全血培养胞质分裂阻断微核(CBMN)实验方法的改进

A modified protocol for the cytokinesis-block micronucleus(CBMN) assay using whole human blood

目的改良全血培养胞质分裂阻断微核(cytokinesis-block micronucleus,CBMN)实验制片法及探讨细胞松弛素B(cytochalasin B,cyt-B)的最佳浓度。方法依据常规微核试验制片法,在低渗液的温度、固定液比例和操作手法等方面改进实验方法;设置不同浓度cyt-B组(4、5、6、7μg/mL),常规外周血淋巴培养至44 h加入cyt-B,继续培养至68 h或72 h,收获细胞。按人类微核计划(HUMN Project)中的识别标准识别双核、多核细胞和微核(micronuclei,MN),确定cyt-B最适浓度,并分析cyt—B对自发微核率的影响。结果改良法所获的双核淋巴细胞的胞膜完整,边界清晰,微核易于辨认。培养72 h后,cyt-B浓度≥5"g/mL时,双核细胞比例较4μg/mL组显著增加(P=0.001);cyt-B浓度≥6μg/mL时,三核以上细胞比例显著增加(P=0.01)。培养68 h后,cyt-B浓度为6μg/mL时,双核细胞比例较高(58.4%)。各浓度组淋巴细胞自发微核率的差异无统计学意义(P=0.32)。结论改良法选用cyt—B浓度为5μg/mL、培养72 h或cyt-B浓度为6μg/mL、培养68 h时收获的细胞,可获得大量双核细胞,且胞膜完整,染色清晰;cyt-B对淋巴细胞自发微核率无显著影响。

Objective To investigate on the optimum concentration of cytochalasin B （cyt-B） and modify protocol for the cytokinesis-block micronucleus （CBMN） assay using whole human blood.Methods Conventional approach was modified in some ways such as temperature of hypotonic solution,operating and proportion of Carnoy fixative.After 44 h of incubation of 37℃ in whole blood cultures with the concentrations of cyt-B at 4,5,6 or 7 μg/mL.Whole blood cultures were harvested 68 or 72 h after initiation using the modified method.Criteria for binucleated,multinucleated cells and micronuclei （MN） cells evaluation were those suggested by the HUMN project.The optium concentration of cyt-B was determined,and the influence of cyt-B on the frequency of spontaneous MN was analyzed.Results Binucleated cells with a well-preserved cytoplasm and MN situated within the cytoplasmic boundary are distinguished easily.The yield of binucleated cells increased significantly （P=0.001） at cyt-B ≥5 μg/mL compared with the lower concenteation,and the yield of multinucleated cells increased significantly （P=0.01） at cyt-B ≥6 μg /mL when whole blood cultures were harvested 72 h after initiation.The proportion of binucleate cells was greater （58.4%） at 6 μg /mL when whole blood cultures were harvested 68 h after initiation.The difference in frequency of spontaneous MN between the 4 groups was not statistically significant （P=0.32）.Conclusions Binucleated cells with an optimal yield and a well-preserved cytoplasm were obtained by adding cyt-B at a dose of 5 μg /mL and harvest 72 h after culture or 6 μg /mL and harvest 68 h after culture.There is no influence on the frequency of spontaneous MN in whole blood cultures treated with various concentrations of cyt-B.