ABSTRACT
Objective To construct BCR-ABL SH3-T79Y mutant recombinant adenovirus vectors and investigate its effects on apoptosis of K562/G01 cells.Methods SH3-T79Y mutant was amplified by overlapping PCR with pMig210 as template and cloned into recombinant adenovirus vectors .After identifying , packaging and amplifying , the recombinant adenovirus vectors containing SH 3-T79Y 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 CrkL phospho-rylation 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 CrkL phosphorylation significantly decreased and so did the expression of BCR-ABL( P<0.05 ) .Conclusions Success-fully constructed the SH 3-T79 Y mutant recombinant adenovirus vectors and it may promote the apoptosis of K 562/G01 cells by inhibiting BCR-ABL and CrkL phosphorylation .
ABSTRACT
Objective To investigate the potential of chronic myeloid leukemia ( CML) cell line KCL22 in indu-cing leukemia in NOD-SCID mice for setting up a basis for constructing a CML mouse transplantation tumor model. Methods 2 ×107 KCL22 cells in logarithmic growth phase were injected via the tail vein into experimental NOD-SCID mice whereas PBS was injected to the mice of control group.General condition of the mice of both groups was observed.Wright staining was used to observe the changes of blood and bone marrow smears.PCR was conducted to detect the transcription level of BCR-ABL, and histology with HE staining was used to evaluate the tumor cell invasion in the liver and spleen. Results Four weeks after the injection of KCL22 cells, the mice in experimental group showed physical signs of decreased reactivity, depression, swollen hindlimb muscles and petechia on the hindlimb femur.Peripheral white blood cells ( WBC) began to increase after 5 weeks, with a significantly increased quantity compared with the control group (P90 days) (P<0.05).Conclusions A NOD-SCID mouse model of CML transplantation tumor is successfully established with leukemia KCL22 cells.