| Objective: Construct plasmids to direct p38MAPK specific siRNA expression in eukaryotic cells and investigate their inhibition efficiency in HUVEC.Methods: Three p38MAPK-siRNA plasmids and one negative control siRNA plasmid were designed and constucted by inserting chemically synthesized ribonucleotide fragment into linearized pGensil-EGFP vector. The green fluorescence protein expression was observed by fluorescent microscope when the plasmids were transfected into HUVEC; The inhibition ratios of siRNA were analyzed by detecting the mRNA and protein levels of p38MAPK through RT-PCR and western blot respectively.Results: Three p38MAPK-siRNA plasmids and one negative control siRNA plasmid were constructed successfully, named as psi1, psi2, psi3, psi4 respectively. After transfected into HUVEC, we got the maximum transfection efficiency of 48% by detecting green fluorescence. RT-PCR and westren blot showed p38MAPK mRNA and protein were significantly depressed after transfection, psi1 plasmid had higher silence efficency.QConlusion: Inhibiting the expression of target gene p38MAPK was demonstrated in HUVEC, especially psi1 has higher inhibition efficiency. Objective: To observe the p38MAPK gene silence by p38MAPK-siRNA and explore its relation with TLR4 in diabetic atherosclerosis.Methods: We mimicked diabetic state in vitro using high glucose, advanced glcosylation end products(AGE), high insulin and H2O2 to stimulate HUVEC separately, analyszed the inhibition of p38MAPK- siRNA in HUVEC by RT-PCR and western blot; the expression and location of TLR4 were respectively observed by RT-PCR, western blot and immunohistochemistry assay before and after stimulation.Results: The increasing of p38MAPK in HUVEC activated by high glucose, AGE, high insulin and H2O2 could be inhibited by p38MAPK- siRNA to normal level; Simultaneously, the expression of TLR4 increased in HUVEC after the stimulation, p38MAPK-siRNA could inhibited the expression of TLR4 induced by 4 stimulating factors.Conclusion: p38MAPK-siRNA could effectively inhibit the expression of p38MAPK and TLR4 in HUVEC cell, which might have a good future in preventing from the development of diabetic atherosclerosis. Objective:To construct pGL3-Basic eukaryotic expression vector containing tissue-specific promoter of FLT-1 and observe the activity of this promoter in HUVEC cells, in order to explore the possibility of FLT-1 promoter to endothelium-targeted gene therapies for vascular disease.Methods:The FLT-1 gene promoter was amplified by polymerase chain reaction and cloned into pGL3-Basic vector to construct pGL3-Basic eukaryotic expression vector containing FLT-1 gene promoter (pGL3- FLT-Basic-luc).Then was transiently transfected into HUVEC cell and HepG2, NIH3T3, HEK293 cell using liposome transfection reagent, and the activity of luciferase was determined 48 h later .After stimulating HUVEC with high glucose, H2O2, advanced glycosylation end products(AGE) and high insulin separately, we detected the expression of FLT-1 mRNA and the transcriptional activity of FLT-1 promoter respectively through RT-PCR and Dual-Luciferase Reporter Assay System.Results:DNA sequencing and digestion confirmed that the recombinant of plamid pGL3-FLT-Basic-luc contained correct FLT-1 promoter sequence. The activity of luciferase in HUVEC was much higher than in HEK293 after transfection of pGL3-FLT-Basic-luc, and little activity of luciferase was detected in other two cells. After stimulated by high glucose, H2O2, AGE and high insulin, HUVEC show high expression of FLT-1mRNA and much higher transciptional activity of FLT-1 promoter in HUVEC, up to 66% when the luciferase activity of pGL3-promoter in HUVEC was considered as 100%.Conclusion: FLT-1 promoter had higher tissue-specific transcr- iptional activity in HUVEC , especially stimulated by high glucose, H2O2, AGE and high insulin, which might be a potential therapeutic reagent for endothelium-targeted gene therapies for AS. |
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