Effect Of Fgl2 Gene Scilencing By Lentivirus-shRNA On Proliferation And Migration Of Myocardial Microvascular Endothelial Cells And The Mechanism Involved

AIM:To isolate and culture Myocardial Microvascular Endothelial Cells primarily,and identify it by immunofluorescence. To construct a lentiviral RNA interferenced vector of human fibrinogen-like protein 2(hfg12) gene.MMVECs were transfectted by lentiviral vector.To investigate the role of fg12 gene in the proliferation and Migration of MMVECs.METHODS:(1) Isolated and cultured Myocardial Microvascular Endothelial Cells primarily,and identified it. In this experiment, we got heart tissues from healthy and clean Sprague-Dawley (SD) rats of seven days old. We removed the heart,washed the blood, cut atrium and right ventricle off, kept the left ventricle, peeled off the endocardial and epicardial. Digested heart tissue with Trypsin and Collagenaseâ…¡, The cell cultures were purified by different speeds of attachment. Removed culture medium and passaged MMVECs when endothelial cells had grown in primary monolayer fusion. Second-generation MMVECs were identified as endothelial Cells by factorâ…§and CD31 related antigen,which were important markers of.MMVECs(2) The nucleotide sequences of hfgl2(NM₀06682) were found in the GeneBank..The four tatget sequences of fgl2 gene could be effectively scilenced with RNA interference were confirmed.The cDNA containing both sense and antisense Oligo DNA fragements of target sequences were designed and cloned into the pGCSIL-GFP vector which was digested by AgeI/EcoRI.The obtained lentiviral vector containing Fg.2shRNA was confirmed by digestion and sequencing.Using lentiviral vector pHelper 1.0,pHelper2.0,pGC-LVfgl2 to transfect 293T cells,then the lentivirus were collected.The titer of virus was tested according to the expression level of GFP. (3) This experiment included three groups:group A,group B and group C. group A:MMVECs(control group); group B:the empty lentiviral vetor (GFP group);groupC: the lentiviral vector containing fgl2shRNA (short hairpin RNA) (fgl2shRNA-LV group). qRT-PCR detected the expression of fgl2,Angl,Ang2,Tie2,AKT2 mRNA, and Westernblot detected the expression of fgl2 protein. Cell proliferation was detected by MTT method and cell migration was detected by transwell methodRESULTS:(1)Cultured myocardial microvascular endothelial cells primarily, and cell purity was about 95%,showed a typical paving pebble-like structure.(2) Immunofluorescence demonstrated that factor VIII was main in the cytoplasm, showed green fluorescence,and CD31 was in the membrane,showed red fluorescence.(3)Digestion and DNA sequencing demonstrate that lentivirus vector pGC-LVFgl2 was constructed successfully which can produce fgl2shRNA. The titer of concentrate virus was 1*109TU/ml. Using the obtained lentiviral vector containing Fg12shRNA to transfect MMVECs,96 hours later,it shows that 70%-80% of MMVECs express GFP. Compared with group A and group B, the qRT-PCR analysis showed that group C the expression of fgl2 was decreased,and the expression of Angl,Ang2,Tie2,AKT2 were increased significantly. Proliferation and migration.of MMVECs was also increased. and there were difference significant (P<0.05) In group A group and group B, the expression of fgl2,Angl,Ang2,Tie2,AKT2 had no change. Proliferation and migration.of MMVECs had no change.CONCLUSION:The lentivirus vector pGC-LVfgl2 was constructed successfully which can produce fgl2shRNA, transfected MMVECs,the expression of fgl2 was decreased,and the expression of Angl,Ang2,Tie2,AKT2 were increased significantly. Proliferation and migration.of MMVECs was also increased. The lentiviral vector containing Fgl2shRNA transfected MMVECs could stimulate the angiogenesis.The possible mechanism may be via Angl/tie2 signal..Fgl2 gene can be a new target for the therapy of impaired angiogenisis.