Influence of pcDU6 vector mediated TGF-β1 shRNA on human peritoneal fibrosis

Objective To Study the inhibition of the TGF β1 expression in human peritoneal mesothelial cells(HPMC) by a vector plasmid containing the shRNA of TGF β1 and compare with the effect of antisense TGF β1 RNA on the expression of TGF β1 in HPMCs. Methods Two selected fragments of coding sequence containing 21 nts started with ggcc. Two pairs of oligos were designed for these two fragments. After annealing double stranded DNA formed,they were ligated to plasmid pcDU6[pcDNA3 1(-) with U6 promoter]separately. The inverted motif contained 6 spacer and four Ts, which made it possible to form short hairpin RNA(shRNA). The recombinant human TGF β1 antisense eukaryotic expression vector was generated. HPMCs were isolated from human omental specimens by trypsin disaggregation to establish a stable culture model. Plasmid pcDU6 mediating the expression of TGF β1 and plasmid pcDNA3 1(-) mediating the expression of antisense TGF β1 RNA were transfected into the third passage HPMC stimulated by 4 25%D glucose and lipopolysaccharide(LPS,10 μg/ml) by lipofectamine 2000. The semi quantification reverse transcriptive PCR(RT PCR) was performed to detect the expression of TGF-β1mRNA and FN,collagenⅠ(ColⅠ)and PAI-1mRNA.The protein level of TGF-β1level and FN and PAI-1in the culture supernatant was measured with a sandwich enzyme-lin ked immunosorbent assay(ELISA).Results The expression of TGF-β1was upregulated significantly in H PMC stimulated by4.25%D-glucose and LPS(P0.01).Recombinant human TGF-β1antisense eukaryotic expression v ector was constructed.The mRNA of FN,ColⅠ,PAI-1in HPMC were repressed by TGF-β1antisense RNA at 24hours after transfection in comparison with the control group,which dec reased by 17%,26%,9.6%respectively.The protein of FN and P AI-1was inhibited at 48hours after t ransfection(P0.05).Both RT-PCR and ELISA showed that pcDU6ve ctor plasmid mediated TGF-β1shRNAs inhibited the expression of TGF-β1in HPMC significantly as compared t o pcDU6vector only(P0.01).PcDNA3.1(-)plasmid-mediated antisense TGF-β1RNA did not remarkably inhibit the e xpression of TGF-β1in HPMC compared with pcDU6group(P0.05).PcDU6vector plasmid mediated TGF-β1shRNAs significantly down-regulated the expression of TG F-β1in HPMC compared with the PcDNA3.1(-)plasmid mediating antisense TGF-β1RNA(P0.01).There was no difference on the expre ssion of TGF-β1between two pcDU6vector plasmid mediated TGF-β1shRNA groups(P0.05).Conclusions PcDU6vector plasmid-mediated shRNA inhibits th e expressin of TGF-β1in HPMC stimulated by 4.25%D-glucose and LPS.These results suggested the possible efficacy of pcDU6vector plasm id mediated shRNA for preventing peritoneal fibrosis in patients rec eiving peritoneal dialysis.[