| Part1:Constructed the A20gene expression vector, Extraced plasmid and identificated in vitroObjective1, We constructed an expression vector with carrying the gene A20.2, We cultured the E. coli, and extracted the plasmid DNA of high concentration and high purity.3, The plasmid DNA were identificated in vitro.Methods1, The construct was designated as pcDNA3.1-A20. The pcDNA3.1empty plasmid was a control. Then the pcDNA3.1-A20and pcDNA3.1empty plasmid were transformed into competent E.coli, then stored at-20℃.2, The E.coli containing of pcDNA3.1-A20or pcDNA3.1empty plasmid were cultured on solid LB plates. After12h ～16h of incubation at37℃one colony was picked, loop-inoculated into a15ml tubes containing5ml of ampicillin-resistant liquid LB medium and incubated at37℃on a rotary shaker at210revolutions per minute of an overnight culture. After harvesting the bacterial strains, the plasmid DNA was extracted according to the plasmid extraction kit (PureLink? HiPure Plasmid DNA Purification Kits, invitrogen) instruction. After restriction endonuclease Nhel and Xbal double digested it, the digestion products and plasmid DNA were verified by1%Agarose Gel Electrophoresis. After pumping large plasmids DNA were according to the kit instructions, and then determined the concentration, saved at-20℃.3, After the recombinant plasmid DNA in vitro transfenction, the expressin of green luciferase gene in cells were observated by the inverted fluorescence microscope.Result1, The results were entirely correct after digestion, extraction, electrophoresis identification.2, The HK-2cells were transfencted in vitro, and then there were green fluorescent expression in cells.Conclusion HK-2cells could effectively transfected of using A20gene expression vector in vitro. Part2:Effect of EGb and Gene transfer-induced A20over-expression on renal ischemia reperfusion injury in ratsObjective1, Establish a stable and reliable rat model of renal ischemia reperfusion injury;2, To study the protection EGb and gene transfer-induced A20over-expression against renal ischemia reperfusion injury in rats, then provided a theoretical basis for experimental clinical prevention of renal IRI.Methods1, Sprague-Dawley (SD) male rats (250～280g body weight) provided by the laboratory animal center of fu jian university of traditional chinese medicine. The rats received a standard diet, with free access tap water. One week later, the model of renal ischemia-reperfusion injury of rats was established. Rats were anesthetized with sodium pentobarbital (50mg/kg) intraperitoneally. Open the abdominal cavity and separate the left renal artery and vein, then renal ischemia reperfusion injury model was done by clamping left renal artery and vein for45min, and cut the right renal before reperfusion of left renal vascular.2, Thirty-two male SD rats were randomly divided into four groups (n=8):saline group, A20group, A20+EGb group, EGb group. Forty eight hours before operation every group was injected with Saline250μl, liposome pcDNA3.1-A20+saline to250μl (15μl of Lipofectamine?2000+10μg of pcDNA3.1-A20, mix well and add saline to250μl). And thirty minute before operation the EGb group and the A20+EGb group were injected with EGb (50mg/kg) by intraperitoneally. Twenty-four hours after operation harvested the rats, One portion of the renal tissue was fixed in10%buffered formalin for twenty-four hours followed by embedding in paraffin. Then the renal function was detected by measuring serum Scr、BUN levels using automatic biochemical analyzer. Renal pathological examination was analyzed by HE staining, renal tubular cell apoptosis was detected by TUNEL assay. Expression levels of A20protein were checked by Western blot, expression of A20mRNA was analyzed by Quantitative reverse transcriptase PCR and EMSA analysis of NF-kB DNA binding activity.Result1, After practice, we established a relatively stable renal ischemia reperfusion injury model, the rate of success was up to95%.2, We observed that the the increase of serum creatinine (a marker of renal injury) and BUN by1RI was reduced in animals pre-treated with pcDNA3.1-A20compared to animals pre-treated with saline group. And the serum Scr、 BUN levels of A20group and the A20+EGb group were significantly lower than the other groups, the difference was statistically significant (p＜0.05). We observed that renal tissue damage after IRI was significant by the light microscope. The levels of renal tubular injury in each group were different:in saline group, renal tubular dilatation; there are cracks during cells and cells become flat; tubular epithelial cells injury or loss of brush border, tubular epithelial cells shedding or necrosis, protein casts or cell debris can be seen in the tube cavity, of which tubular epithelial cells shedding were the most serious. however, the renal tubular damage of EGb group and A20+EGb group was reduced significantly of which the tubular epithelial cells brush border damage was the fundamental changes. Renal tubular epithelial cell apoptosis could seen in the distal tubule, and compared with the saline group, the number of apoptotic cells in each group were significantly reduced, and the difference was statistically significant (p＜0.05); and compared with the A20group, the number of apoptotic cells in the EGb group and A20+EGb group were significantly reduced (p＜0.05); The protein and mRNA expression levels of A20were elevated in all groups, and compared with the saline group, A20group and A20+EGb group increased more significantly, the difference was statistically significant (p <0.05); NF-kB activity in each group were inhibited, compared with the saline group, NF-kB activity were inhibited more obvious in A20group and A20+EGb group.Conclusion EGb and A20transgenic method could increase the A20expression in vivo, EGb can significantly reduce renal IRI, and the two combined effect is more significant, which may be related to the inhibition of renal tubular epithelial cells apoptosis through inhibition of NF-kB signaling pathway. |
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