用多引物PCR分析γ射线引起人外周血淋巴细胞hprt基因突变

正常人外周血用~（６０）Ｃｏ射线分别进行１～８Ｇｙ照射后，在含６－ＴＧ的培养基上克隆和筛选人淋巴细胞ｈｐｒｔ突变细胞。细胞的突变频率与γ射线的照射剂量呈正相关．获得４２个ｈｐｒｔ突变细胞株，用８对ｈｐｒｔ寡核苷酸引物进行多聚酶链反应（ＰＣＲ）从细胞粗提物中分别扩增ｈｐｒｔ各个外显子，分析突变细胞的ｈｐｒｔ基因突变，约６０％（２５／４２）的ｈｐｒｔ基因突变为基因缺失，其中１３个突变是ｈｐｒｔ基因全部缺失，而１２个是部分ｈｐｒｔ基因外显子缺失，约有４０％（１７／４２）的突变无明显的ＰＣＲ扩增变化．同时显示ｈｐｒｔ基因外显子的缺失突变与辐射剂量有关，实验结果提示，此方法有可能用于估计辐射剂量和进行辐射远后效果观察，进而揭示辐射致突的分子机理．

Molecular Analysis of Gamma-Ray-Induced Mutation at hprt Locus in Normal Human Peripheral Lymphocytes

Normal human peripheral blood sample were irradiated by ￣(60)Co gamma-rays in the dose range of 1-8 Gy and the hprt mutants of lymphocytes were cloned in the 6-TG containing selective culture and assayed. The mutation frequency of hprt mutants was found to be related to their radiation dose. A total of 42 independent hprt mutants was recovered and the mutation of hprt exons was analyzed by polymerase chain reaction (PCR3 using 8 pairs of hprt oligonucleotide primers to amplify hprt exons from crude cellular extracts. The result of PCR analysis demonstrated that abount 60% (25/42) of the hprt mutations were deleted,among which 13 mutants were due to total deletion and the other 12 mutants were partial deletion, whereas 40% did not show noticeable changes in the PCR pattern. The percentage of hprt deleted mutants was found to be dependent on the irradiation dose. The results suggest that this assay might be used clinically in the study of acute or chronic effects of gamma-ray irradiation and in the exploration of the molecular mechanism of somatic cell mutation.