Transfection of shRNA expressing plasmid by calcium phosphate nanoparticles inhibits the high glucose-induced TGF-β1 expression in human peritoneal mesothelial cells

LING Guang-hui; LIU Fu-you; PENG You-ming; DUAN Shao-bin; LIU Hong; LI Yin; LI Jun; LIU Ting

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Chinese Journal of Blood Purification ›› 2008, Vol. 7 ›› Issue (3) : 149-152.

Transfection of shRNA expressing plasmid by calcium phosphate nanoparticles inhibits the high glucose-induced TGF-β1 expression in human peritoneal mesothelial cells

LING Guang-hui1, LIU Fu-you1, PENG You-ming1, DUAN Shao-bin1, LIU Hong, LI Yin1, LI Jun1, LIU Ting

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Abstract

【Abstract】Objective Transforming growth factor-β1 (TGF-β1) is one of the key mediators in peritoneal fibrosis. Short hairpin RNAs (shRNA) transcribed under the control of U6 or H1 promoter in plasmid can trigger silence of the target gene in mammalian cells. We have developed TGF-β1 shRNA expression plasmids in a previous study. A novel nonviral vector calcium phosphate nanoparticle (CPNP) was also developed by means of a chemistry method. Transmission electron microscopy and Zeta potential demonstrated that CPNP was of 23.5～34.5 nm in diameter and had positive surface charges of +16.8 mV. In this study, we investigated the transfection of shRNA expressing plasmid mediated by CPNP on high glucose-induced TGF-β1 expression in human peritoneal mesothelial cells (HPMCs). Methods The TGF-β1 shRNA expression plasmid (pcDU6-A1-B1) was transfected into HPMCs mediated by CPNP. Transfected cells were then stimulated with 50mmol/L D-glucose for 48 hours, and the expression of TGF-β1 and fibronectin were evaluated Results TGF-β1 expression was upregulated significantly in control HPMCs stimulated with 50mmol/L D-glucose for 48 hours. Such induction was inhibited by the transfection of shRNA expression plasmid (pcDU6-A1-B1) into the cells mediated by CPNP. Fibronectin expression was also upregulated significantly by the stimulation of 50mmol/L D-glucose for 48 hours. The 50mmol/L D-glucose-induced fibronectin expression became insignificant in HPMCs transfected with the shRNA expression plasmid, as measured by enzyme-linked immunosorbent assay (ELISA). Conclusion The introduction of shRNA expression plasmid into HPMCs effectively inhibited high glucose-induced TGF-β1 expression in these cells. Calcium phosphate nanoparticles were useful for the introduction of this plasmid into HPMCs. shRNA expression plasmid introduced by calcium phosphate nanoparticles may be a promising approach for the gene therapy of peritoneal fibrosis.

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Transforming growth factor-β1 / RNA interference / Nanoparticles

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LING Guang-hui;LIU Fu-you;PENG You-ming;DUAN Shao-bin;LIU Hong;LI Yin;LI Jun;LIU Ting. Transfection of shRNA expressing plasmid by calcium phosphate nanoparticles inhibits the high glucose-induced TGF-β1 expression in human peritoneal mesothelial cells[J]. Chinese Journal of Blood Purification. 2008, 7(3): 149-152