The Protective Effect Of Bone Marrow Stromal Cells Of Children With Leukemia On Leukemic Cells And The Influence Of Cell Adhesion Molecule VLA-4 On The Effect

Objective:To exmine the influence of VLA-4 especial antibody on the protection of BMSC upon leukemic cells through studying the protection of BMSC upon leukemic cells and detecting apotosis rate of leukeimic cells'after interfering by VLA-4 antibody; And by detecting apoptosis-related genes'(survivin bcl-2) expression to further elucidate the potential mechanism about BMSC preventing leukemic cells from apoptosis and offer theory for the research and clinic therapy of childhood luekemia.Methods:Bone marrow samples of childhood leukemia were collected, then mononuclear cells were isolated, bone marrow stromal cells (BMSC) and leukemic cells were got out and cultured at the same time, cells'morphous was observed via inverted phase contrast microscope.The second generation of BMSC and the fourth to sixth generation of leukemic cells were selected for next experiments. Secondly, after co-culturing for 12h or 24h, each groups'leukemic cells were collected respectively to examine by flow cytometry after Annexin V-FITC/PI double stainning and analyze the apoptosis of leukemic cells in each group to evaluate the role of BMSC and VLA-4 antibody upon the apoptosis of leukemic cells. Thirdly, RT-PCR and gel electrophoresis were applied to analyze the expression of survivin and bcl-2 in each group's leukemic cells to illustrate, from gene angle, the relationship of BMSC, VLA-4 antibody and chemotheraeutic agents VP -16 and their impact on apoptosis of leukemic cells.Results:In vitro it was difficult for leukemic cells to survive for a long time, but in the present of BMSC, they were in good state and their survival become long. Flow cytometry results showed that: compared with BMSC, for the leukemic cells group, the apoptosis rate of leukemic cells was significantly increased and the difference has significance,(P<0.05); the apoptosis rate of the VLA-4 antibody group, VP-16 group, VLA-4 antibody plus VP-16 group was higher than BMSC group(P<0.05), moreover, for VLA-4 antibody group, VP-16 group, VLA-4 antibody plus VP-16 group, the difference of any two groups was significant(P<0.05). With the co-culture time was prolonged, the leukemic cells'apoptosis rate of each group was increased. RT-PCR Results: There were 23 cases with survivin gene expression, 28 cases with bcl-2 expression in the 29 cases acute leukemia, the expression rate was 79%(survivin) and 97%(bcl-2) separately, the expression of survivin, bcl-2 in BMSC group was higher than other groups except supernatant group(P<0.05); The expression of survivin, bcl-2 genes in VLA-4 antibody group, VLA-4 antibody plus VP-16 group were significantly lowerer than BMSC's (P<0.05), the comparison of any two groups of the three was statistically significant(P<0.05). The gene expression of VLA-4 antibody plus VP-16 group were the least, compared with the BMSC group, the expression of survivin reduced by 56%, while the expression of bcl-2 reduced even by 71%.Couclusions: BMSC of childhood leukemia play protective role on leulemic cells from childhood leukemia in vitro; VLA-4 antibody can promote leukemic cells'apoptosis; VLA-4 antibody combine with VP-16 can play greater role in increasing the apoptosis of leukemic cells; The VLA-4 antibody can increase leukemic cell's apoptosis and this was realized by down-regulated the expression of survivin, bcl-2.