[Inhibition of TGFbeta1 expression in human peritoneal mesothelial cells by pcDU6 vector mediated TGFbeta1 shRNA].
OBJECTIVE
To investigate the influence of vector plasmid containing the shRNA of TGFbeta1 on the TGFbeta1 expression in human peritoneal mesotnelial cells. the TGFbeta1 expression in human peritoneal mesothelial cells (HPMCs) by a vector plasmid containing the shRNA of TGFbeta1.


METHODS
The 2 selected fragments of coding sequence contained 21 nts, starting 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 4 Ts, which made it possible to form short hairpin RNA. Human peritoneal mesothelial cells were isolated from human omental specimens by trypsin disaggregation to establish a stable culture model. Plasmid pcDU6 mediating the expression of TGFbeta1 and plasmid pcDNA3.1(-) mediating the expression of antisense TGFbeta1 RNA were transfected into the third passage HPMCs stimulated by 4. 25% D-glucose and lipopolysaccharide (LPS, 10 microg/ml) by lipofectamine 2000. The semi-quantification reverse transcriptive PCR (RT-PCR) was performed to detect the expression of TGFbeta1 mRNA. The TGFbeta1 level in the culture supernatant was measured with a sandwich enzyme-linked immunosorbent assay.


RESULTS
The expression of TGFbeta1 was upregulated significantly in HPMCs stimulated by 4.25% D-glucose and LPS (P < 0.01). Both RT-PCR and ELISA showed that pcDU6 vector plasmid mediated TGFbeta1 shRNAs inhibited the expression of TGFbeta1 in HPMCs significantly (P < 0.01). pcDNA3.1(-) plasmid mediated antisense TGFbeta1 RNA did not remarkably inhibit the expression of TGFbeta1 in HPMCs compared with the pcDU6 group (P > 0.05). pcDU6 vector plasmid mediated TGFbeta1 shRNAs may significantly down-regulated the expression of TGFbeta1 in HPMCs compared with the pcDNA3.1(-) plasmid mediated antisense TGFbeta1 RNA (P < 0.01). There was no difference in the expression of TGFbeta1 between the 2 pcDU6 vector plasmid mediated TGFbeta1 shRNAs (P > 0.05).


CONCLUSIONS
pcDU6 vector plasmid mediated shRNAs may inhibit the expressin of TGFbeta1 in HPMCs stimulated by 4.25% D-glucose and LPS, suggesting the possible efficacy of pcDU6 vector plasmid mediated shRNA in preventing peritoneal fibrosis in patients receiving peritoneal dialysis.
Liu
Hong
H
Nephrology Institute, Second Xiangya Hospital, Central South University, Changsha 410011, China.
Liu
Fu-you
FY
Peng
You-ming
YM
Yuan
Fang
F
Liu
Ying-hong
YH
D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000378:metabolism; D005109:Extracellular Matrix / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D010537:Peritoneum / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D016372:RNA, Antisense / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology; D053773:Transforming Growth Factor beta1 / Q000096:biosynthesis* / Q000235:genetics
D017395:Plasminogen Activator Inhibitor 1; D016372:RNA, Antisense; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D053773:Transforming Growth Factor beta1; D005947:Glucose