Aim: To construct the adeno-associated virus(AAV) vectors of Smad 6 and Smad 7 genes and observe their expressions in human renal tubule epithelial cells.
Methods: The plasmids pcDNA3-Smad 6/flag and pcDNA3-Smad 7/flag were digested with BamH I and Xho I. Then the Smad 6/flag and Smad 7/flag gene fragments were cloned into plasmid pAAV-MCS, respectively to construct the recombinant pAAV-Smad 6/flag and pAAV-Smad 7/flag plasmids. The recombinant expression plasmid or pAAV-LacZ plasmid were co-transfected into the HEK 293 cells with pHelper and pAAV-RC by calcium-phosphate precipitation method. Recombinant AAV-2 viral particles were prepared from infected HEK293 cells and then were used to infect human renal tubule epithelial cells (HKCs), The expressions of Smad 6 and Smad 7 in HKCs were demonstrated by immunocytochemical staining.
Results: The recombinant AAV vectors of Smad 6 or Smad 7 genes were constructed and expressed in the HKCs successfully.
Conclusion: These results indicate that AAV can deliver Smad 6 and Smad 7 genes to renal cells in-vitro, suggesting the recombinant AAV can be used for gene therapy of renal fibrosis.
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