流式细胞仪自动化快速初筛环磷酰胺诱导微核作用的研究

目的 利用单一荧光试剂吖啶橙（acridine orange, AO）和简单的单激光流式细胞仪计数骨髓嗜多染红细胞细胞的微核率，达到了解化合物诱导微核作用的目的。方法 采用经典的染色体断裂剂环磷酰胺(cyclophosphamide, CP)腹腔注射小鼠和大鼠，24h后采集骨髓细胞，经过固定和吖啶橙（acridine orange, AO）荧光染色，利用单激光流式细胞仪计数微核，计数结果用WinDMI软件进行分析，作出等高图并设置窗口。结果 随着CP浓度的增高，骨髓细胞的含微核的嗜多染红细胞（micronucleated PCE, MNPCE）也相应增多，呈良好的量效关系。实验结果同时和手工计数比较，没有显著差异。结论 采用单一荧光染色（AO染色）和单激光流式细胞仪快速初筛化合物诱导微核作用是可靠的，较传统的手工方法更为灵敏，并节省了大量时间和成本，操作也更为简单。

A study for automated and rapid screening micronuclei-inducing ability of cyclophosphamide by flow cytometry

Objective This study attempt to discriminate whether chemical compounds are micronuclei-inducing by counting the ratio of bone marrow micronucleus polychromatic erythrocytes (MNPCE) dyed with a single fluorescence reagent (acriding organe, AO) by sigle-laser flow cytometry. Methods The experiment was operated by dosing mice or rats with a classic clastogen agent (cyclophosphamide) by intranperitoneal injection first, then sampling bone marrow cells 24 hours later, counting micronucleus ratio of polychromatic erythrocytes by sigle-laser flow cytometry after fixation and AO-dying, and analyzing data by WinDMI at last. Results The results showed that, along with increasing dose of CP, the ratio of MNPCE also corresponding increase, suggesting significant quantative-efficiency correlation. MNPCE were also counted manually by fluorescence microscopy, and the results showed no significant difference with that by flow cytometry. Conclusions This study proved it is reliable to rapidly screen micronuclei-inducing ability of chemical compounds by sigle-laser flow cytometry, with target cells dyed by AO only. Compared with manual counting, flow cytometric counting is less time-consuming, cost-saving and labor-saving, more objective, and properly more sensitive.