In Vivo Effect Of Recombinant Protein Ptd-od-ha In The Mouse Model Of Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a abnormal clone proliferative disease derived from pluripotent hematopoietic stem cells, The cause of this disease is known that the strong tyrosine kinase activity of BCR / ABL fusion protein activates multiple signal transduction pathways, leading to malignant transformation of blood cells .The N-terminal oligomerization region (oligomerization domain, OD) of the protein mediates BCR/ABL homologous oligomerization,leading to BCR / ABL autophosphorylation and activation of tyrosine kinase.We expressed and purified PTD-OD-HA fusion protein that contains the oligomerization (OD domain).Then we transducted it into leukemia cells.The fusion protein can combine with BCR/ABL competitively in the OD domain,prevent the oligomerization, inhibit BCR/ABL kinase activity,and achieve the purpose of treating leukemia. Our group had expressed and purified PTD-OD-HA fusion protein in prokaryotic cell. In vitro experiments had proved that it can promote apoptosis and inhibit proliferation of leukemic cells. In this study, we further explored the in vivo effect of PTD-OD-HA fusion protein on chronic myeloid leukemia mouse model.The main methods, results and conclusion are as follows:1,The effect of PTD-OD-HA fusion protein on chronic myeloid leukemia tumorigenesis in BALB/C miceWe injected BCR/ABL transformed BaF3-P210 mice cells and BaF3-P210 mice cells treated with PTD-OD-HA fusion protein to BALB/C mice through the tail vein to construct leukemia group model and prevention group model. BALB/C mice without any treatment as normal control group. The growth of mice, body weight, peripheral white blood cell count, cell morphology of peripheral blood and bone marrow with Wright stain were observed;We removed the organs of the mice of each group and observed whether there are swelling, bleeding and other changes, Tissue HE staining have been performed for detecting potential pathological changes and infiltration of leukemic cells. Bone marrow cells were collected for examing the BCR/ABL expression level by western blot. Finally survival curve of mice was made to determine the survival rate changes.BP210 cells transplanted mice exhibited dragged posterior limbs, loss of body weight, elevated white blood cell count; peripheral blood, bone marrow smears staining increased leukemia cells in 3rd week; Spleen significantly swelled.Histopathology examination revealed disordered hepatic cord and a large number of leukemia cell infiltration, splenic nodules disorder, accompanied with infiltration of leukemic cells. PTD-OD-HA treated BP210 cells transplanted mice had a slight decline in body weight and increased leukemia cells in peripheral blood, bone marrow smear . liver and spleen did not swell.Histopathology HE stained showed liver cord and spleen nodules are minor disorder, and a slight infiltration of leukemic cells can be seen.Western blot showed BCR/ABL oncoprotein was highly expression in bone marrow cells of tumor mice.And lowly expression in prevention group mice. Compared with the tumor group mice,prevention group mice had a significantly prolonged survival time in survival curve.Results showed that BaF3-BP210 cells after the treatment of PTD-OD-HA fusion protein significantly decreased its potential of causing leukemia, reduced leukemia cell number in peripheral blood and bone marrow, reduced organ invasion, and extend the lifetime of CML mice.2, The treatment effect of PTD-OD-HA fusion protein on leukemia K562 cell solid tumor in nude mice modelAfter BALB/C nude mice were injected with certain amounts of K562 cells in logarithmic growth phase, leukemia subcutaneous solid tumor model was constructed.We injected PTD-OD-HA fusion protein into the subcutaneous solid tumor(once every two days,the concentration of 10mg/mL of protein 200uL) and observed tumor size. HE histological staining, transmission electron microscopy, TUNEL assay cell apoptosis, immunohistochemistry detect apoptosis related proteins Bcl-2, Bax expression levels. Compared with untreated K562 cell tumor group,tumor growth rate slowed down, tumor size was smaller after PTD-OD-HA fusion protein treatment; Histopathology showed nuclear condensation, cell staining become deeper; TUNEL in situ apoptosis detection of significantly increased green fluorescence is also observed. Transmission electron microscopy detected a slight swelling of mitochondria in tumor cells and apoptotic bodies. Immunohistochemistry detected that expression of Bcl-2 decreased and Bax expression increased.The results shows that PTD-OD-HA fusion protein can inhibit the growth of the K562 cell subcutaneous solid tumors of nude mice, induce apoptosis, and improve the survival state of mice.