Mechanism Of Specific SM22Alpha Promoter Recombinant Lentivirus-mediated P27Gene Overexpression In Carotid Stent Restenosis

Objective:Previous reports showed the major pathological manifestation of in-stent restenosis is intimal hyperplasia and vascular remodeling in which excessive proliferation of vascular smooth muscle cells (VSMCs) plays an important role. This study was designed to construct SM22alpha-p27-EGFP recombinant lentiviral vector driven by SM22alpha promoter and explore their effects on VSMCs specific p27protein overexpression. Then we chose the most appropriate multiplicity of infection (MOI) of recombinant virus for further study.Methods:The SM22alpha promoter was Extracted. SM22alpha-p27-EGFP, negative control lentivirus and SM22alpha-null-EGFP were constructed and infected in primary cultured smooth muscle cells and endothelial cells.Flow cytometry was used to determine the selectivity of the virus infection and the MOI; Immunofluorescence and Western blot werer applied to detect the p27gene expression on smooth muscle cells and endothelial cells.Results:1) SM22alpha promoter was extracted from Rats genome to construct SM22alpha promoter-p27-EGFP and SM22alpha promoter-null-EGFP recombinant lentiviral vectors. The viral titer was5×108U/ml.2) FACs showed the SM22alpha promoter could selectively infect smooth muscle cells with an appropriate MOI=103)Immunofluorescence staining showed SM22alpha promoter could effectively lead to oversxpression of p27protein in smooth muscle cells.4)Western Blot proved the existion of recombinant p27protein in smooth muscle cells.Conclusion:Recombinant lentiviral vector driven by SM22alpha promoter could selectively infect vessel smooth muscle cell and effectively lead to p27protein overexpression. Objective:In the above part, we have showed that the recombinant lentivirus SM22alpha-p27-EGFP could selectively infect smooth muscle cells and over-express p27protein. The purpose of this part is to study whether recombinant lentivirus p27overexpression could inhibite cell proliferation by cell cycle arrest and compared their effects with paclitaxel.Methods:BrdU staining was performed to study the effects of paclitaxel and recombinant lentivirus cause on proliferation of vascular smooth muscle cells and endothelial cell. Flow cytometry was applied to investigate the cell cycle distribution of vascular endothelial cells and vascular smooth muscle cells treated by paclitaxel and recombinant lentivirus;Results:BrdU results showed that both paclitaxel and recombinant lentivirus SM22alpha-p27could effectively inhibit vsmc proliferation (p<0.05). Paclitaxel presented greater inhibiton effect on vsmc proliferation than that of SM22alpha-p27recombinant lentivirus (p<0.01); In endothelial cell group,:paclitaxel can effectively inhibit vsmc proliferation (p<0.05). The negative control virus and SM22alpha-p27recombinant lentivirus showed comparable inhibitory effect (p>0.05). Flow cytometry results showed that both endothelial cells and smooth muscle cells showed a G2/M phase arrest after paclitaxel intervention with the decrease of cell in S-phase and G0/G1phase (p<0.05); SM22alpha-p27recombinant lentivirus induced G1/S phase arrest,increased distribution of G0/G1and decrease of S-phase in smooth muscle cells(p<0.01).Conclusion:Compared with paclitaxel that shows extensive inhibition on cell proliferation, SM22alpha-p27-EGFP recombinant lentivirus can efficiently and specifically inhibit smooth muscle cell proliferation which may be mediated by blocking G1/S phase and cell cycle progression.