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首页> 外文期刊>Mutation Research: International Journal on Mutagenesis, Chromosome Breakage and Related Subjects >Induction of premature chromosome condensation by a phosphatase inhibitor and a protein kinase in unstimulated human peripheral blood lymphocytes: a simple and rapid technique to study chromosome aberrations using specific whole-chromosome DNA hybrid
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Induction of premature chromosome condensation by a phosphatase inhibitor and a protein kinase in unstimulated human peripheral blood lymphocytes: a simple and rapid technique to study chromosome aberrations using specific whole-chromosome DNA hybrid

机译：磷酸酶抑制剂和蛋白激酶在未刺激的人外周血淋巴细胞中诱导染色体过早凝结：一种简单快速的技术，使用特定的全染色体DNA杂交体研究染色体畸变

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摘要

We developed a simple and rapid method to study chromosome aberrations involving specific chromosomes using unstimulated human peripheral blood lymphocytes (HPBL). Premature chromosome condensation (PCC) was induced by incubating unstimulated HPBL in the presence of okadaic acid (OA, a phosphatase inhibitor), adenosine triphosphate (ATP), and p34(cdc2)/cyclin B kinase [an essential component of mitosis-promoting factor (MPF)], which eliminated the need for fusion with mitotic cells. OA concentration and duration of incubation for PCC induction was optimized using mitogen-stimulated HPBL; a final concentration of 0.75 microM incubated for 3 h was optimum, resulting in approximately 20% PCC yield. In unstimulated HPBL, PCC was induced by the addition of p34(cdc2)/cyclin B kinase at concentrations as low as 5 units/ml to a cell culture medium containing OA. Increases in the concentration of p34(cdc2)/cyclin B kinase from 5 to 50 units/ml resulted in a concentration-dependent increase in PCC yield (30% to 42%). We demonstrate that this technique of inducing PCC in unstimulated HPBL is suitable for studying radiation-induced aberrations involving a specific chromosome (chromosome 1) after 24 h repair using a whole-chromosome in situ hybridization probe and chromosome painting. Cells with aberrant chromosome number 1 are characterized with more than two chromosome spots. The frequency of cells with aberrant chromosome 1 increased with 60Co gamma-radiation doses in the region 0-7.5 Gy. The observed dose-effect relationship for the percentage of cells with aberrant chromosome 1 (Y) was explained by using both a linear [Y=(2.77+/-0.230)D+0.90+/-0.431, r(2)=0.966] and a nonlinear power [Y=(5.70+/-0.46)D((0.61+/-0.05)), r(2)=0.9901) model. This technique can be applied to biological dosimetry of radiation exposures involving uniform whole-body low linear energy transfer (LET) exposures.
机译：我们开发了一种简单快速的方法来研究使用未刺激的人外周血淋巴细胞（HPBL）涉及特定染色体的染色体畸变。通过在冈田酸（OA，一种磷酸酶抑制剂），三磷酸腺苷（ATP）和p34（cdc2）/ cyclin B激酶[有丝分裂促进因子的重要组成部分]的存在下孵育未刺激的HPBL，诱导早熟染色体凝缩（PCC）。 （MPF）]，从而无需与有丝分裂细胞融合。使用促细胞分裂剂刺激的HPBL对OA浓度和PCC诱导孵育的持续时间进行了优化。孵育3 h的最终浓度为0.75 microM最佳，导致PCC产率约为20％。在未刺激的HPBL中，通过向含有OA的细胞培养基中添加低至5个单位/ ml的p34（cdc2）/ cyclin B激酶来诱导PCC。 p34（cdc2）/ cyclin B激酶的浓度从5增加到50单位/毫升会导致PCC产量的浓度依赖性增加（30％至42％）。我们证明了这种在未刺激的HPBL中诱导PCC的技术适用于研究使用全染色体原位杂交探针和染色体绘画修复24小时后涉及特定染色体（染色体1）的辐射诱导的像差。 1号染色体异常的细胞具有两个以上的染色体斑点。 1号染色体异常的细胞的频率在0-7.5 Gy范围内随60Coγ射线剂量的增加而增加。通过使用线性[Y =（2.77 +/- 0.230）D + 0.90 +/- 0.431，r（2）= 0.966]解释了观察到的具有异常1号染色体（Y）的细胞百分比的剂量效应关系。以及非线性功率[Y =（5.70 +/- 0.46）D（（0.61 +/- 0.05）），r（2）= 0.9901）模型。这项技术可以应用于涉及统一的全身低线性能量转移（LET）辐射的辐射辐射的生物剂量测定。

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