Role of Liver Sinusoidal Endothelial cell in Liver Regeneration

在实验研究中，越来越多的证据表明乙醇延缓肝损伤后的肝脏再生。然而。与肝细胞相比，对窦状内皮细胞（SECs）的相关机制了解较少。因为在接受乙醇管理的动物中，sec最初暴露于乙醇。我们可以假设SECs的死亡对肝脏再生的延迟起着重要的作用。在体外进行测试。我们制备了大鼠原代培养的内皮细胞，研究了乙醇对细胞凋亡和参与细胞凋亡过程的线粒体通透性转移（MPT）的影响。对Wisse等人的方法进行3次修改，分离出SECs。简单地说，用胶原酶a灌注Wistar雌性大鼠肝脏，用两步Percoll梯度等渗沉淀法纯化细胞。在含血管内皮生长因子（20 ng/ml）的EBM-2中，37℃培养5 d。BrdU掺入抑制了细胞的DNA合成。TUNEL法测定凋亡细胞的百分率，荧光显微镜下观察凋亡细胞的MPT变化，用四甲基罗丹明甲基lester （TMRM）载药。在培养的第5天，SECs显示了融合的细胞片。虽然BrdU标记指数随培养时间的推移而降低，但凋亡细胞的百分比在第2天和第5天之间没有显著差异。然而，在培养基中添加100 mM乙醇6小时，在第2天和第5天，细胞凋亡分别增加2倍和4倍。这种凋亡效应在乙醇（50-150 nM）的剂量依赖性下增强，并且不受ADH抑制剂4-甲基吡唑的影响。乙醇作用3小时后，细胞凋亡率明显升高，但凋亡细胞百分比未见明显升高。此外，环孢素A是一种MPT抑制剂，可抑制暴露于乙醇6小时的sec的凋亡。这些结果表明，乙醇诱导SECs凋亡的过程是独立于其代谢物的，并通过线粒体通透性转变介导。虽然内皮细胞通常比其他细胞类型更容易发生凋亡，但目前的研究结果表明，在高度增殖（培养第2天）的内皮细胞中也可以诱导凋亡。因此，有可能提出一种假设，即乙醇诱导的SECs细胞凋亡可能导致肝脏血管生成受损，从而导致肝脏再生延迟。少

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 … More 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