The Experimental Research On Nuclear Factor-κB SiRNA To Prevent Proliferaion And Restenosis Of Vein Graft In Rats

ObjectiveTo construct NF-кB small interfering RNA plasmid complexes and jugular vein aortic autologous vein graft model of rat, vein graft was processed by NF-кB siRNA transfection in order to research proliferation of intima and smooth muscle cells, measure expression levels of inflammatory cytokines, observe NF-кB siRNA prevention effects on vein graft restenosiS。MethodsAccording to NF-кB (RelA, p65) gene sequence from genetic data, we first design NF-кB siRNA using of RNA interference design software online, and code the length of the 21nt sequence (5'-AAG CAC AGA TAC CAC TAA GAC-3') specific short hairpin RNA (shRNA) and its complementary strand.After DNA sequence identified, extract, purify and amplify NF-кB siRNA plasmid and negative control plasmids for animal experiments.Animal model and the group:Male Wistar rats (weight 300g-350g) were randomly divided into four groups, Group A:no autogenous vein graft for normal vein controls, n= 15. Group B:simple autogenous vein graft group, n= 23. Group C:vein grafts with negative plasmid liposome medical gel complex transfection, n= 30. Group D:vein grafts with NF-кB siRNA liposome plasmid medical gel complex transfection, n=30. The rat model of groups B, C and D need to transplant autogenous external jugular vein to aortic, NF-кB p65 small interfering RNA plasmid liposome medical gel complex soaking, pressure washing and painting the outer membrane of vein graft in groups D for transfection.The Pathomorphology and inflammatory factors levels of vein graft. Each group at four time points after Transplantation(3d,7d,14d,21d), the corresponding time points observe pathomorphology of vein graft,detect proliferating cell nuclear antigen positive cells (PCNA) by Immunohistochemical staining.Measure of inflammatory factors MCP-1 mRNA and TNF-amRNA levels by PCR, determination of NF-кB p65 protein expressions by Western blot.ResultsFirstly vein grafts of groups B, C and D intimal hyperplasia obviously, medial smooth muscle cell proliferation activity, a large number of PCNA-positive neointimal cells.Secondly, inflammatory factor levels at 3d and 7d after vein graft in can be seen in PCR test results, cytokines MCP-1mRNA and TNF-amRNA expression levels of group B and group C was significantly higher than that of group A(P<0.05), but the cytokines level in vein graft tissue of group D was lower than that of group C (P<0.05).Thirdly, to detect the NF-кB p65 protein expression levels in the vascular tissues 7 days after vein graft. The NF-кB p65 protein expression levels of group B and C were significantly higher than normal veins of group A(P<0.05), but the NF-кB p65 protein level in vein graft tissue of group D is lower than that of group C(P<0.05).ConclusionThe MCP-1 mRNA and TNF-amRNA expression levels are Closely related to NF-кB p65 protein expression after vein graft injury. The inflammatory cytokines NF-кB,MCP-1 and TNF-a expression of vein graft have effects on neointima proliferation. Transfection of NF-кB small interfering RNA plasmid liposome complexes can inhibit NF-кB activation, reduc MCP-1 and TNF-amRNA expression levels and inflammation, inhibit the vein graft neointima proliferation and VSMC proliferation and alleviate restenosis to some extent.