微量全血法高通量彗星试验方法的研究与应用  

Experimental method of micro-whole blood high-throughput comet assay and its application

作　　者：王庆荣[1,2] 李红丽 贾效伟 翟庆峰[1,2] 牛勇 戴宇飞[2] 郑玉新[2] 邱玉刚[1] 段化伟[2] 

机构地区：[1]潍坊医学院公共卫生与管理学院,山东潍坊261053 [2]中国疾病预防控制中心职业卫生与中毒控制所,北京100050

出　　处：《工业卫生与职业病》2018年第1期13-17,共5页Industrial Health and Occupational Diseases

基　　金：国家自然科学基金(81573124);国家科技支撑计划(2014BAI12B02)

摘　　要：目的建立一种简易快捷,适合大样本人群生物监测的微量全血法高通量彗星试验技术方法。方法选择1名不吸烟男性,探索微量全血法的彗星试验条件,建立了微量全血法的高通量彗星试验方法。并在柴油机尾气(diesel engine exhaust,DEE)暴露人群中进行验证。结果经过对铺胶量和细胞上样量的探索,发现30μl细胞悬液能够均匀铺设到彗星玻片的小孔中,胶片表面比较平整,细胞基本为单层,经裂解液处理后也不易脱落。上样量中发现5μl抗凝全血与200μl琼脂糖凝胶混合,细胞分散均匀,间距适当,电泳后无细胞重叠,图像清晰容易分辨,对比度较高。在方法学的验证中,高通量彗星试验的结果显示,在DEE暴露组较对照组的彗星尾长、尾矩和尾部DNA百分含量均明显增加,板间及板内变异度小于10%。结论全血法高通量彗星实验适用于化学致癌物和污染物遗传损伤的检测。Objective To establish a simple and rapid method of micro-whole blood high-throughput comet assay for large sample population biomonitoring.Methods A non-smoker was invited as volunteer to explore the experimental conditions and establish the method of micro-whole blood high-throughput comet assay.The comet assay method was used in diesel engine testing workers for validation.Results Through the exploration of the amount of glue and cell suspensions,we found that 30μL cell suspensions were uniformly pipetted into the wells of a 20-well Comet Slide,and the gel surface was smooth,and cells were single layer distribution.After submerging lysis solution,the gel was not easy to fall off.We also found that the image of 5μL of whole blood mixed with 200μL agarose was clear.After running electrophoresis,the cells were dispersed evenly.The contrast ratio was higher.By validating the method of micro-whole blood highthroughput comet assay,it was found that the tail length,the Olive tail moment and percentage of tail DNA was notably increased in DEE-exposed workers.The value of coefficients of variation for inter plate and intraplate were less than 10%.Conclusions Micro-whole blood high-throughput comet assay could be used the detection of chemical carcinogens and genetic damage.

关 键 词：彗星实验 微量全血 高通量 柴油机尾气(DEE) DNA损伤 

分 类 号：R331[医药卫生—人体生理学]