摘要
目的构建能稳定表达BCR/ABL的小鼠转化细胞株BaF3-P210,并初步探讨BCR/ABL对BaF3细胞生物学特性的影响。方法将含有bcr/abl基因的逆转录病毒载体转染到包装细胞,收集病毒感染BaF3细胞,经筛选和亚克隆,获得稳定表达BCR/ABL的转基因BaF3-P210细胞。用PCR、DNA测序、RT-PCR和Westernblot检测bcr/abl基因在靶细胞中的整合和表达;用台盼蓝细胞计数法、流式细胞仪(FCM)和MTT法研究BCR/ABL对BaF3细胞生物学特性的影响。结果bcr/abl基因整合在BaF3细胞基因组中,BaF3-P210细胞能稳定表达bcr/abl基因及其蛋白。与对照组相比,BaF3-P210细胞增殖速度增快(P<0.05);G0/G1期细胞比例下降,S期和G2/M期细胞比例增高(P<0.05);经STI571处理后的细胞增殖抑制率为(54.08±1.90)%(P<0.05),早期凋亡率为(12.35±2.04)%(P<0.05)。结论成功构建的能稳定表达BCR/ABL的小鼠BaF3-P210转化细胞株具有增殖能力增强、细胞周期进程加速和对STI571敏感的恶性表型特征。
Objective To construct the transformed mouse BaF3-P210 cell line stably expressing BCR/ ABL and to initially investigate the influence of BCR/ABL on the cell biological characteristics of BaF3 cell line. Methods The retroviral vector with bcr/abl gene was transfected into the packaging cell line. The BaF3 cells were infected with the collected viral supernatant. The transgenic BaF3-P210 cell line stably expressing BCR/ABL were screened and subcloned. The integration of the bcr/abl gene in the genome of the target cell was determined by PCR and DNA sequencing, trypan blue staining assay, flow cytometry and MTT assay. Results The bcr/abl gene was integrated into the BaF3 cell genome; RT-PCR and Western blot verified the stable expression of the bcr/abl gene and protein in the screened subclone cell line BaF3-P210. Compared with the control group, the cell proliferation rate was promoted （ P 〈 0.05 ） , and the cell proportion in G0/G1 phase was decreased with the increasing of the cell proportion in both S and G2/M phase （ P 〈 0.05 ） ; the cell proliferation inhibition rate and the early apoptosis rate of the BaF3-P210 ceils were respectively （54.08 ± 1.90）% （P 〈 0.05） and （ 12.35 ± 2.04） % （ P 〈 0. 05 ） after the treatment of protein tyrosine kinase inhibitor STI571. Conclusion The transformed mouse BaF3-P210 cell line stably expressing BCR/ABL was successfully constructed. The expression of BCR/ABL not only promoted the proliferation, but also accelerated the cell cycle progression of the BaF3 cells. It endowed the BaF3-P210 transformed cell line with the sensitivity to STI571.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2008年第19期1791-1794,共4页
Journal of Third Military Medical University
基金
教育部高等学校博士学科点专项科研基金(20050631007)~~
