【作者】 何丽芳；

【导师】 王柯敏；

【作者基本信息】 湖南大学 ， 分析化学， 2006， 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的改变具有密切联系。因此,建立快速、准确、灵敏检测基因表达水平的方法,对于肿瘤的发病机理研究、早期诊断和治疗具有重要意义。本论文设计了具有特异识别功能的荧光分子探针,发展了在基因水平上定量研究肿瘤细胞p53 mRNA表达与点突变检测的体系,主要开展了以下三方面的研究工作:1、根据p53抑瘤基因的碱基序列,设计了能特异性检测p53 mRNA的分子信标(molecular beacon,MB),发展了一种快速定量测定细胞内总RNA提取物中p53 mRNA的方法。采用鼻咽癌细胞系(CNE2)和经RNA干扰技术降低p53基因表达的细胞系(CNE2-p53RNAi)为样本,抽提其总RNA并用MB检测。建立了p53/cDNA标准曲线,将其应用于多种肿瘤细胞内p53基因表达水平的分析,表达变化趋势与经典的mRNA分析方法RT-PCR检测结果相符。这种方法简单、灵敏、快速、安全,可以用于不同细胞中p53 mRNA的体外定量检测。2、根据已建立的分子信标定量检测p53 mRNA的方法,考察了用5-氟尿嘧啶(5-FU)作用不同时间后肺腺癌细胞(A549)中p53 mRNA表达水平的变化,并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,验证了分子信标快速检测mRNA方法的可靠性和实用性。结果表明:药物作用后能诱导A549细胞中p53 mRNA表达上调,且作用6小时后p53 mRNA表达最高,但随着药物作用时间的进一步增加,p53 mRNA表达降低。该方法快速、准确、成本低,能够快速地获知该药物对细胞内p53 mRNA表达影响的信息,有望为肿瘤的个体化治疗快速、准确地提供合理用药的参考依据。3、根据p53基因的碱基序列设计荧光基团四甲基罗丹明标记的核酸探针(荧光探针)和熄灭基团4’-(4’-二甲基氨基叠氮苯)苯甲酸标记的核酸探针(熄灭探针),基于荧光共振能量转移原理和RNaseH能降解RNA与DNA杂交体中RNA模板这一特点,发展了一种直接对RNA模板进行点突变检测的新方法。在此基础上,对肿瘤细胞抽提物中p53 mRNA进行点突变检测,检测结果与基因测序结果相符。这种方法简单、快速、安全,能够广泛应用于不同细胞中p53 mRNA点突变的检测。更多还原

【Abstract】 As one of the diseases that threaten human being health, malignant tumors have been studied extensively. A considerable amount of molecular biology data indicates that carcinogenesis process is accompanied by mRNA expression level change of tumor-associated genes. It is essential to establish a quick, accurate and sensitive method for detecting mRNA expression level. In this thesis, A series of molecular probes were developed to detect mRNA expression level of tumor suppressor gene p53 in total RNA quantitatively and mRNA point mutation of tumor cells. This thesis is composed of the following three parts:1. A p53 molecular beacon (p53 MB) was designed according to the base sequence of tumor suppressor gene p53 to detect p53 mRNA in extracted total RNA of tumor cells quantitatively and quickly. The specificity of the p53 MB was tested by hybridizing the MB with total RNA extracted from nasopharyngeal cancer cell lines (CNE2) and CNE2-p53RNAi cell lines in which p53 mRNA expression was reduced by RNA interference. A p53/cDNA standard curve was established to quantify p53 mRNA expression level in various cell lines. The results were in consistent with that obtained from traditional reverse transcription polymerase chain reaction (RT-PCR) method. The proposed method is simple, sensitive and suitable for detecting p53 mRNA expression level in various kinds of cell lines.2. Based on the above MB quantitative assay, the p53 mRNA expression level in lung adenocarcinoma cells (A549) treated with 5-fluorouracil (5-FU) for different time was studied. The reliability of MB detection results was further proved by classical molecular biology methods. The results showed that p53 mRNA expression level in tumor cells was upregulated by 5-FU. The highest expression level of p53 mRNA was observed when the cells treated with 5-FU for 6 hours, then the expression level downregulated for longer treatment time. The method can provide information about the influence of drugs on p53 mRNA expression level in the cells rapidly and accurately. It is expected to provide reasonable drug selectivity for the individualization of cancer treatment quickly and accurately.3. A new method for mRNA point mutation detection using fluorescence resonance energy transfer pairs was developed. Two kinds of nucleic acid probes were designed. One was labeled with tetramethylrhodamine (TAMRA) and the other with 4-[4’-dimethylamino-phenylazo] benzoic acid (DABCYL). The results showed that single base mutation could be identified. The p53 mRNA of tumor cells was also更多还原