Establishment of a new antiatherosclerotic drug and its screening methods using an animal model.

为了建立抗动脉粥样硬化药物的筛选方法，我们建立了去年报道的兔肝细胞原代培养体系。此外，当我们在培养基中加入胶原蛋白时，我们发现兔肝细胞很容易附着在培养皿上。这一发现使得我们的实验很容易完成。正如去年报道的那样，HMG-CoA还原酶抑制剂普伐他汀诱导了肝细胞低密度脂蛋白受体的表达。此外，我们还发现普伐他汀显著减少肝脏载脂蛋白B的分泌。另一方面，当我们用低密度脂蛋白孵育肝细胞时，肝细胞分泌的载脂蛋白B比对照细胞多。当我们在细胞中加入普伐他汀时，细胞胆固醇酯降低。这些结果表明，载脂蛋白B分泌的变化与细胞胆固醇酯含量的变化是平行的。此外，我们还研究了载脂蛋白B在分泌前的细胞内降解，发现普伐他汀的加入加速了载脂蛋白B的细胞内降解，而低密度脂蛋白则减缓了载脂蛋白B的细胞内降解速度。结果表明，细胞内胆固醇酯含量对载脂蛋白B的分泌起关键作用，细胞内载脂蛋白B的降解率可能是细胞内胆固醇酯水平变化调节载脂蛋白B分泌的主要机制。我们正在研究这些发现的详细机制。最后，仍然很难制造出至少具有两种活性的新化合物，例如HMG-CoA还原酶抑制剂和抗氧化作用。这项实验正在进行中。

In order to establish the screening methods for anti-atherosclerotic drug, we could develop the primary culture system for rabbit hepatic cells as reported last year. In addition when we added collagens in the culture medium, we found that rabbit hepatic cells could easily attach to the dish. This finding makes our experiments to be done easily.As reported last year, pravastatin, an inhibitor of HMG-CoA reductase inhibitor, induced the expression of LDL receptor in hepatic cells. Moreover, we found that pravastatin decreased apo B secretion significantly from the liver. On the other hand, when we incubated hepatic cells with LDL, 'hepatic cells secreted apo B more than that of control cells. When we added pravastatin to the cells, cellular cholesteryl ester was decreased. Therefore these results indicated that the change of apo B secretion was in parallel with the change of cellular cholesteryl ester contents. Furthermore we investigated intracellular degradation of apo B prior to secretion and found that the addition of pravastatin accelerated intracellular degradation of apo B, while LDL slowed apo B intracellular degradation rate. However the change of cellular cholesteryl ester could not affect apo B mRNA levels, examined by northen blot analysis.We conclude that intracellular cholesteryl ester contents play a critical role for apo B secretion and intracellular apo B degradation rate could be the main mechanism that regulated apo B secretion in response to the change of intracellular cholesteryl ester level. We are investigating the detail mechanism of these findings.Finally it is still very difficult to make new compounds which have at least two activities, such as an inhibitor of HMG-CoA reductase and an antioxidant action. This experiment is undergoing.