Warning: imagejpeg(C:\Inetpub\vhosts\kidney.de\httpdocs\phplern\27065888
.jpg): Failed to open stream: No such file or directory in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 117 Front+Physiol
2016 ; 7
(ä): 120
Nephropedia Template TP
gab.com Text
Twit Text FOAVip
Twit Text #
English Wikipedia
Culture Model of Rat Portal Myofibroblasts
#MMPMID27065888
El Mourabit H
; Loeuillard E
; Lemoinne S
; Cadoret A
; Housset C
Front Physiol
2016[]; 7
(ä): 120
PMID27065888
show ga
Myofibroblasts are matrix-producing cells with contractile properties, usually
characterized by de novo expression of alpha-smooth muscle actin, that arise in
fibrotic diseases. Hepatic stellate cells (HSCs), known as perisinusoidal cells
containing auto-fluorescent vitamin A, are the major although not exclusive
source of myofibroblasts in the injured liver. Portal myofibroblasts (PMFs) have
been defined as liver myofibroblasts derived from cells that are distinct from
HSCs and located in the portal tract. Here, we describe the protocol we have
established to obtain rat PMFs in culture. In this method, the biliary tree is
(i) separated from the liver parenchyma by in situ enzymatic perfusion of the
liver, (ii) minced and further digested in vitro, until bile duct segments are
isolated by sequential filtration. Bile duct isolates free of HSC contaminants,
form small cell clusters, which initially comprise a large majority of epithelial
cells. In culture conditions (fetal bovine serum) that provide a growth advantage
to mesenchymal cells over epithelial cells, the epithelial cells die and detach
from the substrate, while spindle-shaped cells outgrow from the periphery of the
cell clusters, as shown by video-microscopy. These cells are highly proliferative
and after 4-5 days, the culture is composed exclusively of fully differentiated
myofibroblasts, which express alpha-smooth muscle actin and collagen 1, and
secrete abundant collagen. We found no evidence for epithelial-mesenchymal
transition, i.e., no co-expression of alpha-smooth muscle actin and cytokeratin
at any stage, while cytokeratin becomes undetectable in the confluent cells. PMFs
obtained by this method express the genes that were previously reported to be
overexpressed in non-HSC or portal fibroblast-derived liver myofibroblasts as
compared to HSC-derived myofibroblasts, including the most discriminant, collagen
15, fibulin 2, and Thy-1. After one passage, PMFs retain the same phenotypic
features as in primary culture. In conclusion, this straightforward and
reproducible method of PMF culture, can be used to identify new markers of PMFs
at different stages of differentiation, to compare their phenotype with those of
HSC-MFs and ultimately determine their progenitors and specific functions in
liver wound-healing.
free
free
free
