The Effect And Mechanism Of Injectable Hydrogel Carrying Bcl-2 And VEGF Dual Genetic Co-overexpressing MSC Transplantation In The Treatment Of Rat Myocardial Infarction

Background:Mesenchymal stem cell(Mesenchymal Stem Cell,MSC)transplantation has emerged as a promising treatment for myocardial infarction(MI),but the clinical trials feedback that the poor retention and low survival of transplanted cells limited the therapeutic efficacy.Strategies of genetic modification or using biomaterials to carry are proved to be useful for improvement.Bcl-2(B-cell lymphoma 2,Bcl-2)gene is an anti-apoptotic gene.VEGF(Vascular Endothelial Growth Factor,VEGF)is a gene for angiogenesis.IH(Injectable Hydrogel,IH)is injectable,biodegradable and tissue compatible and can be used for carrying cells when transplantation.Objectives:Injectable hydrogel combined with Bcl-2 and VEGF dual genetic modification to improve therapeutic effects of MSC transplantation for myocardial infarction,to provide experimental foundation and theoretical basis for clinical use.Methods and results:1.PCR was used to amplify and obtain the Bcl-2 gene and VEGF gene.T2A peptide sequence was employed to link and achieve co-overexpression of Bcl-2 gene and VEGF gene.Lentiviral vectors were constructed.SD rat MSC were transfected to obtain cell lines which could co-overexpress the two target genes stably.Results of quantitative real-time PCR and western blot confirmed that the dual genetic modified MSC had higher expression for Bcl-2 and VEGF gene than singly modified MSC.CCK-8 assay showed that the dual genetic modified MSC proliferated more rapidly over 7 days under normal culture condition.2.An in vitro model of OGD(Oxygen and Glucose Deprivation,OGD)was applied to mimic the ischemic microenvironment in vivo.MSC were divided into five groups:MSC-GFP group,MSC-VEGF group,MSC-Bcl-2 group,MSC-BV group,MSC-GFP normal cultured group.Annexin V-FITC/PI flow cytometry and western blot for apoptotic proteins of Bax,Bcl-2,Cleaved-caspase-3 were used to reflect apoptosis of MSC.Results showed that dual genetic modification reduced the apoptosis of MSC under OGD condition.ELISA results showed that the MSC-BV had the highest level of growth factors of VEGF,bFGF,HGF and IGF-1 in all groups.3.Injectable hydrogel was used to carry the Bcl-2 and VEGF dual genetic modified MSC and implanted into the infarcted hearts of SD rats for treatment.Rats were divided into six groups:SHAM group,PBS group,IH group,MSC group,MSC+BV group,MSC+BV+IH group.At 4 weeks after MI and treatment of cell transplantation,transthoracic echocardiography results showed that rats of MSC+BV+IH group promoted cardiac contractile function recovery of infarcted heart best in all groups.HE and Masson stain showed that the rats of MSC+BV+IH group had the smallest infarction size in all groups.The immunofluorescence of cTnT results showed rats of MSC+BV+IH group had a better myocardial viability.TUNEL assay and western blot results showed that rats of MSC+BV+IH group had a reduced apoptosis of myocardial cells.Immunofluorescent of both vWF and a-SMA results showed that rats of MSC+BV+IH group had a stronger angiogenesis.CM-Dil labeling showed that transplanted cells of MSC+BV+IH group had a higher retention in the infarcted hearts.4.Neonatal SD rat cardiomyocytes were cultured in vitro successfully.Cardiomyocytes were cocultured with either MSC-GFP or MSC-BV using Transwell coculture system.The condition of 94%N2,5%CO2,1%O2,24h was used.Cells were divided into four groups:Normal culture group,hypoxia culture group,coculture with MSC-GFP group,coculture with MSC+BV group.Results from flow cytometric assays,western blots and TUNEL assays showed that the apoptosis of cardiomyocytes that cocultured with Bcl-2 and VEGF dual genetic modification MSC under hypoxia condition could be better reduced.Conclusions:1.We constructed a lentiviral vector that overexpress the VEGF and Bcl-2 genes simultaneously and confirmed that the dual genetic modified MSC had a higher expression for VEGF and Bcl-2 gene than singly modified MSC.2.Bcl-2 and VEGF dual genetic co-overexpressing MSC had a better self-protective and paracrine effects in vitro of OGD condition than singly modified MSC.3.Bcl-2 and VEGF dual genetic co-overexpressing MSC transplantation treat myocardial infarction better,and the using of IH for carrying further improved the effects.4.Bcl-2 and VEGF dual genetic co-overexpressing MSC protect cardiomyocytes better under hypoxia in vitro via paracrine effect.