TY  - THES
T1  - The secretome of primary human hepatocytes in an organotypic bioreactor culture for the identification of biomarkers of drug-induced hepatotoxicity
A1  - Tascher,Georg
Y1  - 2012/07/06
N2  - In the presented thesis, a workflow for analysis of the extracellular proteome, or “secretome”, of primary human hepatocytes by a shotgun proteomics approach was developed. The secretome analysis on hepatocytes is of special interest for the pharmaceutical industry as well as medical services because secreted proteins could serve as biomarkers for drug-induced hepatotoxicity. The avoidance of fetal calf serum, a common supplement used for in vitro cell culture was shown to be a prerequisite for efficient secretome analysis because supplemented proteins severely hamper the identification of secreted proteins using mass-spectrometry (MS). The removal of high abundant proteins from the sample by immunodepletion, a common strategy for proteomic analysis of human plasma, was shown to increase the number of identified proteins to almost two-fold the number of identifications obtained without prior immunodepletion, paving the way for in-depth analysis of the secretome. A prefractionation step at the level of proteins and the combination of different MS-types and ionization methods tremendously enhanced the identifications but at the cost of analysis time as well as the fraction of secreted proteins. The elaborated workflow without prior prefractionation was applied to a standard monolayer culture and a promising in vitro cultivation technique in a 3-dimensional bioreactor mimicking the microenvironment of a real liver. With 109 and 160 proteins identified in monolayer and bioreactor cultures, respectively, the number of proteins likely to be secreted by hepatocytes into the extracellular space was much higher than described in the current literature. Furthermore, proteome analysis on extracellular proteins in the applied culture conditions confirmed the tissue-like behavior of primary hepatocytes in the three-dimensional cultivation. Differentially expressed proteins in the bioreactor culture after application of the reference drug diclofenac were detected by label-free quantification and most of the detected differences could be traced back to the underlying mechanism of toxicity proposed for this drug. In summary, the presented work provides a basis for further in-depth analysis of the primary human hepatocytes secretome for in vitro drug-testing and demonstrates the valuable contributions of proteomics to toxicological research.
KW  - Arzneimittelentwicklung
KW  - Proteomanalyse
KW  - Bioreaktor
KW  - Leberepithelzelle
KW  - Biomarker
KW  - Hepatotoxizität
CY  - Saarbrücken
PB  - Universitäts- und Landesbibliothek
AD  - Postfach 151141, 66041 Saarbrücken
UR  - http://scidok.sulb.uni-saarland.de/volltexte/2012/4890
ER  -