MEASUREMENT AND CHARACTERIZATION OF MICRONUCLEI IN EXFOLIATED HUMAN-CELLS BY FLUORESCENCE IN-SITU HYBRIDIZATION WITH A CENTROMERIC PROBE
MEASUREMENT AND CHARACTERIZATION OF MICRONUCLEI IN EXFOLIATED HUMAN-CELLS BY FLUORESCENCE IN-SITU HYBRIDIZATION WITH A CENTROMERIC PROBE
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DOI：
10.1016/0165-1161(94)90007-8
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发表时间：
1994-02-01
期刊：
影响因子：
--
通讯作者：
SMITH, MT
中科院分区：
文献类型：
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作者：
TITENKOHOLLAND, N;MOORE, LE;SMITH, MT
人脱落细胞微核（MN）测定已被广泛用于检测环境诱变剂、传染病和遗传性疾病的遗传毒性效应。不同研究小组报告的MN频率具有显著的可变性。其中一个原因可能是常规使用的Feulgen-Fast-Green染色的分辨率有限。在这里，我们描述了一个新版本的MN测定，采用荧光碘化丙啶染色和荧光原位杂交（FISH）与着丝粒探针。从5名未暴露的健康女性志愿者中收集口腔和尿路上皮细胞，并分析55000个细胞MN频率和异常核事件。Feulgen-Fast-Green和新的荧光染色产生了非常相似的结果。口腔细胞MN的频率为0.145 +/- 0.118%，尿路上皮细胞为0.083 +/- 0.074%。两种脱落细胞中MN的频率无相关性。使用着丝粒探针的FISH允许含有整条有着丝粒的染色体的MN与仅含有无着丝粒片段的MN区分开来。前者是有丝分裂时染色体滞后的结果，而那些没有着丝粒的是由于染色体断裂。在尿路上皮细胞中，43%的MN为着丝粒阴性，颊细胞中为44%。荧光染色比标准Feulgen-Fast-Green染色更准确地评分退行性细胞。纤裂细胞、“破卵”细胞和核裂细胞的合并频率不超过2%，而核分裂和核溶解的合并频率高达21%。发生核溶解和核分裂的细胞频率存在显著的个体间差异。因此，新版本的微核检测可以更精确地对MN进行评分，确定MN形成的机制，并很容易地识别脱落的人类细胞中的异常核事件。因此，使用口腔、膀胱和鼻子上脱落的细胞来研究人类遗传毒性是理想的。
The micronucleus (MN) assay in human exfoliated cells has been widely used to detect the genotoxic effects of environmental mutagens, infectious agents and hereditary diseases. Substantial variability characterizes the MN frequencies reported by different research groups. One reason for this may be the restricted resolution power of the Feulgen-Fast-Green staining that is routinely used. Here we describe a new version of the MN assay that employs fluorescent propidium iodide staining along with fluorescence in situ hybridization (FISH) with a centromeric probe. Buccal and urothelial cells were collected from 5 healthy unexposed female volunteers and 55 000 cells analyzed for MN frequency and abnormal nuclear events. The Feulgen-Fast-Green and the new fluorescent staining produced very similar results. The frequency of MN in buccal cells was 0.145 +/- 0.118% and in urothelial cells 0.083 +/- 0.074%. No correlation was found between the frequencies of MN in the two types of exfoliated cells. FISH with a centromeric probe allowed MN containing whole chromosomes with a centromere to be differentiated from those containing only acentric fragments. The former appear as a result of chromosome lagging in mitosis, while those without a centromere are due to chromosome breakage. In urothelial cells 43% of MN were centromere-negative and in buccal cells - 44%. Fluorescent staining provided more accurate scoring of degenerative cells than standard Feulgen-Fast-Green staining. The combined frequency of pycnotic cells, ''broken eggs'' and cells with fragmented nuclei did not exceed 2%, while that of karyorrhexis and karyolysis together was as high as 21%. Significant interindividual variability was found in the frequency of cells with karyolysis and karyorrhexis. Thus, the new version of micronucleus assay allows for MN to be scored more precisely, the mechanism of MN formation to be determined and abnormal nuclear events to be readily identified in exfoliated human cells. It is therefore ideal for studying genotoxicity in human populations using exfoliated cells from the mouth, bladder and nose.