Grawé J, Abramsson-Zetterberg L, Eriksson L, Zetterberg G
Department of Genetics, Uppsala University, Sweden.
Mutagenesis. 1994 Jan;9(1):31-8. doi: 10.1093/mutage/9.1.31.
A fixation method for mouse bone marrow erythrocytes was developed which allows both flow cytometric enumeration of micronucleated polychromatic erythrocytes (MPCEs) and detection of centromeres using fluorescent in situ hybridization (FISH) with a mouse gamma satellite probe on flow-sorted MPCEs. Male CBA mice were treated with clastogens or spindle poisons: X-irradiation (XR; 0.5 Gy), cyclophosphamide (CPA; 80 mg/kg), vincristine sulphate (VCR; 0.125 mg/kg) and colchicine (COL; 1 mg/kg). At 30 and 50 h after treatment bone marrow suspensions were prepared and subsequently analysed by flow cytometry to enumerate and determine the DNA content of induced MPCEs. The mean DNA content in MPCEs was found to be higher after treatments with the two aneugens, VCR and COL, than with the two clastogens, CPA and XR. The mean DNA content of MPCEs was positively correlated with the mean proportion of micronuclei (MN) containing centromeres, indicating that determination of the mean DNA content alone can give information about the mechanism of MN induction. When the MPCEs induced with VCR were outsorted according to four classes of increasing DNA content and the presence or absence of centromeres was determined with FISH, it was found that only the class with the lowest DNA content (0.8-1.7% of the diploid DNA content) had a low proportion (< 20%) of centromere-containing micronuclei while in the three classes with higher DNA content (1.7-10.2% of the diploid DNA content) > 80% of the MN had centromeres. This was in contrast to the results from treatments with CPA where the proportion of centromere-containing MN did not increase with increasing DNA content.
开发了一种用于小鼠骨髓红细胞的固定方法，该方法既允许对微核多色红细胞（MPCEs）进行流式细胞术计数，又能在对流式分选的MPCEs上使用小鼠γ卫星探针通过荧光原位杂交（FISH）检测着丝粒。雄性CBA小鼠用致断裂剂或纺锤体毒物处理：X射线照射（XR；0.5 Gy）、环磷酰胺（CPA；80 mg/kg）、硫酸长春新碱（VCR；0.125 mg/kg）和秋水仙碱（COL；1 mg/kg）。处理后30和50小时制备骨髓悬液，随后通过流式细胞术分析以计数并确定诱导的MPCEs的DNA含量。发现用两种非整倍体诱导剂VCR和COL处理后，MPCEs中的平均DNA含量高于用两种致断裂剂CPA和XR处理后的含量。MPCEs的平均DNA含量与含着丝粒的微核（MN）的平均比例呈正相关，表明仅测定平均DNA含量就能提供有关MN诱导机制的信息。当根据DNA含量增加的四类对流式分选的VCR诱导的MPCEs进行分类，并通过FISH确定着丝粒的有无时，发现只有DNA含量最低的类别（二倍体DNA含量的0.8 - 1.7%）含着丝粒的微核比例较低（< 20%），而在DNA含量较高的三类（二倍体DNA含量的1.7 - 10.2%）中，> 80%的MN含有着丝粒。这与CPA处理的结果形成对比，在CPA处理中，含着丝粒的MN比例并未随DNA含量增加而增加。
