热休克蛋白70在乳腺癌中表达的意义及其对体外乳腺癌细胞株生物学功能的影响

发布时间：2018-02-24 00:28

本文关键词： 乳腺癌热休克蛋白无疾病进展预后乳腺肿瘤热休克蛋白转染细胞培养凋亡　出处：《武汉大学》2015年博士论文　论文类型：学位论文

【摘要】：第一部分热休克蛋白70在乳腺癌组织乳腺中的表达及临床价值分析乳腺癌目前是我国女性最常见的恶性肿瘤,占癌症死亡原因中位的第六位,严重威胁女性身心健康。随着分子病理和分子生物学的发展,为进一步提高乳腺癌早期诊断、综合治疗以及预后判断的水平,寻找与乳腺癌复发、转移相关生物学标志物,在乳腺肿瘤领域开展了大量的临床研究。与此同时,研发针对生物学标志相关靶点的抗癌药物,也成为乳腺癌基础与临床研究热点,其中就包括热休克蛋白在乳腺癌中的作用。本文通过定性和定量的方法,分析了热休克蛋白70在不同乳腺组织类型中的表达,及其与分子生物学之间的特征和关系,探讨其是否为客观预测乳腺癌转移和预后的生物学指标,为乳腺癌的诊治及预后判断提供一定的实验依据。目的：观察热休克蛋白70在乳腺癌、不典型增生、癌旁组织和正常组织中的表达,分析其与乳腺癌相关因素之间的关系。方法：收集经手术切除病理确诊的乳腺癌病例160例,获得完整的临床资料,分别应用定性和定量方法,检测HSP70在不同乳腺组织中的表达,并分析其与乳腺癌相关因素的关系。结果：1.HSP70蛋白在乳腺癌组织中存在一定水平的表达,且表达与肿瘤大小、腋窝淋巴结转移数目、HR状态和Her-2状态呈现明显相关性,但和病理类型、月经状态、组织学分级无明显相关性；同时HSP70的表达对无疾病进展时间和总生存时间存在相关性：在HSP70阳性组(n=70),无疾病进展时间为24.0月(95%CI：22.0-26.1月),而在HSP70阴性组(n=50),无疾病进展时间33.0月(95%CI：29.8-36.2月),Log Rank=11.6, P=0.001。Kaplan-Meier分析发现,在HSP70阳性组(n=70),总体生存时间为32.0月(95%CI：26.8-37.2月),而在HSP70阴性组(n=50),总体生存时间为39.0月(95%CI：29.7-48.3月),Log Rank=6.9, P=0.012。2. Real-timePCR方法检测HSP70 mRNA在乳腺癌和不典型增生中的表达明显高于单纯乳腺增生(P0.05),但从不典型增生至乳腺癌的过程中HSP70的表达无明显变化(P=0.82); HSP70mRNA的表达与肿瘤大小,病理类型,组织学分级,PR状态,Her2状态,腋窝淋巴结转移状况无关,与ER表达呈正相关(相关系数rs=0.792,P0.001)。结论：HSP70在乳腺癌组织中有一定程度的表达,HSP70阳性表达与乳腺癌病人预后较差有关。第二部分HSP70基因的表达对体外乳腺癌细胞株生物学功能的影响我们前期的研究显示,HSP70的表达和乳腺癌的临床病理特征有关,是乳腺癌病人预后不良的指标。为进一步了解其作用的机制,我们应用了基因转录方法检测HSP70基因表达对体外乳腺癌MDA-MB-231细胞株的抗增殖作用和生物学功能的影响：通过Western-blot和Real-timePCR技术检测HSP70mRNA在MDA-MB-231细胞中的表达水平,并观察比较转染后MDA-MB-231细胞的迁移能力,黏附能力和形态学改变等生物学功能指标,为进一步揭示HSP70在乳腺癌发生发展中的作用机制提供一定的实验依据。目的：探讨热休克蛋白70基因表达对体外乳腺癌细胞系生物学功能的影响。方法：构建HSP70mRNA慢病毒载体,应用基因转录方法检HSP70基因表达对体外乳腺癌MDA-MB-231细胞系的抗增殖作用和生物学功能的影响；通过Western-blot和Real-time PCR技术检测HSP70mRNA在MDA-MB-231细胞中的表达水平,并通过MTT法,Transwen小室法、流失细胞技术和倒置显微镜等观察比较转染HSP70mRNA基因的MDA-MB-231细胞的迁移能力,黏附能力和形态学改变。结果：1.含HSP70mRNA慢病毒载体转染的MDA-MB-231细胞,HSP70的表达在mRNA和蛋白水平均明显增强,其HSP70蛋白的水平是未转染组的2.7和3.5倍.2.MTT法检测转染组MDA-MB-231细胞的增殖活性,粘附能力明显增强(p0.05)；倒置显微镜下观察,转染组细胞,72h后生长状况良好,未见明显细胞减少,并可见阳性克隆；流式细胞仪检测不同转染组MDA-MB-231乳腺癌细胞在加入药物后24h、48h、72h后细胞凋亡率,其中48h后转染组细胞凋亡率为1.17%,未转染组为17.81%,空转染组为19.80%(P0.05)；转染组细胞培养48h后,划痕边缘的细胞逐渐进入空白区域,培养72h后细胞“划痕”基本愈合；高表达HSP70mRNA的转染组细胞具有明显的迁移和侵袭能力.结论：HSP70基因转染对体外乳腺癌MDA-MB-231细胞系具有促进细胞增殖、侵袭和抗凋亡的作用,导致肿瘤的复发和转移,有可能是影响乳腺癌患者预后的因子之一。
[Abstract]:The first part and the clinical value of heat shock protein 70 in breast cancer breast tissue expression analysis of breast cancer is currently one of the most common malignant tumors in women, the sixth leading cause of cancer deaths accounted for, a serious threat to women's physical and mental health. With the development of molecular pathology and molecular biology, to further improve the early diagnosis of breast cancer. The level of comprehensive treatment and prognosis, and for breast cancer recurrence, metastasis related biomarkers in breast cancer has carried out a large number of clinical studies. At the same time, the anti-cancer drug development target for biological markers related to breast cancer, has become the basic and clinical research, including the effects of heat shock protein in breast cancer in this paper, through qualitative and quantitative methods, analysis of the heat shock protein 70 in different breast tissue type expression and molecular biology The characteristics and relationships, to discuss whether or not the objective prediction of breast cancer metastasis and prognosis of biological indicators, and provide experimental basis for diagnosis and prognosis of breast cancer. Objective: To observe the effect of heat shock protein 70 in breast cancer, atypical hyperplasia, the expression of adjacent tissues and normal tissues, analyze the relationship between related and the factors of breast cancer. Methods: surgically resected pathologically diagnosed 160 breast cancer patients were obtained with complete clinical data, using qualitative and quantitative methods, to detect the expression of HSP70 in different breast tissues, and analyze the relationship between the related factors of breast cancer. Results: the expression of 1.HSP70 protein in a certain level in breast cancer, and the expression and tumor size, number of metastatic lymph nodes, HR status and Her-2 status showed significant correlation, but with pathological type, menstrual status, histological grade