Overexpression Of Rictor Gene Adenovirus Construction And Toll - Like Receptor 2 In Angiotensin â…¡ - Induced Hypertensive Mice Cardiac Fibrosis

ObjectiveUsing the Ad-Easy system for construction of recombinant adenovirus expression vector containing rictor. It provids a basis further study of application of rictor gene in endothelial cell senescence.MethodsThe rictor was inserted into the shuttle vector pAdTrack-CMV which contains a separated expression box of green fluorescence protein, the shuttle vector was then transformed into E. coli(BJ5183)which was already contained adenovirus backbone vector pAdEasy-1to achieve the homologous recombination and obtain recombinant adenovirus genomee plasmid. This recombinant construct was then linearlized and transfected into293cells. The level of rictor mRNA was detected by realtime PCR and rictor protein was detected by Western blot.ResultsRecombinant adenovims named Ad-rictor was obtained. The CPE of293cells was observed after being transfected. A gene integration of rictor in continuously multiplied Ad-rictor confirmed by PCR and the green fluorescence was observed in the293cells under fluorescent microscope. We got high titer virus and the infection efficiency of Eahy926cell can be80%～90%. The expression level of rictor mRNA and protein increased.ConclusionsThe Ad-Easy system can be used to construct recombinant adenovirus with high titer and infection efficieacy rapidly and simply, meanwhile, it is also an ideal vector with great security for good use of gene therapy, which would provide experimental basis for the research in endothelial cell senescence by rictor. Objective:we aimed to explore the relationship of TLR2and angiotensin Ⅱ (Ang Ⅱ)-induced cardiac fibrosis in hypertensive mice.Methods:Eighteen Male wild-type C57mice were randomly divided into three groups:blank control group, AngⅡ group, and TLR2blocking group(6in each group). Mice were infused for7days either vehicle(saline)or a pressor dose of angiotensin Ⅱ (Ang Ⅱ,1500ng·kg-1·min-1) with an osmotic mini-pump implanted subcutaneously to established a hypertension model. Blood pressure was measured by tail-cuff plethysmography method to evaluate the effect of infused AngⅡ. At day7, mice were anesthetized and hearts were excised and immediately processed for morphological analysis After injection of TLR2neutralizing antibodies. Masson staining and immunohistochemical were used to analyze cardiac fibrosis.Results:Compared with blank control group, the expression of Collagen deposition, Collagen Ⅰ and TGF-β was higher than in AngⅡ group. The difference was statistically significant (P<0.05). Expressions of Collagen deposition, Collagen Ⅰ and TGF-β were decreased by71.2%,75.5%and77.7%in TLR2blocking group than those in Ang Ⅱ group, respectively (P<0.05).Conclusion:TLR4Participates in Ang Ⅱ-induced cardiac fibrosis in hypertensive mice.