| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1999: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1998: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
In experimental studies, accumulating evidence suggests that ethanol delays liver regeneration in response to Liver injury. However. The responsible mechanisms have been less understood in sinusoidal endothelial cells (SECs) than hepatocytes. Since SECs are initially exposed to ethanol in animals receiving ethanol administration. It is possible to assume that death of SECs plays an important role in delay of liver regeneration. To test this in vitro. We prepared primary cultured SECs in rats and investigated the effect of ethanol on apoptosis and mitochondrial permeability transition (MPT), which is involved in the process of apoptosis.
SECs were isolated based on 3 modification of the method of Wisse et al. Briefly, Wistar female rat liver was perfused with collagenase A. and cells were purified by isopynic sedimentation in a two-step Percoll gradient. SECs were cultured in EBM-2 containing vascular endothelial growth factor (20 ng/ml) at 37℃ for 5 days. DNA synthesis of cells was dete
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rmined by BrdU incorporation. Percent of apoptotic cells vas assessed by TUNEL assy. After loading with tettramethylrhodamine methylester (TMRM), MPT was monitored in SECs with fluorescence microscope.
SECs displayed confluent cell sheet at day 5 in culture. While BrdU labeling index was decreased during the time course in culture, percent of apoptotic cells was not significantly different between day 2 and 5. However, addition of 100 mM ethanol to media for 6 hr induced 2- and 4-fold increase in apoptosis at day 2 and 5, respectively. This apoptotic effect was augmented in a dose dependent manner of ethanol (50-150 nM), and was not affected by an ADH inhibitor, 4-methylpyrazole. When SECs were exposed to ethanol for 3 hr, MPT was observed, but not significant increase in percent of apoptotic cells. Further, cyclosporin A an MPT inhibitor, suppressed apoptosis in SECs exposed to ethanol for 6 hr.
These results indicate that ethanol induces apoptosis in SECs, the process of which is independent or its metabolites and mediated by mitochondrial permeability transition. Although endothelial cells are generally susceptible to apoptosis rather than other cell types, the present finding reveals that apoptosis could be induced in SECs, which are highly proliferating (day 2 in culture) as well. It may be therefore possible to raise a hypothesis that ethanol-induccd apoptosis in SECs may cause impaired angiogenesis in liver, thus contributing to delay of liver regeneration. Less
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