TY  - GEN
CY  - Heidelberg
AV  - public
Y1  - 2015///
TI  - Gene expression recovery during an acute toxic damage in the liver
ID  - heidok18873
A1  - Ghafoory, Shahrouz
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/18873/
N2  - Specific structures and cell types in the organization of the liver are the key for its variant functions, like protein production, glucose homeostasis and detoxification. In the present work, liver damage from an acute toxic injury caused by intraperitoneal injection of a mixture of CCl4 and mineral oil in Balb/c mice and its subsequent recovery was studied using different methods to investigate specific cellular functions in the liver. The analysis by in situ hybridization and RT-qPCR showed how expression of liver specific enzymes and proteins in mouse hepatocytes is changed over a period of 6 days following injection. The genes investigated included Albumin, Arginase, Glutaminase2, Glutamine synthetase, Glucose-6-phosphatase, Glycogen synthase2, Gapdh, Cyp2e1 and Glucagon receptor genes. Interestingly, a significant change in gene expression of enzymes involved in nitrogen and glucose metabolism and their local distribution in different areas of the liver were observed following CCl4 injury. Cyp2e1, an essential metabolizing enzyme in CCl4 metabolism, was strongly expressed in the pericentral zone during recovery. In comparison to hepatocytes in livers from untreated mice, liver cells from treated animals displayed distinct gene expression profiles in the damaged area around the pericentral vein during the analyzed time course and showed a complete recovery with strong albumin production at day 6 post CCl4 injection. The results obtained indicate that despite of the severe damage, liver cells in the damaged area do not simply die but instead locally adjust gene expression to deal with the damage effect and thereby ensure survival.
In order to optimize the preparation of cRNA hybridization probes and enable the rapid synthetize of the large number of probes used in this study, a new rapid method for antisense cRNA preparation was established. The development of this rapid and efficient protocol for the generation of labeled cRNA probes was an important pre-requisite for the project. The new protocol is based on the preparation of DNA templates in vitro by PCR using primers that include RNA polymerase promoter sequences and size based purification of PCR fragments containing the target gene specific cDNA and promoter elements for T7 and SP6 RNA-polymerase. Purified PCR fragment based in vitro transcription enables the preparation of in situ hybridization probes, which can be used for the detection of the respective gene and visualization of the distribution of gene expression in tissue slices for any gene of interest. The optimized synthesis and purification protocols ensure high transcription efficiency and target specificity of the labeled cRNA and the obtained cRNA hybridization probes are compatible with established in situ hybridization protocols.      
This study proved that with a single dose of CCl4 injection in mouse, liver pericentral hepatocytes are the main cell type responsible for neutralizing the toxic agent, and the main consequence of this damage is not simply to induce cell death due to apoptosis, but instead these damaged hepatocytes seem to reduce any unnecessary activities in favor of processes needed for recovery from damage.
ER  -