Study On GATA-4Gene In Improving The Paracrine Effect Of Bone Marrow Mesenchymal Stem Cells

Objective:To investigate the effect of over-expressed GATA-4on the paracrine effect ofmesenchymal stem cells (MSCs).Methods:1. MSCs were isolated and cultured by adherent culture method and identified byflow cytometer (FCM).2. MSCs were transfected with GATA-4(MSCGATA-4) by using a pMSCV retroviralexpression system, and null vector transfection (MSCnull) were taken as the control.3. GATA-4mRNA and protein of MSCs were analyzed by Real-time quantitativePolymerase Chain Reaction (RT-PCR), Western blotting and immunehistochemistry.4. MSCs were divided into：normoxic groups: MSCs, MSCnulland MSCGATA-4group;hypoxic groups: MSCs, MSCnulland MSCGATA-4group.5. Growth factors secreted in MSCs medium, such as vascular endothelial growthfactor(VEGF), hepatocyte growth factor(HGF) and insulin growth factor-1(IGF-1) weredetected by ELISA.6. After culturing for48hours, the cellular survival was evaluated by rate of MTT cellproliferation, positive rate of Annexin V and lactate dehydrogenase (LDH) release.7. After adding VEGF and IGF-1neutralization antibodies for48hours, the survivalof MSCs was evaluated by the same method again.Results:1. MSCs have been separated and purified successfully. Spindle or polygonal primarycells could be screened.2. Over-expression of GATA-4mRNA and protein in MSCGATA-4group were obtainedby RT-PCR, Western blotting and immunehistochemistry.3. Hypoxic cells had a higher amount of VEGF、 HGF and IGF-1than normoxic cells; under normoxic condition， the amount of VEGF,and IGF-1in MSCGATA-4groupsignificantly higher than that of MSCs and MSCnullgroup（P <0.05）,and there is nosignificance in HGF secretion（P>0.05）;under hypoxic condition，the amount of VEGF,andIGF-1in MSCGATA-4group significantly higher than that of MSCs and MSCnullgroup（P<0.05）,and there is no significance in HGF secretion（P>0.05）.4. After adding VEGF,and IGF-1neutralization antibodies, compared with the before,the MTT uptake ratio remarkably decrease, both the positive rate of Annexin V and theamount of LDH all increase in three groups; under hypoxic condition，compared to theMSC and MSCnullgroup, the MTT uptake ratio obviously increase, the positive rate ofAnnexin V and the amount of LDH significantly decrease in MSCGATA-4group (p<0.05);while there were no significant difference between hypoxic MSCs and hypoxic MSCnull（P>0.05）.Conclusion:1. The recombinated GATA-4vector was constructed successfully, and stable MSCsclones with GATA-4over-expressed were obtained.2. MSCs over-expressed GATA-4could promote the secretion of VEGF, HGF andIGF-1.3. The survival of three groups’ MSCs were all apparently inhibited after adding theneutralization antibodies，while over-expression of GATA-4could improve the cellularsurvival, which infers that GATA-4could affect cellular survival by strengthening itsparacrine effects.4. GATA-4gene over-expressed could improve MSCs survival, which could providea new way for MSCs transplantation treatment of ischemic myocardium.