Construction Of Recombinant Plasmid PBudCE4.1-dll4-16 And Its Effect On Leukemic Cells K562

Objective:This article constructs a vector containing human suppressor gene dll4 and p16 INK4a , uses the Lipofectamine 2000 to dry the dll4 and p16INK4a into the Leukemic cell line K562,and observing its expression and function in Leukemic cells.To further study many kinds of genes to use in the tumor treatment laying the foundation.Methods:Recombinant plasmid pBudCE4.1-dll4-16 was designed and constructed for simultaneous expression of human suppressor genes dll4 and p16 INK4a in mammalian cell line. After transfected into Leukemic cells K562 with lipfectamine TM 2000,divides into three stages: First, the extension dyes 48 hours later, collects each group of cells, decomposes the cells with RIPA, the expression of dll4 and p16 INK4agenes was detected by Western-immunobltting ; Second, The cell proliferation was assessed by CCK-8; Third, the changes in cell cycle was evaluated by flow cytometry.Results:These recombinant plasmids pBudCE4.1-dll4-16,pBudCE4.1-dll4 and pBudCE4.1-16 were constructed successfully and were verified by restriction enzyme digestion methods. Western blotting was used to detect dll4 gene and p16 gene expression at protein levels 48 hours after transplantation. By immunoblotting method, the cells cotransfected with the two tumor suppressor genes showed detectable levels of p16INK4a and dll4 protein. The extension dyes 48 hours latter , after PI dyeing processing, compared with the control groups, the number of cells at G0 /G1 stage was increased . the difference has statistics significance (P<0.001).The proliferation of the experimental group cells was inhibited according to CCK-8 method.Conclusion:Expression of Recombinant plasmid pBudCE4.1-dll4-16 in leukemic cells K562 results in reduced proliferation, G0/G1 arrest and not induction of apoptosis.