MRP-1 can inhibit CHG-5 cells and U251 cell proliferation;We speculate CD82/KAI, CD9/MRP-1 clinical testing will contribute to glioma treatment options and prognosis.更多还原