BCR-ABL SH3-T79Y突变体重组腺病毒载体的构建及促进白血病K562/G01细胞凋亡

Construction of BCR-ABL SH3-T79Y mutant recombinant adenovirus vectors and its promotion on apoptosis of K562/G01 cells

目的探讨构建BCR-ABL SH3-T79Y突变体(简称SH3-T79Y突变体)的重组腺病毒载体及其对白血病耐药细胞株K562/G01细胞凋亡的影响。方法以p Mig210质粒为模板,用重叠延伸PCR扩增SH3-T79Y突变体片段,将其克隆入重组腺病毒载体,通过鉴定、包装、扩增后得到含SH3-T79Y突变体的重组腺病毒。将重组腺病毒感染白血病K562/G01细胞株,测定其感染效率,瑞氏染色检查细胞形态学,流式细胞术检测细胞凋亡,Western blot检测BCR-ABL、Crk L磷酸化及总蛋白水平。结果重组腺病毒载体构建成功。SH3-T79Y突变体转染K562/G01细胞株72 h效率大于80%,可见明显的凋亡小体、核聚集等凋亡现象,凋亡率为32.46%,较对照组显著增加(P<0.05);明显抑制BCR-ABL和Crk L的磷酸化水平,降低BCR-ABL总蛋白表达(P<0.05)。结论成功构建SH3-T79Y突变体重组腺病毒载体,并证实其通过抑制BCR-ABL及底物Crk L磷酸化水平促进K562/G01细胞凋亡。

Objective To construct BCR-ABL SH3-T79 Y mutant recombinant adenovirus vectors and investigate its effects on apoptosis of K562 / G01 cells. Methods SH3-T79 Y mutant was amplified by overlapping PCR with p Mig210 as template and cloned into recombinant adenovirus vectors. After identifying,packaging and amplifying,the recombinant adenovirus vectors containing SH3-T79 Y mutant was collected. Recombinant adenovirus vectors were transferred into K562 / G01 cells. Then transfection efficiency was determinated,changes of cell morphology were observed by Wright＇s staining,cell apoptosis was evaluated by flow cytometry,BCR-ABL and Crk L phosphorylation was detected by Western blot. Results The vectors were successfully constructed. Transfection efficiency was more than 80% after transferring into K562 / G01 cells for 72 h; there was obvious apoptosis phenomenon,cell apoptosis significantly increased to 32. 46% compared with the control groups（ P 0. 05）,BCR-ABL and Crk L phosphorylation significantly decreased and so did the expression of BCR-ABL（ P 0. 05）. Conclusions Success-fully constructed the SH3-T79 Y mutant recombinant adenovirus vectors and it may promote the apoptosis of K562 /G01 cells by inhibiting BCR-ABL and Crk L phosphorylation.