Cryostat: Preparation of liver
tissue for dissociation
Microscope: Separation of active
and Proteomics of Fibrosis
Fibrosis Group: Geraldine Grant, Mike
Estep, Linda O'Reilly
Liver fibrosis is a central feature of chronic liver
injury due to metabolic, genetic, viral disease. Extensive
liver fibrosis, resulting from the deposition of collagen
type I within the liver, can ultimately lead to cirrhosis
and chronic liver failure, due to an unnatural hardening of
the organ, thus inhibiting its function. Currently, there
is no known treatment, with the exception of replacing the
affected organ via transplant.
Hepatic stellate cells play a central role in the process
of fibrosis, as they are the major source of collagen in the
liver. Under normal conditions, stellate cells are “inactive”,
and are mainly involved in retinol storage. Following liver
injury, stellate cells become “activated” fibroblast-like
cells. These cells dump their retinol store, mobilize to the
site of injury, and promote fibrosis via collagen deposition,
forming a scar. Scar formation is a natural defense against
injury and under normal circumstances should resolve with
the dissolution of the scar, once the injury is removed. The
stellate cells should also be signaled to die at this point.
However, in fibrosis the scar remains. The exact steps involved
in this fibrogenic process and the associated molecular pathways
are not entirely clear. We hope to gain insight into the fibrotic
processes by determining the differential gene expression
of fibrotic liver tissue.
To investigate the molecular mechanisms of fibrosis, we will
examine the transcriptional profile of the inactive and active
stellate cells from patients with end stage cirrhosis, by
differential gene expression, utilizing a custom cDNA microarray.
The hepatic transcriptome is second only to the brain in size,
with 25-40% of human genes expressed in the liver. This could
make total genome analysis too complex. By custom building
our microarray of 5700 genes, to include genes involved with
fibrotic-specific pathways, such as apoptosis, and cell proliferation,
we can focus specifically on fibrosis data.
Liver specimens from 20 patients undergoing orthotopic liver
transplantation due to idiopathic fibrosis. The tissue is
dissociated; the hepatic stellate cells are collected and
segregated to their active and inactive populations. These
samples are then hybridized to the microarray. The data obtained
is then further investigated by RT-PCR, and cell culture experiments
Liver tissue is dissociated by collagenase perfusion for 1
hour. The resulting released cells are harvested by centrifugation.
This cell pellet is then separated on a nycodenz density gradient.
Due to the additional boyant density of the stored retinol,
the inactive stellate cells will be at the top of the gradient,
the activated stellate cells present in the pellet fraction.
The active stellate cells are then separated from other liver
cells, by plating on tissue culture treated plastic culture
flasks for 1 hour. The active stellate cells, due to the fibroblast-like
nature, bind to the flask surface, and the other cells can
be washed away. The RNA will be extracted from both cells
populations and investigated using the custom cDNA microarray,
and by semi-quantitative RT-PCR.
Microarray: Examination of microarray data