We investigated the effect of transforming growthfactor beta (TGFbeta1)
short hairpin RNA (
shRNA) mediated by pcDU6 plasmid on TGFbeta1 expression in human peritoneal mesothelial cells (HPMCs) and compared that effect with the effect of antisense TGFbeta1
RNA. We designed two pairs of
oligonucleotides for two selectedfragments of coding sequence containing a 21-nucleotide (nt) TGFbeta1 sequence starting with GGCC. After annealing,
double-stranded DNA was formed and separately ligated to plasmid pcDU6 [pcDNA3.1(-) with U6 promoter). The inverted motif contained six spacers and four Ts, which made it possible to form
shRNA (TGFbgeta1 shRNA1 and TGFbeta1 shRNA2). We generated recombinant human TGFbeta1 antisense mammalian expression vector, and we isolated HPMCs from human greater omentum by
pancreatin disaggregation to establish a stable cell-culture model. We used
Lipofectamine 2000 to transfect third-passage HPMCs with plasmid pcDU6 mediating the expression of TGFbeta1 and plasmid pcDNA3.1(-) mediating the expression of antisense TGFbeta1
messenger RNA (
mRNA). The resulting transfected cells were then stimulated with 4.25%
D-glucose and 10 microg/mL
lipopolysaccharide (GS+LPS). We used semi-quantitative
reverse-transcriptase polymerase chain reaction to detect the expression of TGFbeta1,
fibronectin (FN),
collagen 1, and
plasminogen activator inhibitor type 1 (PAI-1)
mRNA by the stimulated cells. The TGFbeta1, FN, and PAI-1
protein levels in the culture supernatant were measured with a sandwich
enzyme-linked
immunosorbent assay. Expression of TGFbeta1 was significantly upregulated in HPMCs stimulated with GS+LPS (p < 0.01). As compared with control HPMCs in serum-free F12 medium, HPMCs transfected with TGFbeta1
antisense RNA showed inhibited expression of FN,
collagen 1, and PAI-1
mRNA (17%, 26%, and 9.6% respectively after 24 hours). Forty-eight hours after transfection, the FN and PAI-I
proteins were inhibited by 54.55% and 61.13% respectively (p < 0.05). In the pcDU6 plasmid vector-mediated TGFbeta1
shRNA groups, TGFbeta1 expression was obviously downregulated as compared with the GS+LPS group and the pcDU6 void vector group (p < 0.01). No significant difference was observed between the pcDU6 plasmid vector-mediated TGFbeta1
shRNA groups (p > 0.05). No significant difference was observed between the pcDNA3.1(-) vector-mediated
antisense RNA group and the pcDU6 void vector group (p > 0.05). The expression of TGFbeta1 in pcDU6 plasmid vector-mediated TGFbeta1
shRNA groups was obviously downregulated as compared with the pcDNA3.1(-) plasmid vector-mediated
antisense RNA group (p < 0.01). In HPMCs stimulated with GS+LPS, pcDU6 plasmid vector-mediated
shRNA can significantly inhibit the induced expression of TGFbeta1. These results suggest the possible application of pcDU6 plasmid vector-mediated
shRNA in preventing
peritoneal fibrosis in patients receiving
peritoneal dialysis.