Analysis Of C-kit+cardiac Progenitor Cells Expressing And Secreting Endothelial Cell-specific Proteins

Objective:C-kit+cardiac progenitor cells（CPCs）play an important role in the treatment of ischemic cardiomyopathy,but the properties of this cell are still not fully understood.This experiment will mainly explore:1.Prove from the protein level that c-kit+CPCs can express and secrete proteins related to endothelial cell characteristics;2.After hypoxic treatment of c-kit+CPCs,detect the changes of c-kit+CPCs expression and secreted cytokines.Methods:1.Adult male C57BL/6 mouse hearts were cut into approximately1mm3 pieces,digested with type II collagenase and trypsin into a single cell culture and amplified,immunomagnetic beads labeled for c-kit+CPCs sorting,immunofluorescence,flow The purity was detected by cytometry.Cardiomyocyte control group（Car group）and c-kit+CPCs normal culture group（c-kit group）were set up.Protein chip was used to detect the expression of endothelial cell specific protein and related cytokines.2.To detect the effect of hypoxia treatment on the secretion function of c-kit+CPCs:hypoxia chamber was used to perform hypoxia treatment on the sorted third-generation c-kit+CPCs.The hypoxia treatment group（c-kit hyp group）was cultured in 37℃,5%carbon dioxide,95%N₂,and the normal group（c-kit nor group）was cultured in37℃,5%carbon dioxide and 21%O₂.Each group is set for 3,6,12 hours.The cell culture supernatant was collected for protein chip detection,and the cell lysate was collected for protein blot（WB）detection.Results:1.Positive sorting based on cell surface marker c-kit to obtain c-kit+cells.After sorting,c-kit+cells grew well,and the cell morphology was significantly more uniform than before sorting,showing a fusiform and short rod shape.2.After sorting,the third-generation cells were subjected to surface c-kit antibody identification.Almost all cells showed red fluorescence labeled with c-kit antibody.Flow cytometry identification found that the proportion of c-kit+cells in the mixed cells before sorting was 1.34%.After immunomagnetic bead positive sorting,the cell purity is about 95%,and the surface markers can be stably expressed under our culture system.3.Factors with increased expression in the c-kit group compared to the Car group are:endothelin-1（Endothelin-1）,cysteine protein 61（Cysteine-rich 61,Cyr61）,FGF basic,placental growth factor 2（Placenta Growth Factor-2,Pl GF-2）（P<0.05）,these factors have the characteristics of endothelial cells secreted and play a role in angiogenesis.4.The increased expression of c-kit hyp 3h group compared to c-kit nor 3h group are:CXC chemokine ligand 16（CXC chemokine ligand 16,CXCL16）,Angiopoietin-1,FGF basic,IL-1beta,human platelets Derived growth factor BB（Platelet derived growth factor,PDGF-BB）,hepatocyte growth factor（HGF）,neuroregulin-1（Neuregulin-1,NRG-1）（P<0.05）;c-kit Hyp 6h group and c-kit nor 6h group showed no significant difference in protein chip results;c-kit hyp12h group compared with c-kit nor 12h group increased expression factors:vascular endothelial growth factor（Vascular endothelial growth factor,VEGF）（P<0.05）.Conclusions:1.c-kit+CPCs can express and secrete a variety of endothelial cell characteristic related proteins and angiogenic factors.2.The appropriate time of hypoxia treatment can enhance the secretion ability of c-kit+CPCs,which may help to improve the therapeutic effect of c-kit+CPCs secretion function on ischemic cardiomyopathy.3.Different proteins have different reaction time to hypoxia.NRG-1 secretion increases after 3 hours of hypoxia,and VEGF increases after 12 hours of hypoxia.