【作者】 王炜；

【导师】 王柯敏；

【作者基本信息】 湖南大学 ， 分析化学， 2005， 硕士

【摘要】 恶性肿瘤是严重威胁人类健康的疾病之一,越来越多的研究表明恶性肿瘤发生过程与细胞内相关基因表达水平改变具有密切联系。因此建立快速、准确、灵敏检测基因表达水平的方法,对于肿瘤的早期诊断、发病机理研究和治疗具有重要意义。本论文从分子信标定量检测肿瘤抑制基因mRNA表达水平入手,发展了在基因水平上研究基因间的相互作用和基因表达与细胞药物敏感性关系的体系。第一部分,首先建立高表达p33ING1鼻咽癌HNE1细胞模型,以细胞内一种重要的肿瘤抑制基因——p21的序列设计分子信标,发展了基于分子信标直接在总RNA中定量检测靶mRNA的方法。利用建立的p21/cDNA标准曲线,初步考察了肿瘤抑制基因p33ING1转染和化疗药物5-氟尿嘧啶(5-FU)处理对于p21mRNA表达水平的影响。结果显示基因转染和5-FU处理都能诱导p21mRNA表达水平的上调,利用p21分子信标检测的样品中p21mRNA拷贝数在1.92×109至8.30×1010/μg总RNA之间。该方法快速、准确、成本低的优点有望用于肿瘤相关基因的定量检测和对各种药物的疗效评价。第二部分,在上一章建立的分子信标定量检测mRNA的方法基础上,考察了鼻咽癌HNE1细胞p33ING1转染前后该基因mRNA表达水平的变化,结合传统分子生物学方法研究该基因转录水平变化对细胞增殖的影响及可能机制,并从蛋白质水平验证分子信标快速检测mRNA方法的可靠性和实用性。结果表明:p33ING1基因能通过增强p53信号通路相关蛋白表达和抑制STAT3信号通路蛋白的表达水平两种途径来实现抑制细胞增殖。第三部分,本研究利用分子信标定量检测mRNA的方法,系统考察了5-FU作用浓度和时间对不同鼻咽癌细胞系中p33ING1mRNA表达水平的影响。并通过传统分子生物学手段,考察了p33ING1基因高表达的细胞对于5-FU的敏感程度和产生的分子机制。发现增加药物浓度和延长作用时间都能诱导p33ING1 mRNA水平上调,并且其上调程度与初始p33ING1基因表达水平相关,表达高的细胞中这种诱导作用更明显。MTT结果表明p33ING1基因表达水平上调能增强鼻咽癌细胞对5-FU敏感性。表明该方法可望为肿瘤的个体化治疗提供快速、准确、合理用药提供参考依据。更多还原

【Abstract】 As one of the gene diseases that threaten human being health, malignant cancer has been studied extensively during past years. A considerable amount of data on molecular biology indicates that carcinogenesis process notably accompanied by mRNA expression level change of tumor-associated genes. So to establish a quick, accurate and sensitive method for detecting mRNA expression level is essential. Based on molecular beacon, the thesis has developed application systems to detect mRNA expression level of tumor suppressor gene p21 and p33ING1 in total cellular RNA quantitatively and quickly. The thesis is composed of three major parts: In the first part, a new method for detecting p21mRNA in trace total RNA samples was developed based on molecular beacon (MB). p21 MB was synthesized for the determination of a well-known p53 activating tumor suppressor gene p21 mRNA in p33ING1 overexpression nasopharyngeal carcinoma( NPC) cell line. Furthermore, The effect of p33ING1 gene transfection and chemical drug Fluorouracil( 5-FU ) treatment on p21 mRNA expression was quantitatively measured combining with p21/cDNA standard curve. The data that consistent with results obtained from experiment employing traditional reverse transcription polymerase chain reaction (RT-PCR) method showed that p21 mRNA expression level could be enhanced not only through p33ING1 plasmid transfection but also by 5-FU treatment. The copies of p21mRNA were between 1.92×109 and 8.30×1010/μg. The proposed method is suitable for cancer diagnosis and drug evaluation. In the second part, the p33ING1mRNA expression level in NPC cells with plasmid transfection or not were studied based on MB quantitative detection assay. The reliability of MB detection results was further proved from protein level by molecular biology method. Results suggested that p33ING1 could inhibit cell proliferation not only by inducing the expression of proteins on p53 pathway but also by restraining the expression of proteins on STAT3 pathway. In the last part, p33ING1 mRNA level in cellular total RNA was detected quantitatively based on MB assay in order to conduct the fundamental investigation of the effect of 5-FU on HNE1 cells. During the operation, the effects of 5-FU concentrations and treatment time in HNE1 cells and HNE1 cells transfected by p33ING1 were measured in vitro. Traditional molecular biochemistry methods were applied to explore whether p33ING1 could enhance 5-FU induced apoptosis in HNE1 cells and its molecular mechanism. The results were as follows: p33ING1 mRNA expression level in tumor cells was enhanced not only by the 5-FU concentration but also by lapse of time. The MTT results also proved that high expression of p33ING1 mRNA could increase cell’s sensitivity to chemical drug 5-FU. The detection method based on molecular beacon can be used to provide useful evidence quickly and quantitatively for clinical individual therapy and new chemical drugs development.更多还原