荧光原位杂交技术分析大剂量照射后Calyculin A诱导的早熟凝集染色体

Analysis of Calyculin-A-induced prematurely condensed chromosome after high-dose irradiation by using fluorescence in situ hybridization （FISH）

目的 探索用荧光原位杂交（FISH）技术分析大剂量照射后Calyculin A（CA）诱导的早熟凝集染色体的可行性.方法 采用X射线照射离体外周血,吸收剂量为0、1、5、10、15和20 Gy.RPMI 1640培养基培养,CA诱导染色体早熟凝集,1、4号全染色体探针荧光原位杂交,荧光显微镜下观察,计数畸变阳性细胞数及两条染色体断片数,拟合剂量效应曲线.结果 以阳性细胞作为观察对象,剂量范围在0 ～15 Gy时,阳性细胞数和照射剂量呈现良好的剂量-效应关系,Y=0.008＋0.065D＋1.858x10-5D2（R2 =0.994）.以断片数作为观察对象,剂量范围在0～ 20 Gy时,同样呈现良好的剂量-效应关系：Y=-0.032＋0.216D-0.01D2（R2 =1.0）.结论 用FISH分析CA诱导的早熟凝集染色体,可用于估算大剂量照射后的生物剂量.

Objective To explore the feasibility of using fluorescence in situ hybridization （FISH） technology to detect the Calyculin A （CA）-induced prematurely condensed chromosome（PCC） after high-dose irradiation of X-rays.Methods Human peripheral blood was irradiated by X-rays at 0,1,5,10,15 and 20 Gy.Whole blood was cultured with RPMI 1640 medium,thereafter the CA was used to induce premature chromosome condensation.The No.1 and No.4 whole chromosome probe was used in the FISH,and the chromosomes were observed under the fluorescence microscope.The aberration positive cell quantity and the quantity of fragments of two chromosomes were counted,and dose-effect curve fitted was performed.Results The ratio of chromosomal aberration positive cells had a good dose response （0-15 Gy） with an equation Y =0.008 ＋ 0.065D ＋ 1.858 x 10-5D2 （R2 =0.994）.The PCC fragments also had a good dose response within 0-20 Gy with an equation Y=-0.032 ＋0.216D-0.01D2（R2 =1.0）.Conclusions Analysis of Calyculin-A-induced PCC by using FISH technique could be used to estimate the biological dose after high-dose irradiation.