Establishment of an immortalized human hepatic stellate cell line to develop antifibrotic therapies.
Because human hepatic stellate cells (HSCs) perform a crucial role in the progress of hepatic fibrosis, it is of great value to establish an immortalized human cell line that exhibits HSC characteristics and grows well in tissue cultures for the development of antifibrotic therapies. Thus, we engineered an immortalized human hepatic stellate cell (HSC) line TWNT-4 by retrovirally inducing human telomerase reverse transcriptase (hTERT) into LI 90 cells established from a human liver mesenchymal tumor. Parental LI 90 entered replicative senescence, whereas TWNT-4 showed telomerase activity and proliferated for more than population doubling level (PDL) 200 without any crisis. TWNT-4 expressed platelet-derived growth factor-beta receptor (PDGF-betaR), alpha-smooth muscle actin (alpha-SMA), and type I collagen (alpha1) and was considered to be an activated form of HSCs. Treatment of TWNT-4 cells with either 100 U/ml of IFN-gamma or 1 ng/ml of rapamycin (Rapa) for 14 days led to lower expression of type I collagen (alpha1) at RNA and protein levels. Exposure of TWNT-4 cells to both of IFN-gamma (10 U/ml) and Rapa (0.1 ng/ml) for 14 days effectively decreased the expression of type I collagen (alpha1), PDGF-betaR, and alpha-SMA expression and suppressed TGF-beta1 secretion of TWNT-4 cells. We successfully induced apoptosis by transducing TNF-related apoptosis-inducing ligand (TRAIL) into TWNT-4 cells using adenovirus vectors Ad/GT-TRAIL and Ad/PGK-GV-17. These findings suggested that immortalized activated HSC line TWNT-4 would be a useful means to develop antifibrotic therapies.
Shibata
Norikuni
N
Division of Gastroenterology, Department of Internal Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0114, Japan.
Watanabe
Takamasa
T
Okitsu
Teru
T
Sakaguchi
Masakiyo
M
Takesue
Michihiko
M
Kunieda
Takemi
T
Omoto
Kenji
K
Yamamoto
Shinichiro
S
Tanaka
Noriaki
N
Kobayashi
Naoya
N
D000199:Actins / Q000378:metabolism; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D002455:Cell Division; D002460:Cell Line; D002478:Cells, Cultured; D016922:Cellular Senescence; D024042:Collagen Type I / Q000378:metabolism; D004268:DNA-Binding Proteins; D005355:Fibrosis / Q000188:drug therapy*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D008024:Ligands; D008099:Liver / Q000166:cytology*; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053221:TNF-Related Apoptosis-Inducing Ligand; D019098:Telomerase / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000199:Actins; D051017:Apoptosis Regulatory Proteins; D024042:Collagen Type I; D004268:DNA-Binding Proteins; D008024:Ligands; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D012313:RNA; D007371:Interferon-gamma; D020797:Receptor, Platelet-Derived Growth Factor beta; D019098:Telomerase