基于流式细胞术的大鼠骨髓网织红细胞微核实验方法的建立

The Establishment of a Three-color Flow Cytometry Approach for the Scoring of Micronucleated Reticulocytes in Rat Bone Marrow

目的建立并验证大鼠骨髓网织红细胞(reticulocytes,RETs)微核的流式细胞术检测方法。方法选用CD71-异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记骨髓网织红细胞,CD45-藻红蛋白(phycoerythrin,PE)标记白细胞,核酸染料DRAQ5标记DNA,建立骨髓微核的流式细胞术检测方法。SD大鼠随机分为甲基磺酸乙酯(ethyl methanesulfonate,EMS)、环磷酰胺(cyclophosphamide,CP)、N-乙基-N-亚硝基脲(ethyl nitrosourea,ENU)和秋水仙素(colchicine,COL)染毒组,各组大鼠按染毒剂量不同再分为4亚组,每亚组5只。以染毒剂量为0,作为各组溶剂对照。染毒方案为间隔24 h两次经口给药。末次给药后24 h采集双侧股骨骨髓,采用本研究建立的基于流式细胞术的骨髓微核检测方法测定骨髓中的RETs微核率和RETs比例,并同时使用人工显微镜下计数方法进行检测。结果成功建立基于流式细胞术的骨髓微核检测方法。采用该方法检测20只溶剂对照大鼠骨髓微核率(背景微核率)为0.83‰±0.12‰,采用人工显微镜下计数微核的方法检测的溶剂对照大鼠背景微核率为1.43‰±0.44‰,基于流式细胞术计数骨髓微核的方法背景微核率更低,且更为稳定。两种方法均可检测到EMS、CP、ENU、COL染毒后RETs微核率呈染毒剂量依赖性上升趋势,RETs比例呈染毒剂量依赖性下降。两种方法检测4种受试物诱导的RETs微核率相关性良好(相关系数为0.834 3~0.913 7)。结论本实验建立的基于流式细胞术的骨髓微核检测方法具有操作简便、灵敏快捷、背景微核率较低等优势,是人工微核计数方法的良好替代,可应用于化学物的遗传毒性评价。

Objective To develop and verify a flow cytometric measurement of reticulocytes RETs micronucleus in rat bone marrow. Methods In our flow cytometric protocol, reticulocytes, leukocytes and DNA were labeled by anti-CD71-fluorescein isothiocyanate(FITC), anti-CD45-phycoerythrin(PE) and DRAQ5, respectively.Sprague-Dawley(SD) rats were assigned to four treatment groups randomly, and were exposed to ethyl methanesulfonate(EMS), cyclophosphamide(CP), ethyl nitrosourea(ENU) and colchicine(COL) respectively. Each treatment group was divided into four subgroups(5 rats per subgroup) according to different exposure dosage. A exposure dose of 0 was used as vehicle control for each group. Rats were administered with testing mutagens by gavage twice with a 24 h interval. Bone marrow from both femurs were collected 24 h after the last administration. The frequency of micronucleated reticulocytes(MN-RETs) and the percentage of reticulocytes(RETs%) were determined by flow cytometric measurement established in this study. And the manual counting method with microscope(by Giemsa staining) was conducted at the same time.Results A method for detection of reticulocyte micronucleus in bone marrow based on flow cytometry was successfully established. The MN-RETs in rat bone marrow of 20 SD rats treated by vehicle(i.e., background value of MN-RETs) was 0.83‰±0.12‰ by this method. The background value of MN-RETs in manual enumeration method was 1.43‰±0.44‰.It was obvious that the flow cytometric method had lower background value and more stable results. The trend, in which MN-RETs ascended and RETs% descended with increasing dose, can be detected by both methods in rats that exposed to EMS, CP, ENU and COL. Both methods were good to detect the correlation of induced-MN-RETs with four testing mutagens(the correlation coefficients were ranged from 0.834 3 to 0.913 7). Conclusion With its sensitivity, rapidity,easy operation and low background value, the three-color flow cytometric enumerative protocol established in our laboratory can be used as a good substitute for manual micronucleus counting method and used in genotoxicity assessment of chemical substances.