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  Director : Christian Bréchot et Valérie Thiers (brechot@necker.fr et vthiers@pasteur.fr)



Together with our activity of Reference Center on Molecular Epidemiology of Viral Hepatitis B and C, we are interested in new technologies, such as laser-based microscopic techniques (LCM), allowing the opportunity to study pure populations of cells in their native tissue environment. The use of LCM as the starting point for molecular genetic analysis might facilitate the identification of factors and mechanisms involved in HBV and HCV disease progression.



1 - Microdissection and viral genome B and C analysis

For hepatitis B we focused our study on HBx gene. Point mutations and deletions of HBx gene are suspected to play a role in the multistep process of liver carcinogenesis. In particular, 3' -end deletions of HBx gene have been more frequently observed in patients with hepatocellular carcinoma (HCC). However, a direct role of these mutants in the carcinogenesis process has not been established. To clarify this issue, we have studied HBx sequences heterogeneity in groups of 1000 morphologically characterized hepatocytes captured from distinct zones of tumorous and non-tumorous sections. The analysis of sequence data has shown that mutations in HBx gene were heterogeneous and equally distributed between tumor and non-tumor. However, deletions of the 3' part of the X gene were mostly observed in the tumor. These results are consistent with the selection of deleted HBx sequences in tumorous cells. Recent data, obtained by the group directed by D. Kremsdorf (Inserm U370), have demonstrated by using natural and artificial mutants that these HBx truncated proteins had lost their pro-apoptotic function. This result suggest that abrogation of the apoptotic effect may endow a growth advantage on cells containing these particular HBx mutants (Tu et col., 2001). The analysis of HBx gene genetic variability on different stage of liver disease (minimal lesion, chronic hepatitis, and cirrhosis) is under process.

The pathogenesis of liver injury caused by hepatitis C virus (HCV) chronic infection is not well understood. Furthermore, it is still debated whether or not sera and intrahepatic HCV viral load are correlated. To examine this issue, our project developed a direct measure of intra-hepatic HCV viral load by quantitative approaches. Groups of 2000 hepatocytes were harvested and intra-hepatic HCV RNA was measured by means of a quantitative test (b-DNA3). Using an adjacent slide the percentage of infected hepatocytes was determined by immunohistochemical (IHC) detection of HCV proteins expression by using polyclonal anti-HCV antibodies. Our preliminary results showed a positive correlation between the amount of viral RNA in the two compartments. Thus indicating that the levels of vireamia reflected the amount of virus present in the liver. However, our results support the hypothesis that the severity of liver lesions is not directly correlated with the level of HCV genome replication and expression.

2 - Microdissection and genome expression

The laboratory have started a more technically challenging approach aiming to combine microdissection to array or proteome analysis. For the transcriptomic aspect, as the amount of RNA from microdissected material is limited our goal will be to set up an accurate preamplification technique. Two different approaches, shown to preserve expression profile are currently evaluated: the T7-based linear amplification and a PCR-based amplification technique (SMART, Clontech). Concerning the proteomic part we have started to define the experimental conditions to extract and solubilise the proteins from the microdissected cells.


puce Publications of the unit on Pasteur's references database


  Office staff Researchers Scientific trainees Other personnel

APRIL France, Inserm april@necker.fr

BRÉCHOT Chistian, PUPH-AP U/ParisV/IP, brechot@necker.fr

TUVERI Rosella, Post-Doc rtuveri@pasteur.fr

IAVARONE Massimo PhD student iavaron@pasteur.fr

TRABUT Jean-Baptiste DEA trabut@necker.fr

THIERS Valérie, Ingénieur, IP, vthiers@pasteur.fr

PINON Gregory, Technician, CDD Faculté Necker


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