哺乳类动物细胞株HGPRT基因突变电化学试验方法的建立及机制研究

For a long time, only genetic end point was employed as detection point of gene mutation in the traditional detection method of the gene mutation, and the free markers, fast, in-situ, and real-time detection was not realized. In this project, using the cell electrochemical method, basing on four purine bases and uric acid as biomaker, the multi-signals electrochemical detection system of purine nucleotide metabolism was established. The electrochemical spectrogram of relation of purine nucleotide metabolism and HGPRT gene mutation as the target genes was built based on the above detection system. Variation law of purine nucleotide metabolism caused by HGPRT gene mutant was analyzed. The mechanisms of detecting HGPRT gene mutation using electrochemical method are revealed. The electrochemical test of HGPRT gene mutation of mammalian cell lines was established. It was realized that the nucleotide metabolism changes are evaluated using the electrochemical method with multiple-index, low-cost, real-time druing the gene mutation assay, the false-negative from traditional test methods was eliminated, and environment pollutions are evaluated. The subject should open up a new way to detecting gene mutation by electrochemical method, provide a new technology for mammalian mutagenicity tests, and create the new pathway for pharmacological studies related to purine nucleotide metabolism.

长期以来，传统基因突变检测法仅能采用遗传学终点作为基因突变检测点，无法实现无标记、快速、原位、实时监测。本课题拟采用细胞电化学技术，以四种嘌呤碱基及尿酸为生物标记物，建立细胞嘌呤核苷酸代谢的多信号电化学检测系统；采用该系统，以HGPRT基因为靶基因，构建HGPRT基因突变与嘌呤核苷酸代谢关系的电化学图谱，分析HGPRT基因突变导致的嘌呤核苷酸代谢的变化规律，揭示电化学法检测HGPRT基因突变机理，建立哺乳动物细胞株HGPRT基因突变电化学试验方法。实现对基因突变试验过程中核苷酸代谢变化的多指标、低费用、快速、实时检测，消除传统试验方法出现假阴性结果的可能，并对环境致突污染物进行评价。本课题的研究开辟了电化学法检测基因突变的新途径，可为哺乳动物致突变试验提供新的技术方法，同时为嘌呤核苷酸代谢相关通路的药理研究提供新的研究手段。

随着工业化和城市化的快速发展，诱发基因突变的因素急剧增加，传统基因突变检测法仅能采用遗传学终点作为基因突变检测点，无法实现无标记、快速、原位、实时监测。本课题采用细胞电化学技术，以四种嘌呤碱基及尿酸为生物标记物，建立了原位细胞嘌呤核苷酸代谢的三信号电化学微检测系统；采用该系统，以HGPRT基因为靶基因，构建HGPRT基因突变与嘌呤核苷酸代谢关系的电化学图谱，分析HGPRT基因突变导致的嘌呤核苷酸代谢的变化规律，揭示电化学法检测HGPRT基因突变机理，建立哺乳动物细胞株HGPRT基因突变电化学试验方法。检测结果与突变克隆检测法检测结果基本一致，试验周期由传统方法的15天缩短至5天，在分子水平上快速检测HGPRT基因突变。实现对基因突变试验过程中核苷酸代谢变化的多指标、低费用、快速、实时检测，并对环境致突污染物进行评价。本课题的研究开辟了电化学法检测基因突变的新途径，可为哺乳动物致突变试验提供新的技术方法，利用HGPRT基因突变电化学试验方法评价环境毒物的致突性，具有重要的科学意义和应用前景，同时为嘌呤核苷酸代谢相关通路的药理研究提供新的研究手段。