.pi3-k/akt Shrna Rat Thy-1 Nephritis Hyperplasia

Partâ… Construction and identification of eukaryotic vectors expressing shRNA targetting PI3-K/Akt geneObjective:To construct PI3-k/Akt shRNA expression vectors for observing the effect of silencing PI3-k/Akt signal pathway on preventing renal cell proliferation of rats with Thy-1 nephritis(Thy-1 N). Methods:Eight 19～21bp reverse repeated motifs targeting of PI3-k/Akt gene were synthesized and cloned into eukaryotic expression plasmid pGenesil-1.After being screened and sequencing confirmed,the recombinant plasmids were transfected into rat glomerular mesangial cells (GMCs),then the levels of P-Akt and T-Akt protein in rat GMCs were measured using Western blotting to select the optimal shRNA.Results:It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the constructed eukaryotic vector expressing shRNA of PI3-K/Akt were correct.Western blotting results showed that the optimal shRNA plasmids which could effectively silence the target genes were shPik3c3-2 and shAkt1-4 respectively.Conclusion:The eukaryotic vectors expressing shRNA of PI3-k/Akt were constructed successfully.The results of the study lay the foundation for further studying on biological functions of PI3-k/Akt signal pathway in Thy-1 N. Partâ…¡Suppression of mesangial cell proliferation and extracelluar matrix production in Thy-1 nephritis rats by knockdown of PI3-k/Akt with shRNAObjective:To explore the effect of silencing PI3-k/Akt signal pathway on suppressing of mesangial cell proliferation and extracelluar matrix production in Thy-1 nephritis rats.Methods:shPik3c3-2 or/and shAkt1-4 plasmids were transferred into the kideny of Thy-1 nephritis rats by usinghydrodynamic injection method.The expressions of Tsp-1,TGF-β₁, cyclin D₂ and FN mRNA were assessed by Real-time PCR and the protein levels of P-Akt,T-Akt,Tsp-1,TGF-β₁ cyclind₂ and FN weredetected by Western blotting or Immunohisto- chemistry.In addition,histopathology and Urine protein were both observed.Results:In the renal cortex of Thy-1 nephritis rats transferred with shPik3c3-2,shAkt1-4, shPik3c3-2+shAkt1-4,the expressions of P-Akt and T-Akt were successfully reduced.And the expressions of Tsp-1,TGF-β₁,cyclin D₂ and FN were also inhibited.Meanwhile,the glomerular cell proliferation and extracellular matrix accumulation were markedly suppressed in concomitant with downregulation excretion of Urinary protein.Conclusion: The amelioration of the pathological findings of Thy-1 nephritis by silencing PI3-k/Akt signal pathway suggests that PI3-k/Akt may serve as a potential therapeutic target for mesangial proliferation nephritis in the future.