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Tesi etd-03292012-094645

Tipo di tesi
Tesi di laurea magistrale
Protein Bex1 and Liver Regeneration after Tissue Damage
Corso di studi
relatore Prof.ssa Bisgaard, Hanne Cathrine
relatore Prof. Paolicchi, Aldo
Parole chiave
  • siRNA
  • Bex1
  • Real Time PCR
  • liver regeneration
Data inizio appello
Riassunto analitico
Recent studies suggest the involvement of the BEX genes in cancer biology; for example Bex1 (Brain Expressed, X-Linked) is overexpressed in neuroendocrine tumors and is downregulated in glioblastoma cells compared to normal tissue. Genome-wide analysis of epigenetic silencing identifies BEX1 and BEX2 as candidate tumor suppressor genes in malignant glioma. Moreover Bex1 is over-expressed as a result of peripheral axonal damage and some studies also show an altered skeletal muscle regeneration in Bex1 knock out mice; therefore Bex1 could be considered as regeneration-associated gene. It is an adapter molecule involved in p75NTR/NGFR signaling and plays a role in cell cycle progression and neuronal differentiation.
Many aspects of Bex1 are unknown so it is a an exciting field of research. Bex1 is expressed in central nervous system, lung, skeletal muscle, peripheral blood leukocytes, stomach, lymph node, trachea, bone marrow; highly expressed in pituitary, cerebellum, temporal lobe. Our attention was placed on the expression of Bex1 in normal and pathological liver tissues. Our study was focused on defining the mechanism by which Bex1 might regulate proliferation and differentiation of hepatic progenitor cells into the hepatic or cholangiocytic lineages during regeneration after tissue damage and in development of liver cancer.
I used different methods in this project: Real Time-PCR, Immunohistochemistry, immunofluorescence and confocal microscopy, culturing of human hepatocellular carcinoma cell lines, Knock-Down in Huh7 cell line using Dharmacon® Accell® siRNA with subsequent protein harvest, Bradford assay and Western Blot.
The results of Real Time PCR show that there is an up regulation of Bex1 during liver regeneration after all liver diseases compared to donor liver. The immunolocalization in hepatic tissue shows a presence of Bex1 mostly in the bile duct and also in reactive ductular cells (RDCs) and hepatic progenitor cells (HPC). The knock down of Bex1 in Huh7 cell line shows a doubtful result, because we cobserved a reduction of our protein compared to control, but we expected Bex1 at ~ 23kDa, while we found it at ~ 62kDa. There are maybe three explanations: or the antibody recognizes an isoform of Bex in the cell line, or the protein have not been reduced sufficiently, or modifications of the protein are involved.
For the future my aim is to examine the function of Bex1 as tumor suppressor in hepatocellular carcinoma and the role in signaling pathways regulating apoptosis. This whould likely allow a better understanding of the role that Bex1 plays in liver regeneration.