Effects And Mechanism Of Hedgehog-Gli Signaling Pathway On The Apoptosis Of Acute Myeloid Leukemia Cells In Bone Marrow Stromal Cells

Objective: To investigate the apoptosis effects of HS-5 humun bone marrow stromal cells on acute myeloid leukemia cells HL-60 and elucidate the underlying mechanisms. to provide a theoretical basis for the treatment of acute myeloid leukemia.Method:(1)Application of bone marrow stromal cells HS-5 and acute promyelocytic leukemia cells HL-60 in vitro setting up co-culture model to simulate the effect of bone marrow microenvironment on apoptosis of leukemic cells in vivo. CCK-8 analysis was performed to detect the proliferation of HL-60 cells, Apoptosis of HL-60 cells stained with Annexin V-FITC/PI were detected by flow cytometry, PI single staining flow cytometry detected in HL-60 cell cycle distribution of HL-60 cells was analyzed by flow cytometry after cell stained with PI; co-culture of bone marrow stromal cells HS-5 and acute myeloid leukemia HL-60 cells were detected by scanning electron microscope(SEM); Agarose gel electrophoresis of semi-quantitative RT-PCR showed the composition Gli of hedgehog signaling pathway of HL-60 cells in co-culture model and anti-apoptosis gene BCL-2 and BCL-XL m RNA were up-regulated; The expression of Gli 1 protein of acute myeloid leukemia HL-60 were detected by immunofluorescence assay.Results:(1) the rapid proliferation of HL-60 cells,co-cultured with HS-5 cells,GLI as a target of Hedgehog signaling pathway inhitor GANT61 treated co-cultures slowing HL-60 cells proliferaion after the system(2) HL-60 cells co-cultured with HS-5 apoptosis increases, GLI as a target of Hedgehog signaling pathway inhibitor GANT61 processing co-cultured HL-60 increased apoptosis rate system.(3) co-cultured 48 h, HL-60 cells were arrested in G0/G1 phase,the proportion of cells G0/G1 phase increased significantly, this situation continues to co-culture 72 h.(4)Bone marrow stromal cells HS-5 with acute myeloid leukemia cells HL-60 cultivation, the HL-60 cells and bone marrow stromal cells HS-5 adhesive growth,some cells embedded in bone marrow stromal cell layer under scanning electron microscope.(5) RT-PCR gel electro phoresis results showed that co-culture system in HL-60 cells GLI 1 m RNA upregulation of antiapoptotic gene BCL-2 and BCL-XL expression of m RNA upregulation.(6)immunofluorescence GLI 1 protein expression levels: co-culture system in HL-60 cells GLI 1 protein increased.Conclusion:(1) bone marrow stromal cells can promote the proliferation of acute myeloid leukemia cells.(2) bone marrow stromal cells can inhibit the apoptosis of acute myeloid leukemia cells.(3) Hedgehog signaling pathway in acute myeloid leukemia cells was activated by bone marrow stromal cells and up-regulated downstream target genes BCL-2 and BCL-XL.