Construction Of ShRNA-SIfn1 Recombinant Adenovirus Vector In Rat And Its Effects On Proliferation Of Vascular Smooth Muscle Cells

Objective: To construct recombinant adenovirus containing the Enhanced Green fluorescent protein and an shRNA(short-hairpin RNA)to silence Slfn1 mRNA,package it in an adenovirus vector,and then use it to transfect primary rat VSMCs for investigation of its effects on proliferation of the VSMCs 48 h later.Methods: 1.Three pairs of shRNA-Slfn1,i.e.shRNA-Slfn1(1),shRNA-Slfn1(2)and shRNA-slfn1(3),and a pair of control shRNA sequence(NC-shRNA)were synthesized.The single primer was annealed into double-stranded oligo sequences and connected into the U6 promoter downstream of linearized RNA interference carrier of pDKD-CMV-shRNA to replace the original ccdB gene.Three shuttle vectors and a control shuttle vector were respectively transfected into HEK293 cells with backbone plasmid using the Admax system.The obtained recombinant adenoviruses were amplified in a little quantity and determined for their titers.Plasmids were identified using Western Blot with a flag antibody.2.The primary culture and subculture were carried out with a tissue-piece inoculation.Cell morphology was observed with an inverted phase contrast microscope and identified with immunofluorescence.3.VSMCs were infected with the shRNA-Slfn1(1)recombinant adenovirus at the optimal multiple infections(MOI).EGFP was observed under the inverted phase contrast microscope after 48 h,with its transfection efficiency detected using a flow cytometer.The Slfn1 mRNA level was detected using RT-PCR and the change in cell proliferation was determined using CCK-8.Results: Results: 1.Restriction-enzyme digestion and sequencing of the PCR products showed that the shRNA-Slfn1(1),shRNA-Slfn1(2),shRNA-Slfn1(3)and the NC-shRNA were successfully constructed and the titers of their corresponding viruses were 1.0×1011(ifu/ml),2.5×1011(ifu/ml),6.3×1010(ifu/ml),5.5x1010 ifu/ml,respectively.2.The VSMCs grew on days 4~6 and showed fusiform;On days 7~9,some cells formed mesh;more cells grew into whorled pattern and formed compact sarciniform.On days 10~14,cells showed a typical “peak-valley” structure and became nearly 80% integrated.In this time period,cells grew faster and became larger,with their refractivity weakened.A typical ?-Smooth muscle actin(?-SM-actin)in VSMCs could be discerned with an immunofluorescent staining,with a positive rate of ?-SM-actin in VSMCs being more than 95%.3.After VSMCs were infected with the shRNA-Slfn1(1)recombinant Adenovirus for 48 h,the transfection efficiency was above 80% as determined under the inverted phase contrast microscope or with flow cytometry.Transfection with shRNA-Slfn1(1)recombinant Adenovirus significantly decreased the mRNA expression when compared with the control shRNA.After this Slfn1 knockdown,proliferation of the VSMCs was increased efficiently compared with the counterparts infected with the control shRNA.Conclusion: 1.A recombinant adenovirus vector of rat Ad-shRNA-Slfn1 was successfully constructed.2.The shRNA-Slfn1(1)recombinant adenovirus can efficiently interfere with the Slfn1 mRNA level in the primary VSMCs from rats,which in turn can enhance the cell proliferation.