Experimental Study Of Recombinant Adenovirus Vector Mediated MiR-27b Infection To Endothelial Progenitor Cells

BackgroundEndothelial progenitor cells (EPCs) are precursors of vascular endothelial cells, and involve in vascular regeneration and repairing process after endothelial injury. Senescence and dysfunction of EPCs can induce vascular endothelial dysfunction which is closely associated to the development of atherosclerosis. In patients with postprandial hypertriglyceridemia, the increased plasma remnant-like particles (RLPs)-cholesterol levels are closely related to endothelial dysfunction. RLPs can accelerate the senescence process of human EPCs derived from peripheral blood mononuclear cells, however, the mechanism is still not completely clarified. MicroRNAs (miRNAs) are a class of endogenous small non-coding single-stranded RNAs that are highly reserved during evolution, and play important roles on regulating a series of complicated pathophysiological process such as cell differentiation, growth and development, apoptosis and tumorigenesis, via inhibiting the expressions of eukaryotic genes at the posttranscriptional level. Recently, it has been found that miRNA involves in the regulation of cell senescence. Our previous study discovered the expression level of miRNA-27b (miR-27b) decreased markedly in the RLPs-induced senescent EPCs, from which we conjectured that miR-27b may regulate RLPs-induced EPCs senescence.Objective Construct recombinant adenoviral vector using enhanced green fluorescence protein (EGFP) as reporter gene and carrying mmu-miR-27b gene by homologous recombination in bacteria, infect mouse EPCs, lay foundation for future research on the role of miR-27b during the RLPs-induced EPCs senescence.MethodsMononuclear cells were isolated from mouse bone marrow by density gradient centrifugation and differential adhesion method. The remaining cells were cultured and differentiated to EPCs in EBM-2. EPCs were identified by flow cytometer analysis. Plasmid contain target gene miR-27b and target vector pDC316-siRNA liberated via enzyme digestion, respectively, the products recovered from agarose electrophoretic gels were connected directionally and co-transformed into BJ5183cells. The bacteria clones were identified by polymerase chain reaction (PCR). DNA sequencing and comparison analysis would be performed toward positive clones and the right clones were successfully-constructed target plasmids. The recombinant plasmid was transfected to293cells to package and amplify recombinant adenovirus vector (Ad-miR-27b). Detect the titer of the adenovirus. Ad-miR-27b, together with Ad-EGFP as negtive countrol, infected EPCs respectively and real-time PCR (RT-PCR) was performed then to test the expression of miR-27b in EPCs. ResuLtsCells obtained from mouse bone marrow by density gradient centrifugation and differential adhesion method formed clusters at4d. At12d, positive ratios of CD34+、CD133+、Flk-1+and CD31+were (65±4)%、(48±3)%、(37±3)%and (51±4)%, respectively. Positive recombinant plasmid was obtained from plasmid containing target gene miR-27b and target vector pDC316-siRNA. After double-enzyme-digestion, two plasmids were cotransformed into BJ5183cells. The positive clones were chosen by DNA sequencing and comparison analysis. Ad-miR-27b was successfully constructed after recombinant plasmid was packaged and amplified in HEK293. The titer of the adenovirus was1.5×109pfu/mL. Expression of miR-27b upregulated significantly in24h after the infection of Ad-miR-27b into the EPCs when compared with the negtive control group.ConclusionsEPCs effectively express target gene in vitro after being infected by successfully-constructed recombinant adenoviral vector.