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Construction of Recombinant shRNA-espressing Vectors Targeting to Two Different Hepatic Fibrosis-related Cytokines and Expression in Hepatic Stellate Cells

Author: ZhouDeJiang
Tutor: ZhangJianJun
School: Luzhou Medical College
Course: Internal Medicine
Keywords: RNA interference Liver fibrosis Plasmid vector Connective tissue growth factor Tissue inhibitor of metalloproteinase factor-1 Hepatic stellate cells
CLC: R575.2
Type: Master's thesis
Year: 2010
Downloads: 58
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Abstract


Objective: CTGF (connective tissue growth factor, CTGF) is a recently discovered an important cytokine promoting liver fibrosis, transforming growth factor-β (transforming growth factorβ TGF-β) signaling pathway downstream effects of media . CTGF can directly mediated activation of primary hepatic stellate cells (hepatic stellate cell, HSC) proliferation and migration, but also promote the activation of HSC synthesis and secretion of extracellular matrix (extracellular matrix, ECM). Tissue inhibitor of metalloproteinase factor 1 (tissue inhibitor of metalloproteinase 1, TIMP-1) TGF-β signaling pathway downstream effects of the media, in liver fibrosis occurs, it is mainly expressed by activated HSC. TIMP-1 not only inhibits the activity of matrix metalloproteinases (matrix metalloproteinase, MMPs) to prevent degradation of collagen, and play a key role in the progression of liver fibrosis also promote collagen secretion by inhibiting apoptosis of HSC. Therefore, to reduce the expression of CTGF and TIMP-1, can reduce the degree of liver fibrosis. This study aimed to construct the the rat CTGF or TIMP-1 gene targeting short hairpin RNA (short hairpin RNA, shRNA) expression vector, to provide a powerful tool to further explore the gene therapy of liver fibrosis. Methods: RNA interference target CTGF and TIMP-1 gene is the most effective pre-screened, that CTGF gene 1560 ~ 1580nt and TIMP-1 gene 412 ~~ 432nt RNA interference (RNA interference, RNAi), sequence design principles each design containing short hairpin RNAi target sequences, annealing to form a double-stranded DNA, were cloned into the constructed recombinant plasmid vector containing the purpose of the target gene fragment by double digestion after the plasmid vector psiRNA-DUO-GFPzeo psiRNA -GFP-CTGF, psiRNA-GFP-TIMP-1 and psiRNA-GFP-Com (containing CTGF and TIMP-1), and the recombinant plasmid double digested agarose gel electrophoresis and sequencing. Recovery in cultured rat hepatic stellate cells in vitro, to take the logarithmic growth phase cells with 0.25% trypsin digestion, 3 × 105/ml were seeded in six-well plates until the cells grow to about 80% fusion, serum-free Opti-MEM medium synchronization mediated liposomes (TransFast Transfmlection Reagent) 2000, will build a successful recombinant plasmid (of psiRNA-GFP-of CTGF, psiRNA-GFP-TIMP-1 and psiRNA-GFP-Com (containing CTGF and TIMP -1) and the empty vector psiRNA-DUO-GFPzeo instantaneous transfected rat hepatic stellate cells, fluorescence microscope plasmid transfection, the transfection efficiency measured by flow cytometry. Results: 1 confirmed to rat CTGF or TIMP-1 gene to target the expression of the shRNA target gene fragment has been successfully cloned into the plasmid vector psiRNA-DUO-GFPzeo sequencing results prove that the recombinant plasmid vector of a target gene fragment of the insertion sequence and design exactly 2: fluorescence microscopy hepatic stellate cells transiently transfected plasmid transfected into cells all green fluorescent preliminary proved to be transfected into hepatic stellate cells. Conclusions: The successful construction targeting rat CTGF and TIMP-1 most RNA interference target shRNA expression plasmid recombinants. transient transfected rat hepatic stellate cells transfected into cells with fluorescence, fluorescence microscopy can find successful transfection of hepatic stellate cells such as new way to lay the experimental basis for further exploration of hepatic fibrosis gene therapy.

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CLC: > Medicine, health > Internal Medicine > Digestive and abdominal diseases > Liver and gall bladder disease > Cirrhosis
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