Pancreas · 2011
In vitro transdifferentiation of HepG2 cells to pancreatic-like cells by CCl₄, D-galactosamine, and ZnCl₂.
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Kanoh Yoshiya, Tomotsune Daihachiro, Shirasawa Sakiko, Yoshie Susumu, Ichikawa Hinako, Yokoyama Tadayuki, Mae Shin-Ichi, Ito Jun, Mizuguchi Masahiro, Matsumoto Ken, Yue Fengming, Sasaki Katsunori
Аннотация
OBJECTIVE: The objective of the study was to induce transdifferentiation of human hepatoma HepG2 cells into pancreatic-like cells without direct genetic intervention.
METHODS: HepG2 cells were transfected with plasmids for the hepatocyte marker protein green fluorescent protein (albumin-GFP) and the pancreatic cell marker Discosoma spp red fluorescent protein (elastase-DsRed) to create FAE-HepG2 cells. Fluorescent marker expression was used to monitor in vitro transdifferentiation stimulated 100 mM CCl₄, 2 mM D-galactosamine, or 200 μM ZnCl₂. Concentrations were selected for optimal cell survival rate. Transdifferentiation was also characterized by immunohistochemical detection of amylase, glucagon, and insulin and by polymerase change reaction analysis of amylase and insulin mRNA production.
RESULTS: Control cells expressed albumin-GFP but no elastase-DsRed. By 30 days of culture, all 3 agents induced expression of pancreatic-like cell marker elastase-DsRed. ZnCl₂ was the most effective as most cells expressed elastase-DsRed in the absence of simultaneous expression of albumin-GFP. For CCl₄ and D-galactosamine, elastase-DsRed was expressed in the same cells as albumin-GFP. Cells treated by each agent also expressed amylase, insulin, and glucagon proteins and mRNAs.
CONCLUSIONS: Without direct genetic intervention, select low small molecules can induce in vitro transformation of hepatoma cells into pancreatic-like cells.
METHODS: HepG2 cells were transfected with plasmids for the hepatocyte marker protein green fluorescent protein (albumin-GFP) and the pancreatic cell marker Discosoma spp red fluorescent protein (elastase-DsRed) to create FAE-HepG2 cells. Fluorescent marker expression was used to monitor in vitro transdifferentiation stimulated 100 mM CCl₄, 2 mM D-galactosamine, or 200 μM ZnCl₂. Concentrations were selected for optimal cell survival rate. Transdifferentiation was also characterized by immunohistochemical detection of amylase, glucagon, and insulin and by polymerase change reaction analysis of amylase and insulin mRNA production.
RESULTS: Control cells expressed albumin-GFP but no elastase-DsRed. By 30 days of culture, all 3 agents induced expression of pancreatic-like cell marker elastase-DsRed. ZnCl₂ was the most effective as most cells expressed elastase-DsRed in the absence of simultaneous expression of albumin-GFP. For CCl₄ and D-galactosamine, elastase-DsRed was expressed in the same cells as albumin-GFP. Cells treated by each agent also expressed amylase, insulin, and glucagon proteins and mRNAs.
CONCLUSIONS: Without direct genetic intervention, select low small molecules can induce in vitro transformation of hepatoma cells into pancreatic-like cells.
Предметные рубрики MeSH
AlbuminsAmylasesCarbon TetrachlorideCell TransdifferentiationChloridesGalactosamineGene ExpressionGlucagonGreen Fluorescent ProteinsHep G2 CellsHumansImmunohistochemistryInsulinLuminescent ProteinsMicroscopy, FluorescencePancreasPancreatic ElastaseRecombinant Fusion ProteinsReverse Transcriptase Polymerase Chain ReactionTime FactorsTransfectionZinc Compounds