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，我们可以建立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时，我们发现兔肝细胞可以很容易地附着在培养皿上。去年报道，HMG-CoA还原酶抑制剂普伐他汀可诱导肝细胞LDL受体表达。此外，我们发现普伐他汀显著降低肝脏apo B的分泌。另一方面，当我们用LDL孵育肝细胞时，肝细胞比对照细胞分泌更多的apo B。当我们向细胞中加入普伐他汀时，细胞胆固醇酯减少。因此，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，我们研究了分泌前apo B的细胞内降解，发现加入普伐他汀加速apo B的细胞内降解，而LDL减慢apo B的细胞内降解速率。细胞内胆固醇酯含量的变化对载脂蛋白B mRNA的表达无影响，提示细胞内胆固醇酯含量的变化对载脂蛋白B的分泌起重要作用，细胞内载脂蛋白B降解速率的变化可能是细胞内胆固醇酯含量变化调节载脂蛋白B分泌的主要机制。最后，要合成具有HMG-CoA还原酶抑制剂和抗氧化剂两种活性的新化合物仍然非常困难。这个实验正在进行中。

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.