CHO细胞微核的全自动化检测技术研究

发布时间：2018-06-10 16:09

本文选题：CHO细胞微核 + 自动化　；参考：《昆明理工大学》2012年硕士论文

【摘要】：微核实验是一种以观察细胞中微核的形成来检测遗传毒性的毒理学分析方法,能经济、简便、准确地检测各种理化因子对细胞的遗传损伤,在化合物遗传毒性检测方面发挥着重要作用。传统微核实验耗时费力、检测结果易受主观因素影响,很难满足快速、大量检测化合物的需要,而基于仪器的微核自动化检测技术可以达到快速、高通量检测化合物的遗传毒性的目的。本论文选择了国内外毒理学检测常用的细胞株之一中国仓鼠卵巢细胞(CHO细胞)作为材料,利用流式细胞仪和激光扫描细胞仪来研究体外微核的全自动检测技术,旨在建立CHO细胞体外微核的流式细胞仪自动化检测方法和激光扫描细胞仪自动化检测方法,以达到快速准确地进行化合物遗传毒性检测的目的。流式细胞仪方法利用体外微核分析试剂盒对染色体断裂剂环磷酰胺和非整倍体剂秋水仙碱引起的CHO细胞微核进行了分析和研究,并与传统的显微镜检测方法相比较,参考美国国家临床实验室实验室标准化委员会EP9-A文件的方法,对两种方法的微核检测结果进行离群值检验、取值范围判断以及回归分析。研究结果显示,流式细胞仪自动化检测方法和传统的显微镜检测方法具有良好相关性,由此建立了一种CHO细胞体外微核的流式细胞仪自动化检测方法,为了考察该方法的可靠性和适用性,并将所建立的流式细胞仪方法应用于食品添加剂、化妆品(染发剂)、药品、农药、水体、重金属离子、卷烟烟气等诱发的CHO细胞微核检测,同时与传统的微核显微镜检法相比较分析。结果表明,二种方法的测定值无明显离群值,相关性良好,说明该方法可以适于食品添加剂、化妆品(染发剂)、药品、农药、水体、重金属离子、卷烟样品总粒相物诱发的体外细胞遗传损伤的检测,结果准确可靠。激光细胞仪方法结合细胞胞质分裂阻断微核技术,采用两种荧光染料,对环磷酰胺(CP)诱导的CHO细胞微核进行了研究,并将该方法与传统的微核显微镜检法进行比较分析,结果显示两种方法的测定值无明显离群值,二者相关性良好,由此建立了一种CHO细胞体外微核的激光扫描细胞仪自动化检测方法,并利用该方法对13种不同类型的物质处理的CHO细胞进行微核检测,同时与显微镜方法相比较,研究结果表明该方法评价结果稳定可靠,能够快速高通量检测多种类型的化合物所诱导的微核。本研究所建立的体外细胞微核流式细胞仪方法和激光扫描细胞仪方法最大的优点是方法简便、检测速度快、准确性高,可适用性强,为体外细胞微核的自动化检测方法的研究提供参考,同时也为化合物诱变性的筛选、食品毒理学检测和环境污染检测等提供了快速检测方法。
[Abstract]:Micronucleus assay is a toxicological analysis method to detect genotoxicity by observing the formation of micronucleus in cells. It can be used to detect the genetic damage caused by various physical and chemical factors in an economical, simple and accurate way. It plays an important role in the detection of genetic toxicity of compounds. The traditional micronucleus experiment is time-consuming and laborious, and the test results are easily influenced by subjective factors, so it is difficult to meet the needs of rapid and mass detection of compounds. However, the instrument-based micronucleus automated detection technology can achieve rapid results. The aim of this study was to detect the genotoxicity of compounds by high throughput. In this paper, Cho cells, one of the commonly used cell lines of Chinese hamster ovary, were selected as materials. Flow cytometry (FCM) and laser scanning cell analyzer (LSCC) were used to study the automatic detection technique of micronucleus in vitro. The aim of this study was to establish an automatic detection method for in vitro micronucleus of Cho cells by flow cytometry and laser scanning cell analyzer. In order to detect the genotoxicity of compounds quickly and accurately, the micronucleus of Cho cells induced by chromosome breaking agent cyclophosphamide and aneuploidy colchicine was detected by flow cytometry with in vitro micronucleus analysis kit. Has carried on the analysis and the research, Compared with the traditional microscopical methods and referring to the EP9-A document of the National Committee of Clinical Laboratory Standardization, the outlier values, the range of values and the regression analysis of the results of the two methods were tested. The results showed that there was a good correlation between the flow cytometry automated detection method and the traditional microscope detection method, so a flow cytometry automatic detection method for Cho cell micronucleus in vitro was established. In order to investigate the reliability and applicability of the method, the flow cytometry was used to detect the micronucleus of Cho cells induced by food additives, cosmetics (hair dye, drugs, pesticides, water, heavy metal ions, cigarette smoke, etc.) At the same time, it is compared with the traditional micronucleus microscopy. The results show that there is no obvious outlier and the correlation between the two methods is good. The results show that the method is suitable for food additives, cosmetics (hair dye, medicine, pesticide, water, heavy metal ions). The detection of in vitro cell genetic damage induced by total granulocytes in cigarette samples was accurate and reliable. Two kinds of fluorescent dyes were used in combination with cytoplasmic division blocking micronuclear technique. The micronucleus of Cho cells induced by cyclophosphamide (CPN) was studied and compared with the traditional micronucleus microscopy. The results showed that there was no significant outlier between the two methods and there was a good correlation between them. A laser scanning cytometer (LSCC) method was developed to detect the micronucleus of Cho cells in vitro. The method was used to detect the micronuclei of Cho cells treated with 13 different types of substances, and the results were compared with the microscopic method. The results show that the evaluation results of this method are stable and reliable. The method of in vitro micronucleus flow cytometry and laser scanning cytometer was developed to detect micronucleus induced by many kinds of compounds. The advantages of this method are that the method is simple, the detection speed is fast, and the accuracy is high. It provides a reference for the study of automated detection of micronucleus in vitro, as well as a rapid detection method for the screening of mutagenicity of compounds, the detection of food toxicology and the detection of environmental pollution.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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