Inhibition of TGF-β_1 expression and extracellular matrix secretion in human peritoneal mesothelial cells by pcDU_6 vector mediated TGF-β_1 shRNA and by pcDNA3.1(-) mediated antisense TGF-β_1RNA

Objective To investigate the effect of shRNA of TGF-β_1 carried by a vector plas mid and antisense TGF-β_1 RNA no the TGF-β_1 expression in human peritioneal mesothelial cells (HPMC) and extracellular matrix secretion. Methods Two pairs of oligos were designed for the 2 selected fragments. After annealing double stranded DNA formed,they were ligated to plasmid pcDU_6 [pcDNA3.1(-) with U6 promoter] separately to form short hairpin RNA. The recombinant human TGF-β_1 antisense eukaryotic expression vector was generated. Human peritoneal mesothelial cells were isolated from human omental specimens by trypsin digestion toestablish a stable culture model. Plasmid pcDU_6 mediating the expression of TGF-β_1 and plasmid pcDNA 3.1(-) mediating the expression of antisense TGF-β_1RNA were transfected into the third passage HPMC stimulated by 4.25% D-glucose and lipopolysaccharide(LPS,10(g/ml) by lipofectamine 2000. The semi-quantification reverse transcriptive PCR (RT-PCR) was performed to detect the expression of TGF-β_1mRNA and FN,collagenⅠ(colⅠ)and PAI-1 mRNA. The TGF-β_1 level in the culture supernatant was measured with a sandwich enzyme-linked immunosorbent assay. Results The expression of TGF-β_1 was upregulated significantly in HPMC stimulated by 4.25% D-glucoseand LPS(P0.01). The mRNA of FN, colⅠ,PAI-1 in HPMC were repressed by TGF-β_1 antisense RNA at 24 hours after transfection in comparison with the control group, they decreased 17%, 26%, 9.6% respectively.PcDNA3.1(-) plasmid mediated antisense TGF-β_1RNA did not remarkably inhibit the expression of TGF-β_1 in HPMC compared with pcDU_6 group(P0.05). PcDU6 vector plasmid mediated TGF-β_1 shRNAs significantly down-regulated the expression of TGF-β_1 in HPMC compared with the PcDNA3.1(-) plasmid mediated antisense TGF-β_1RNA(P0.01). Conclusion PcDU6 vector plasmid mediated shRNA inhibit the expressin of TGF-β_1 in HPMC stimulated by 4.25%D-glucose and LPS. These results suggested the possible efficacy of pcDU_6 vector plasmid mediated shRNA for preventing peritoneal fibrosis in patients receiving peritoneal dialysis.