gilz基因沉默增强脓毒症血清诱导成肌细胞合成代谢的实验分析word论文.docxVIP

AbstractObjective To construct the siRNA adenovirus vitors targeted rat GILZ, research the effects of GILZ gene silencing on metabolism of septic serum-induced myoblast.Methods (1)Sepsis model was manufactured by cecal ligation and puncture, then septic serum was collected. (2)The specific siRNA targeted rat GILZ mRNA were designed and cloned, the double standed DNA Oligo were inserted into vector GV119 which hasdouble-digested by AgeⅠ and EcoRⅠ, then shuttle plasmid and backbone plasmid pBHGlox ΔE1, 3 Cre were co-transfected into HEK293 cells after sequencing analysis, the titer of the recombinant adenovirus were detemined after propagate and purify. (3)The fluorescence and morphological changes of L6 myoblast were observed after infected with recombinant adenovirus, the infection efficiency was detemined, and the silencing efficiency of GILZ was established via real-time quantitative polymerase chain reaction. (4)The expression of GILZ, MyHC, MyoD and myogenin were measured via real-time quantitative polymerase chain reaction after induced by septic serum.Results (1)Sepsis model manufactured by cecal ligation and puncture was successful, the general state, weight, rectal temperature, inflammatory markers, bacterial culture and laparotomy results were corresponding to the diagnostic criteria of sepsis. (2)Recombinant adenovirus was successfully constructed, sequencing analysis showed that siRNA was inserted into GV119 correctly, real-time quantitative polymerase chain reaction results showed that the expression of GILZ in Ad-siRNA-1-GILZ group was least, there wassignificant difference compared with the control group(P＜0.05), the silencing efficiencyis about 59.8%, the titer of is 2.0×1010PFU/mL. (3)The infection efficiency of Ad-siRNA-1-GILZ is 80% in Enhanced Infection Solution, and MOI is about 20. (4)The expression of GILZ and MyHC is decreased, while MyoD and myogenin are increased after induced by septic serum, and the difference was significant compared with