The Expression Of The PTD-bcr/abl Fusion Protein And It's Effect On The Growth Of K562 Cells

Chronic myeloid leukemia (CML) is a malignant disease of human hematopoietic stem cell, accounting for 20% of leukemias in adult?The common symptoms are asthenia,weight loss,sweating,and the discovery of abdominal mass(the spleen) ?There are Philadelphia chromosome(Ph)-the cytogenetic hallmark of CMI. in most cases?The Ph is a shortened chromosome 22 that arises from a reciprocal translocation, t(9;22) (q34;ql 1), which gives origin to a hybrid bcr/abl oncogene, encoding a p210-BCR/ABL fusion protein with elevated tyrosine kinase and transforming activities ?Most clinic and laboratory research provided direct evidences that bcr/abl is the main cause of CML.The prognosis of CML is improved after the application of bone marrow transplantation (BMT)o Only a few cases would get this therapy because of the restriction of age, matched bone marrow, and so on?Most of the patients who get the treatment of BMT died from infection, graft versus host disease (GVHD)or leukemia relapse ?STI571 (Glivec,Gleevec or Imatimb), a designed drug specifically inhibit tyrosine kinases have been introduced which has entered clinical trials and shown promising activities in chronic phase (CP), accelerated phase (AP) and blast crisis (BC) as evidenced by significant hematological and cytogenetic responses in CML patients<> But the effect still be dissatisfying 0The other function domains and relating signial transduce molecules beyond the tyrosine kinases in Bcr/Abl fusion protein are in focus recently?i e: Hawk et al had proved the ser-354, in BCR region of BCR/ABL fusion protein, can down-regulated the activity of tyrosine kinases when it was phosphorylated by gene mutation technology; others, such as Farnesyl protein transferase inhibitors, which block the RAS way; Wotmannin, which block the PI-3K way a But some shortcomings can not be overcome in all above methods 0PTD(Protein Transduction Domain) is a peptide in the TAT(transactivator gene)which is encoded by HIV-1 (Human immunodeficiency virus)0 Its special transmembrane action was noticed by its high speed and high efficiency ?After fused with PTD , the protein(Mr!5X 103-2X105) get the ability of transmembrane o we constructed a vector ,in which the PTD gene was linked with bcr梐bl sh3 segment ?The vector was expressed in E.coli, and the Mr of the PTD-BCR/ABL SH3 fusion protein we got was 23kDo It was vertified that this protein could entry into cells when cultivated with HL60 or injected into mice from the tail vein,, To learn more about this protein, we have done the following work:1 The rabbit was immunized with the PTD-bcr/abl and the polyclonal antibody to the PTD-bcr/abl was obtained, the quality of the antibody was verified to be high affinity and high speciality by Elisa,Western-Blot, immunohistochemistry.2 It has been report that the PTD-bcr/abl would entry into cells when cultivated together In this experiment we discover when the K562 cells cultivated together with the PTD-bcr/abl, the cells would change shape,the growth of cells would be restrained, even be apoptosis if the protein's concentration is high.3 After the K562 cells were cultivated together with the PTD-bcr/abl, we detect the cell cycle by flow cytometry (FCM) We found the ratio of cells in S phase was reduced, while the ratio of the G2 phase was ascended This result indicated the PTD-bcr/abl could block the cells in G2 phase The cyclin Dl was not affected o4 To learn about the operation mechanism of the PTD-bcr/abl, we detect the telomerase of the cells cultivated together with the PTD-bcr/ab in different timeo The result is that the telomerase activity did not change In this research, the polyclonal antibody to the PTD-bcr/abl was got, it's affinity and speciality was verified to be suited to the latter research ?The PTD-bcr/abl would restrain the growth of the K562 cells,even induce the apoptosis of cells if the protein concerntration is high With the Flow cytometry, the telomerase activity was detected and no change was found The cyclin Dl was n...