Utilizing The Transgenic Model To Explore The Function Of BMP4 In Bone Marrow Hematopoiesis Stem Cell Regulation

Objective: 1.Compare the hematopoiesis of BMP4 overexpressed mice to Wild-Type(WT)mice under physiological condition 2.Observation on BMP4 in cell cycle and apoptosis regulation on hematopoietic stem/progenitor cells(HSPC)after myelosuppression induced by 5-fluorouracil and preliminarily postulate the mechanism underlie it.3.Explore the impact of BMP4 on the homing efficiency and reconstitution capability of hematopoietic stem cell(HSC).Methods: 1.Design the BMP4 global expressed mice.2.Blood routine test of the peripheral blood and flow cytometric analysis of the each hematopoietic lineage progenitors.3.Detect the m RNA expression of several hematopoietic factors,include Csf2? Csf3? CXCL12? SCF? ANG-1?Hif-1? by q RT-PCR.4.Analyze the expression of ITGA4 protein through Western-blot.5.Myelosuppression on mice through 5-fluorouracil at the dose of 150mg/kg and extract the bone marrow nucleated cell(BMNC).6.Count the peripheral blood cell and BMNC dynamically.7.Utilize c-kit/sca-1 double stain to label the hematopoietic stem/progenitor cells,and use Annexin V/PI and Ki-67/DAPI double stain to analyze the apoptosis and cell cycle respectively.8.Sort the HSC through flow cytometry,labeled with CFSE and then transplanted through tail vein into mice under lethal irradiation,the homing efficiency was tested by flow cytometry 16 hours later.Capacity of Reconstitution was evaluated by observation on survival duration post-transplantation.Results: 1.The number of the granulocyte in BMP4 mice is significant higher than WT mice under physiological condition(P <0.01).2.The m RNA and protein expression of Csf3 and ITGA4 is promoted in BMP4 mice(P<0.05)3.Peripheral blood cell and BMNC are seriously impaired at 4 days post-myelosuppression.4.Under physical condition,the apoptosis and cell cycle of Hematopoietic stem/progenitor cell(HSPC)between BMP4 mice and WT mice is similar,but after myelosuppression induced by 5-FU,the transgenic mice perform significant higher proportion of G1/S/G2/M(P <0.01)and increased apoptosis rate(P <0.05).5.After myelosuppression,the m RNA expression of HIF-1? and CXCL12 in bone marrow stroma of BMP4 mice is significantly downregulated(P <0.01).6.Through HSC transplantation after lethal radiation,homing efficiency and survival duration of HSC is impaired in BMP4 mice(P<0.01).Conclusions: 1.BMP4 could promote the proliferation of granulocyte under physiological condition,and might exert this function by Csf3.2.During myelosuppression or stress hematopoiesis,the m RNA expression of CXCL12 and HIF-1? is impeded,which propel the HSPC into cell cycle and lift the apoptosis rate.3.BMP4 could impair the ability of hematopoietic reconstitution and homing efficiency of HSC after lethal radiation.