Protection Of SiRNA In The Injury Effects Of Human Cytomegalovirus On Rabbit Vascular Smooth Muscle Cells

Protection of rabLOX-1 in the injury-effects caused by HumanCytomegalovirus on rabbit Vascular smooth muscle Cells The incidence of atherosclerosis(AS) has increased in developed countries, even in China. Inflammation has been verified to play a key role in the progression of AS. Epidemiological and molecular biological studies indicate that Human cytomegalovirus(HCMV) antigen and its DNA sequence exist in AS plaque, especially in Vascular Smooth Muscle cells (VSMC) and vein endothelial cell(ECV). In addition, positivity of HCMV antibody in surum from AS patients is much higher than that in the healthy strongly indicating that HCMV infection involves in the development of AS but the precise mechanism remains unknown. Thus it is very important to investigate the mechanism by which HCMV takes part in the formation of AS. In present study, siRNA for rabbit Lectin-like oxidized low density lipoprotein receptor-1(LOX-1) was cloned into lentivirus vector (rabLOX-1) which was adopted to examine the expression of LOX-1 in SMC stimulated with HCMV. monocyte chemoatt ractant protein-1 (MCP-1) is very important during the monocyte adhere to endothelium and adopt fatmelt into foam cell , which also stimulate VSMC proliferation to accelerate the pathogenesis of AS. Methods 1. The design and generation of siRNA plasmidThe chemically synthesized target sequence was cloned into pGCsi-H1/Neo/DsRed vector; the ORF of target gene was cloned into pdsRED2-Nl. The highest efficient plasmid was combined with lentivirus vector and proliferated in 293 cells.2. Cell cultureTissues from media of blood vessels was cultured and identified and passaged. Passages of 3-8 cells were utilized for vitro experiments.3. Gene transfectionCells were cultured in 5 groups. Control group : Cells were cultured in conditional media containing 2% new born serum(NBS) without any treatment. rabLOX-1+VSMC group: Cells were transfected with rabLOX-1 plasmid at the concentration of 100MOI; HCMV group: 70-80% confused VSMC was infected with HCMV at the concentration of 100TCID₅₀/0.1ml. HCMV+ rabLOX-1 group: Cells were transfected with rabLOX-1 plasmid at the concentration of 100MOI after being infected with HCMV. HCMV+vector: Cells were infected with HCMV followed by transfection of empty vectors at the concentration of 100MOI.4. RT-PCRTotal RNA was extracted from VSMC with Trizol for reverse transcription and proliferation of cDNA according to the manuscriptures' instruction. Data were analyzed by computer and the results were expressed in optical density ratio of LOX-1,MCP-1,HCMV IE gene to GAPDH.5. Western blottingTotal protein from cell lysis was eletrophoresied followed by membrane transferation. After incubation with primary and sencodary antibodies the protein bands were developed and analyzed by computer. Results1.The effectively siRNA plasmid was successfully combined with Lentivirus vector in the 293T cells confirmed by enzyme digesting and DNA sequencing. 2.Primary culture of VSMC was confirmed by immune histochemistry 3.RT-PCR result indicate LOX-1mRNA, MCP-1mRNA in HCMV+ rabLOX-1 group was a little higher than control group and rabLOX-1 group , LOX-1mRNA in HCMV group and empty vector group was much higher. HCMV IE72mRNA is much higher in HCMVgroup and empty group than in HCMV+ rabLOX-lgroup. which prompt siRNA plasmid can efficiently inhibit LOX-1mRNA, MCP-1mRNA expression and HCMV multiplication in VSMC .4.Western blotting result indicate protein LOX-1 was expressed at a low level both in control group and HCMV+ rabLOX-lgroup and rabLOX-1 group, protein LOX-1 was much higher in HCMVgroup and empty vector group, which indicate siRNA plasmid plasmid can efficiently inhibit protein LOX-1 expression in VSMC induced by HCMV. ConclusionsRNA interference can effectively down-regulate the LOX-1 and MCP-1 expression and inhibit HCMV multiplication in VSMC.