[Construction of recombinant adenovirus expressing shRNA targeting human leukocyte-derived arginine aminopeptidase gene].

AIM: To construct the recombinant adenovirus expressing small hairpin RNA (shRNA) targeting human leukocyte-derived arginine aminopeptidase (LRAP) gene and silence the expression of LRAP in human retinal microvascular endothelial cells (HRMECs). METHODS: Three pairs of oligonucleotides coding for shRNAs targeting human LRAP gene were designed and synthesized, and were cloned into the shuttle vector pRNAT-H1.1/Adeno after annealing. The three constructed shuttle plasmids, through homologous recombination with adenoviral backbone vector pAdEasy-1, were transformed into E.coli BJ5183-AD- to produce recombinant adenoviral plasmids. And then the recombinants linearized with Pac I were transfected into 293a cells to package adenoviruses. After amplification and titter determination, the recombinant adenoviruses were used to infect the primary HRMECs. Quantitative real-time PCR was taken to determine the relative amount of LRAP mRNA to screen the adenovirus with the highest silencing efficiency. The level of LRAP protein after RNA interference in HRMECs was further determined by Western blotting. RESULTS: PCR, restriction digestion and DNA sequencing confirmed that the purpose shRNA-coding sequences were correctly inserted into the shuttle vectors and adenoviral plasmids, and the recombinant adenoviruses were packaged successfully in 293a cells. The most effective adenovirus with the silencing efficiency up to 79% was selected by quantitative real-time PCR, which significantly lowered the expression of LRAP in HRMECs. CONCLUSION: The recombinant adenovirus expressing shRNA effectively silencing LRAP gene was constructed successfully, which would facilitate further study of the role that LRAP plays in the development of diabetic retinopathy.

[Dexamethasone intensify the tight junctions in rat retinal vascular endothelial cells in vitro].

OBJECTIVE: To investigate the effect of dexamethasone on the expression, distribution and function of tight junctions in rat retinal vascular endothelial cells in vitro. Try to explain the mechanism of glucocorticoid in the treatment of macular edema from a new viewpoint. METHODS: Rat retinal vascular endothelial cells were isolated and purified by magnetic beads coated with anti-CD31. Cells were identified by vW factor indirect immunofluorescence staining. The fourth-passage cells were used to investigate the effect of dexamethasone on the tight junctions in rat retinal vascular endothelial cells. Cells were separated into two groups, one was treated with 500 nmol/L dexamethasone and the other was used as the control. Transepithelial electrical resistance (TER) was measured to estimate the changes in the treated group. Indirect immunofluorescent stain and RT-PCR were used to observe the difference of tight junction protein distribution and mRNA expression level between these two groups. RESULTS: Rat retinal vascular endothelial cell monolayer showed positive immunofluorescent staining for vW factor. The dexamethasone treated group showed greater TER than that of the control (P < 0.01). Tight junction protein in the dexamethasone treated group localized closer to the borders of retinal vascular endothelial cells than that of the control. Claudin-1 mRNA level of the dexamethasone treated cells were greater than that of the control. CONCLUSIONS: Dexamethasone intensifies the tight junctions in rat retinal vascular endothelial cells. Therefore this is one of the mechanisms of treatment of macular edema by glucocorticoid.