稳定表达BCR/ABL-T315I的小鼠BP210-T315I细胞株的建立

目的构建能稳定表达T315I的BCR/ABL的小鼠BP210-T315I细胞株,并观察该细胞株对酪氨酸激酶抑制剂STI571的敏感性。方法采用PCR法将重组逆转录病毒载体MIGR-P210中的abl基因第944位碱基C定点突变为T,再将该位点突变成功的逆转录病毒载体转染到包装细胞内,收集病毒,感染BaF3细胞,经筛选和亚克隆获得稳定表达T315I的BCR/ABL蛋白的小鼠BP210-T315I细胞。采用RT-PCR、DNA测序和Western blot鉴定abl基因第944位碱基是否发生突变;MTT法检测BP210-T315I细胞对STI571的耐药性。结果 T315I的bcr/abl基因已整合到BaF3细胞基因组中,B210-T315I细胞能稳定表达T315I的bcr/abl基因与蛋白;与BaF3-P210细胞株相比,经STI571处理后,BP210-T315I细胞对STI571明显耐药,其耐药倍数约为100。结论已成功构建稳定表达小鼠T315I的转化细胞株,该细胞株具有对STI571耐药的恶性表型特征。

Establishment of Mouse BP210-T315I Cell Strain for Stable Expression of BCR/ABL-T315I

Objective To construct a mouse BP210-T315I cell strain for stable expression of BCR / ABL-T315I and observe its sensitivity to tyrosine kinase inhibitor ST1571.Methods The base C at site 944 of abl gene of recombinant retrovirus vector MIGR-P210 was mutated to T by PCR,then the vector was transfected to packaging cells.The BaF3 cells infected with the packaged virus were collected,from which the mouse BP210-T315I cells for stable expression of BCR / ABL-T315I were obtained by screening and subcloning.The mutation at site 944 of abl gene was identified by RT-PCR,DNA sequencing and Western blot.The resistance of BP210-T315I cells to STI571 was determined by MTT method.Results The bcr / abl gene of T315I was integrated to the genome of BaF3 cells,and the bcr / abl gene and protein of T315I were stably expressed in B210-T315I cells.Compared with BP210 cell strain,the resistance factor of BP210-T315I cells after treatment with STI571 was about 100 to STI571.Conclusion The transformed cell strain for stable expression of mouse T315I was successfully constructed,which showed a malignant phenotype character of resistance to STI571.