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Knocking Down of Osteopontin in Vivo Inhibits Tubular Epithelial to Mesenchymal Transition in Rats with Chronic Allograft Nephropathy

Author: ZhangTing
Tutor: GuMin
School: Nanjing Medical University
Course: Surgery
Keywords: Osteopontin Epithelial to mesenchymal transdifferentiation RNA interference Electroporation Interstitial fibrosis / tubular atrophy Chronic Allograft Nephropathy
CLC: R692
Type: Master's thesis
Year: 2011
Downloads: 18
Quote: 0
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Abstract


Objective: To explore osteopontin (Osteopontin, OPN) shRNA interference plasmid in rat chronic allograft nephropathy (CAN) model rats transplant kidney epithelial cells to mesenchymal quality transdifferentiation (EMT). Explore the role and mechanism of OPN in chronic allograft nephropathy. Methods: Rat CAN model: F344 and Lewis inbred rats were used as for the receptor, whichever donor left kidney as a donor kidney removal of receptors left kidney first double binocular microscope, rows donor renal vein and receptors renal vein anastomosis donor renal artery with receptor renal artery line end to end anastomosis for renal the bladder flap receptor bladder anastomosis line orthotopic kidney transplantation. Postoperative 10d, receptor right kidney removed. All rats were treated with cyclosporine A (CsA) 10 days to prevent acute rejection, 1.5mg/kg? D, to subcutaneous injection. Rat CAN model. Groupings: F344 rats donor Lewis rats as recipients, line orthotopic kidney transplantation, establish a rat the CAN model of (Allo group), donor and recipient Lewis rats as isograft control ( Syn group). Kidney to fluid mechanics supplemented by electroporation-based gene transfection transfected plasmid to the transplanted kidney (RNAi group). The negative control plasmid as a control (NC) group. 40 rats in each group of ten. 12 weeks after the animals in each group were sacrificed. Taken transplanted kidney specimens HE and Masson staining observed histological changes. And the Banff 97 standard score and statistical analysis. Kidney allograft α-SMA, E-Cadherin expression were observed using western blotting and immunohistochemistry methods. At the same time using the above method to detect kidney allograft OPN expression, and its gray scale for statistical analysis. Collected and to detect rat serum creatinine, blood urea nitrogen, 24-hour urinary protein. Results: CAN model in rats: through surgical training technology is mature and conducted a total of 100 cases of surgery, surgical success rate of 85%, the surgery failed mainly due to anastomotic bleeding and thrombosis. The donor and recipient operation time was 70 ± 20,65 ± 15min, warm ischemia time the 1min around, cold ischemia time was 45 ± 16min. After renal transplantation 12 weeks, found that the transplanted kidney OPN expression was detected by immunohistochemistry and Western blot method, significantly lower than Allo the RNAi group OPN expression. The the Allo / NC rats transplanted kidney tissue morphology complies with chronic allograft nephropathy change. RNAi group transplanted kidney morphological changes than Allo alleviate. Banff 97 score, the the Allo group of tubular atrophy score was significantly higher than the Syn group (1.6 ± 0.81 vs 0.1 ± 0.20), the the Allo group of tubulointerstitial degree of fibrosis was more severe in the Syn group (2.1 ± 0.78 vs 0.2 ± 0.46) , the differences were significant (P <0.01); the RNAi group transplanted kidney tissue visible minor CAN performance, meantime interstitial fibrosis and tubular atrophy score were 0.7 ± 0.54,1.1 ± 0.71, significantly low Allo group (P <0.05); Allo NC groups Banff score difference was not statistically significant (P> 0.05). Immunohistochemistry and western blot results showed that, compared with Syn, Allo group, mainly in tubular epithelial renal interstitial quality strong expression of α-SMA, while the Allo group tubule epithelial E-Cadherin expression compared with the control group decreased. In contrast, α-SMA RNAi group downregulation, upregulation of E-cadherin. Kidney transplant after 12 weeks, the the Allo group of serum creatinine (Scr) Syn group (116.9 ± 3.1 vs 64.6 ± 5.7) was significantly higher than that, the difference was statistically significant (p lt; 0.05). The RNAi group Scr compared Allo group decreased significantly (79.4 ± 3.2 vs 116.9 ± 3.1), and the difference was statistically significant (p lt; 0.05). Conclusion: OPN upregulation in the rat the CAN model of transplantation kidney OPN in vivo RNA interference to reduce the expression of OPN in renal transplantation. OPN RNA interference can alleviate the deterioration of renal function, and ease the transplanted kidney, pathological changes, and can inhibit transplantation renal tubular epithelial to mesenchymal transition differentiation (EMT). The results suggest the potential occurrence of OPN and CAN. Body of its RNA interference can be a therapeutic effect in rats with chronic allograft nephropathy. OPN to affect CAN onset may by promoting tubular epithelial EMT pathway occurs.

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CLC: > Medicine, health > Surgery > Urology ( urinary and reproductive system diseases) > Kidney disease
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