| Background Genetic modification T cell immune therapy is more and more shows its superiority, it has played a positive role in the treatment of cancer patients. The "supernatural" T cell regulation, however, is the important problem need to solve in clinical gene and cellular therapy, regulation of the genetic modification cells with suicide gene system is one of the security measures of gene therapy. Researchs show that inducible Caspase9(iCasp9) suicide gene system can induce cell apoptosis effectively and rapidly, and almost all of the iCasp9 come from human, so the transduction cells have lower risk of immune response significantly, Therefore, the iCasp9 system has received the widespread attention.Objective To establish an inducible apoptosis cell model, containing iCasp9 suicide gene system, whose conditional dimerization can induce Jurkat T cell have an biological effect of apoptosis. Exposing to a synthetic dimerizing drug, the iCasp9 becames activated and leads to the rapid death of cells expressing this construct. The obtaining of ralative datas on T cell melting condition can provide security for genetic modification of T cell immunotherapy.Methods1. The constructed fusion gene (FKBP12/Caspase9) was inserted into the lentiviral vector named pCDH-CMV-MCS-EF1-puro by molecular cloning technology, build piCasp9 expression plasmid. Three plasmid (plasimd piCasp9,packaging plasimd pDel8.9 and helper plasimd pVSVG) transfection 293T cells with calcium phosphate to intense preparation lentivirus (LV-KLC9) and the drop degree detersation.2. Jurkat T cells were infected with the virus collected from the 293 T cells. Then, we selected the transfected cells expressing iCasp9 by means of puromycin screening. To detect the integration and expression of fusion gene in the transfected cell clones selected, Western blot was used respectively. The effect of inducible apoptosis on cell clones by AP20187 was analyzed by Annexin V detection.Results1. The piCasp9 expression plasmid was build successfully, Three plasmid transfection 293 T cells with calcium phosphate to intense preparation lentivirus (LV-KLC9) has a high efficiency,95% of the 293T cells can be observed green fiuorescence 72h later. Used by hole dilution drops of determination method of determination of virus drops, virus drops was 3.0X107IU/ml.2. Efficiency of virus infection Jurkat T cell up to 65%; Western blot found there is an apparent specific band in the 49 kD, and the control group do not have the stripe, which show that virus take fusion genes into Jurkat cells, and expressed successfully in the cells. To detect apoptosis at different time points, give cell lOnM AP20187 to induce apoptosis, Flow cytometry showed that:with the extension of time, the cell apoptosis rate increased gradually, show the time dependence of cell death.Conclusions The inducible apoptosis cell model was successfully established, AP20187 can induce apoptosis of selected cells effectively in vitro. Jurkat cell with the purpose gene present time dependent apoptosis. Our results may provide security for genetic modification of T cell immunotherapy. |
