Effections Of SiRNA Interfering Plasmids And Eukaryotic Expression Vector For GDF-15 Gene On HUVECs Expression And Proliferation

Objective:To observe the Effections of siRNA interfering plasmids and eukaryotic expression vector for GDF-15 gene on HUVECs that proliferation and expressions of VEGF and bFGF in vitro.Methods:The siRNA interfering plasmids and eukaryotic expression vector for GDF-15 gene targeting on human GDF-15 gene were constructed and transfected into HUVEC cells respectively. The relative expression of mRNA in GDF-15 gene was detected by real time PCR, then screened out the optimal siRNA interference sequence.Divided the cells into 5 groups: black control group;interference group;negative control of interfering group;eukaryotic expression group;negative control of eukaryotic expression group.Then we detected expressions of GDF-15 protein by Western blot,concentrations of VEGF and bFGF in cell culture supernate by ELISA,and detected cell cycles by FCM to learn proliferation of cells.Results:1.Outcomes of real-time fluorescent quantitative PCR showed that, expressions of mRNA in HSH022853-24-CU6 plasmid group significantly lower than other groups,the inhibition rate was about 92%,HSH022853-24-CU6 was selected out as the optimal interference plasmid.Expressions of mRNA in eukaryotic expression group wassignificantly increased,the number was 1062.5 times as that of its negative control group,significantly lower than other groups.2.The results of Weston blot showed that, the expression of GDF-15 protein in eukaryotic expression group was highest,and significantly higher than other groups. The interference group was lowest,and significantly lower than its negative control group and the black control group;3.The results of ELISA showed that concentrations of VEGF in the the black control group was significantly higher than other groups.But compared with their corresponding negative control group, expressions of VEGF in eukaryotic expression group and in the interference group differences were nearly equal.We almost couldn’t detected concentrations of bFGF in any group;4.Outcoms of FCM showed that the percentage of cells entering to G2 and S phase in eukaryotic expression plasmid group was highest,and significantly higher than that in black control group and interference group.But compared with their corresponding negative control group, outcomes of the eukaryotic expression group and the interference group were similar.Conclusion:For normal cultivated HUVEC cells,GDF-15 gene has no effections on HUVEC cells,capacity for proliferating and generating VEGF and bFGF.