of ignorance Significant correlation; and correlation of progression free and overall survival time and the expression of HSP70 in HSP70 positive group (n=70), progression free time was 24 months (95%CI:22.0-26.1 months), while in HSP70 negative group (n=50), progression free time of 33 months (95%CI: 29.8-36.2 months), Log Rank=11.6, P=0.001.Kaplan-Meier the analysis found that in HSP70 positive group (n=70), overall survival time was 32 months (95%CI:26.8-37.2 months), while in HSP70 negative group (n=50), overall survival time was 39 months (95%CI:29.7-48.3 months), Log Rank=6.9, HSP70 mRNA Real-timePCR method to detect the expression of P=0.012.2. in breast cancer and atypical hyperplasia was significantly higher than that of control breast hyperplasia (P0.05), but the expression of HSP70 from atypical hyperplasia to breast cancer in no significant change (P=0.82); the expression of HSP70mRNA and tumor size, pathological type, histological grade, PR status, He R2 status, axillary lymph node metastasis, it was positively correlated with ER expression (correlation coefficient rs=0.792, P0.001). Conclusion: HSP70 has a certain degree of expression in breast cancer tissues, the positive expression of HSP70 correlates with poor prognosis in breast cancer patients. Our previous expression of partial HSP70 gene second influence on the biological function of breast in vitro cancer cell lines showed that the expression of HSP70 and clinicopathological characteristics of breast cancer and poor prognosis of patients with breast cancer. In order to further understand the mechanism of the effect, we use the method to detect the gene transcription of HSP70 gene on the expression of MDA-MB-231 in breast cancer cells in vitro antiproliferative effect and biological function through the expression: to detect the level of HSP70mRNA Western-blot and Real-timePCR in MDA-MB-231 cells, and to observe the migration ability of MDA-MB-231 cells after transfection, cell adhesion Biological function index ability and morphological change, in order to further reveal the HSP70 in breast cancer in the development and provide experimental basis. Objective: To investigate the effect of heat shock protein 70 gene expression on the biological features of breast cancer cells in vitro. Methods: HSP70mRNA lentivirus vector construction, expression of application of gene transcription method to detect HSP70 the gene of breast cancer MDA-MB-231 cells in vitro anti proliferation and biological function; by detecting the expression level of HSP70mRNA Western-blot and Real-time PCR in MDA-MB-231 cells, and by MTT method, Transwen assay, cell migration ability loss of technology and inverted microscope observation and comparison of HSP70mRNA gene transfected MDA-MB-231 cells, changes of adhesion ability results: 1.. The morphological and HSP70mRNA containing lentiviral vector transfected MDA-MB-231 cells, HSP70. As at mRNA and protein levels were significantly increased, the level of HSP70 protein was 2.7 in non transfection group and 3.5 times by.2.MTT transfection group MDA-MB-231 cell proliferation and adhesion ability was significantly enhanced (P0.05); inverted microscope, transfected cells, 72h growth in good condition, no obvious cells decreased, and visible positive clones; flow cytometry in different transfection group MDA-MB-231 breast cancer cells in 24h after adding the drug, 48h, the rate of apoptosis of 72h cells, 48h transfected cells apoptosis rate was 1.17%, the untreated group was 17.81%, blanktransfected group was 19.80% (P0.05); transfected cells after 48h culture, scratch the edge of the cells gradually into the blank area, "scratch" 72h cells after healing; high expression of transfected cells with HSP70mRNA migration and invasion significantly. Conclusion: the transfection of HSP70 gene in breast cancer MDA-MB- in vitro 231 cell line has the effect of promoting cell proliferation, invasion and anti apoptosis, leading to tumor recurrence and metastasis. It may be one of the factors that affect the prognosis of breast cancer patients.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9

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