pcDU6载体介导的TGF-β1短发夹RNA对人腹膜间皮细胞中TGF-β1表达的抑制作用

Inhibition of TGF-beta1 expression in human peritoneal mesothelial cells by pcDU6 vector-mediated TGF-beta1 shRNA.

作者信息

Liu Fuyou, Liu Hong, Peng Youming, Liu Yinghong, Li Jun, Chen Xing

机构信息

Nephrology Department, 2nd Xiangya Hospital, Central South University, Changsha City, Hunan Province, China.

出版信息

Nephrology (Carlton). 2006 Feb;11(1):23-8. doi: 10.1111/j.1440-1797.2006.00530.x.

DOI:10.1111/j.1440-1797.2006.00530.x

PMID:16509928

Abstract

BACKGROUND AND AIM

Transforming growth factor-beta(1) (TGF-beta(1)) is an important mediator of the fibrosis process. High expression of TGF-beta(1) is closely related to peritoneal fibrosis. RNA interference using short hairpin RNA (shRNA) can mediate sequence-specific inhibition of gene expression in mammalian cells. The aim of this study was to assess the effect of shRNA targeting TGF-beta(1) on the expression of TGF-beta(1) in human peritoneal mesothelial cells (HPMC).

METHODS

TGF-beta(1) specific shRNA expression vectors were constructed and introduced to HPMC stimulated with 4.25% D-glucose (Gs) and 10 microg/mL of lipopolysaccharide (LPS). Expression of TGF-beta(1) mRNA was assessed by semiquantification reverse transcription polymerase chain reaction (RT-PCR). The TGF-beta(1) protein level in the culture supernatant was determined by sandwich enzyme-linked immunosorbent assay.

RESULTS

The expression of TGF-beta(1) was upregulated significantly in HPMC stimulated with 4.25% D-Gs and 10 microg/mL LPS P < 0.01. TGF-beta(1) expression in pcDU6 plasmid vector-mediated TGF-beta(1) shRNA groups were obviously downregulated when compared to the 4.25% D-Gs and 10 microg/mL LPS group (P < 0.01) and the pcDU6 void vector group (P < 0.05), with no significant difference among pcDU6 plasmid vector -mediated TGF-beta(1) shRNA groups (P > 0.05). No significant difference was found between teh pcDNA3.1(-) vector plasmid-mediated TGF-beta(1) antisense RNA group and pcDU6 void vector group (P > 0.05). The expression of TGF-beta(1) in pcDU6 plasmid vector-mediated TGF-beta(1) shRNA groups were obviously downregulated when compared to the pcDNA3.1(-) plasmid vector-mediated TGF-beta(1) antisense RNA group (P < 0.05).

CONCLUSION

TGF-beta(1)-specific shRNA can significantly inhibit the expression of TGF-beta(1) in HPMC stimulated with 4.25% D-Gs and 10 microg/mL LPS in HPMC. These results suggest the possible application of TGF-beta(1)-specific shRNA in preventing peritoneal fibrosis in patients receiving peritoneal dialysis.

摘要

背景与目的

转化生长因子-β1（TGF-β1）是纤维化过程的重要介质。TGF-β1的高表达与腹膜纤维化密切相关。使用短发夹RNA（shRNA）的RNA干扰可介导哺乳动物细胞中基因表达的序列特异性抑制。本研究旨在评估靶向TGF-β1的shRNA对人腹膜间皮细胞（HPMC）中TGF-β1表达的影响。

方法

构建TGF-β1特异性shRNA表达载体，并将其导入用4.25% D-葡萄糖（Gs）和10μg/mL脂多糖（LPS）刺激的HPMC中。通过半定量逆转录聚合酶链反应（RT-PCR）评估TGF-β1 mRNA的表达。用夹心酶联免疫吸附测定法测定培养上清液中TGF-β1蛋白水平。

结果

在用4.25% D-Gs和10μg/mL LPS刺激的HPMC中，TGF-β1的表达显著上调（P < 0.01）。与4.25% D-Gs和10μg/mL LPS组（P < 0.01）和pcDU6空载体组（P < 0.05）相比，pcDU6质粒载体介导的TGF-β1 shRNA组中TGF-β1的表达明显下调，pcDU6质粒载体介导的TGF-β1 shRNA组之间无显著差异（P > 0.05）。pcDNA3.1（-）载体质粒介导的TGF-β1反义RNA组与pcDU6空载体组之间无显著差异（P > 0.05）。与pcDNA3.1（-）质粒载体介导的TGF-β1反义RNA组相比，pcDU6质粒载体介导的TGF-β1 shRNA组中TGF-β1的表达明显下调（P < 0.05）。