The Research On Inhibition The Ppif Gene Protects Kidney From Ischemia-reperfusion Induced Injury

Objective:(1) To establish an injury model of ishchemia-reperfusion in normal rat kidney cells (NRK cells) and investigate optimum condition of modeling in vitro. To verify the expresssion of Ppif gene in normal rat kidney cells (NRK cells); (2) To screen the siRNAs that could inhibit the expression of Ppif in normal rat kindey(NRK)cell; (3) To construct the lentiviral vector transferred plasmid pGCL-Ppif mediating rat Ppif gene silencing and set up the basis for further packaging the lentiviral vector; (4) To study the protection and its mechanisms of Ppif gene in normal rat kidney cells (NRK cells)renal ischemia-reperfusion injury. Methods:(1) Cultured normal rat kidney cells (NRK cells) were subjected to the oxygen-glucose deprivation (Krebs solution and sodium dihionite) and recovery of oxygen-glucose, which simulated in vitro ischemia and reperfusion injury(IRI), normal rat kidney cells (NRK cells) apoptosis was assayed by flow cytometry after Annexinâ…¤/PI double staining. The expression of Ppif(coding gene of CypD) in mRNA were detected by RT-PCR method; (2) Three Ppif siRNAs and one negative control siRNA were designed and synthesized and labeled with FAM carboxy fluorescein at the 5'end for the measurement of transfection rate. siRNAs were transfected into normal rat kidney cells (NRK cells) with LipofectamineTM 2000. RT-PCR and Westernblotting were performed to detecte the expression of Ppif affected by the siRNAs at the level of gene and protein, respectively; (3) Taking rats Ppif as a target gene and according to the principle of designing RNA interference sequence, four pairs of two DNA sequencescontaining small hairpin structure were designed, and then were formed into double-stranded DNA by annealing.The obtained products were cloned into the lentiviral vector transferred plasmid pGCL-GFP after double digestion. The recombinant lentiviral vector transferred plasmids pGCL-Ppif containing four target gene fragments were constructed normal rat kidney cells (NRK cells) Finally the plasmids that were identified by PCR were used for sequence analysis; A renal ischemia-reperfusion model was made in 60 rats, which were evenly divided into three groups, i. e. non-control group, control group, and Ppif gene inhibitor group (treated group). Ppif gene inhibitor was given to treated group; normal saline,0.3 mL, to I-R group while reperfusion being done; (4) for non-control group, no renal pedicles were clamped. The renal cell apoptosis index (AI), creatinine (Cr) and blood urea nitrogen (BUN), Histopathological analysis of HE staining. Results:(1) After the oxygen-glucose deprivation(40 min) and recovery of oxygen-glucose, normal rat kidney cells (NRK cells) apotosis reached a peak at 16 h(P<0.05). Ppif mRNA was expressed at a low level at 0 h, began to increase at 4 h and reached a peak at 16 h, after 16h, it began to decrease, it significantly decreased at 48 h, but it was higher than that at 0 h(P< 0.05); (2) The transfect rates of normal rat kidney cells (NRK cells) three siRNAs and the negative control siRNA were all more than 90%. The siRNAs located in had no significant inhibition to Ppif the 405 and 556 gene positions (P> 0.05). However, the siRNA located in significantly decreased the expression of Ppif the 198 gene position at gene (decreased by 75.25%, P< 0.05) and protein levels (decreased by 72.13%, P< 0.05); (3) The Ppif shRNA fragments were successfully cloned into the lentiviral vector pGCL-GFP transferred plasmid. And the shRNA coding consistent with the designed fragments sequences of the four obtained recombinant plasmids were exactly; (4) A comparison between treated group and control group showed that for treated group, the levels of Cr and BUN were both decreased, the cell apoptosis index decreased, (P <0.05). The differences of the above items were of statistically significant.the results of Histopathological analysis of HE staining is significantly. Conclusions:The model well simulated in vitro ischemia-reperfusion, and the oxygen-glucose deprivation (40 min) and recovery of oxygen-glucose (16 h) was the optimum condition of modeling, simultaneously, it verified Ppif gene expressions in normal rat kidney cells (NRK cells);The siRNA located normal rat kidney cells (NRK cells)in the 198 gene position could effectively inhibit the expression of Ppif in NRK cells;The normal rat kidney cells (NRK cells) four recombinant lentiviral vector transferred plasmids of Ppif shRNA were successfully constructed. It laid the the lentiviral vector mediated Ppif gene silencing foundation for further packaging; Ppif gene-targeted RNAi lentiviral vector Inhibit expression Ppif gene, Inhibiting the mitochondrial apoptosis and reducing normal rat kidney cells (NRK cells) renal ischemia-reperfusion injury.