Mechanism Study On The Inhibition Of Rat Renal Allograft Fibrosis Via Smads Pathway By TGF-β1 ShRNA

PART ONEObjective: To construct rat Transplant Kidney-sclerosis accelerated model and transfected with the plasmid shRNA- TGF-β1, and to observe the inflammatory cells infiltration and fibrosis extent of shRNA-TGF-β1 plasmid on renal allograft.Methods: Adopot the enhanced ischemia reperfusion injury method, and the SD to Wistar rat Transplant Kidney-sclerosis accelerated model was constructed. The receptors were divided into four groups: Group T (plasmid group) injected with shRNA-TGF-β1; Group H (vacant plasmid group) injected with vacant plasmid; Group Y (transplantation group) injected with no plasmid; Group J (sham-operated group) only removed the right kidney with no transplantation. Transfected with the plasmid based on the hydromechanics. The pathological changes and infiltrated inflammatory cells were assessed by HE staining. Results: Rat Transplant Kidney-sclerosis accelerated model was constructed successfully, and they presented fibrosis in a short time. There were large amount of inflammatory cells infiltration and severe deposition of ECM displayed HE staining. There were also less infiltrated chronic inflammatory cells and extracellular matrix deposition in the plasmid group.Conclusion: Rat Transplant Kidney-sclerosis accelerated model could be constructed via enhanced ischemia reperfusion injury. Renal gene transfection technology based on hydromechanics is feasible. The plasmid could inhibit the fibrosis of rat renal allograft.PART TWOObjective: To investigate the inhibitory effcts of shRNA-TGF-β1 plasmid on the expression of TGF-β1 and ECM in rat renal allograft.Methods: Transplanted renals were collected at the first, second and third months after transplantation. The gene transcriptional level of TGF-β1 by RT-PCR and the protein variation was examined by western blot, and the ECM deposition was assessed by Masson staining.Results: In group T, the expression of TGF-β1 was inhibited by the plasmid, and it was significantly lower than group H/Y (P﹤0.01 or P<0.05). The ECM depositon of Group T was much less than Group H/Y.Conclusion: The shRNA- TGF-β1 plasmid could inhibit the expression of TGF-β1 and reduce the synthesis of extracellular matrix, which could prevent fibrosis of renal allograft at some extent.PART THREEObjective: To explore the possible mechanism on the inhibition of rat renal allograft fibrosis by shRNA-TGF-β1 plasmid.Methods: RT-PCR and Western blot were used to detect the expression of phosphorylated Smad3/7 (p-Smad3/7), type I collagen and E-cadherin. Immunohistochemical stainings of E-cadherin andα-SMA were used to label tubular epithelial cells and fibroblast respectively in order to assess the cells'change.Results: In the plasmid group, the signal proteins of p-Smad3 was down-regulated and p-Smad7 was up-regulated(P﹤0.01 or P<0.05); Type I collagen was also inhibited and its expression was lower than Group H/Y (P﹤0.01 or P<0.05); the expression of E-cadherin was higher than Group H/Y(P<0.01 or P<0.05). Epithelial cells were much more and fibroblast was much less than that of control groups, and there was less occurrence of EMT.Conclusion: Our results suggest that shRNA- TGF-β1 plasmid could prevent the fibrosis of renal allograft. The mechanism may be associated with its effects of down-regulating p-Smad3, and up-regulating p-Smad7, maintaining the expression of E-cadherin, leading to the suppression of epithelial-myofibroblast transdiferentiation and extracellular matrix synthesis.