A study on p450-related detoxication of hepatocytes in primary culture.

我们先前已经发现，在肝源性蛋白多糖存在的情况下，分离的大鼠肝细胞组装成多细胞球体，并在球体中保留了各种分化的肝脏特异性功能。本研究旨在解决以下几个问题：1)肝源性蛋白多糖中的什么分子负责球体的形成；2)球体中是否保留了P450相关的解毒能力；3)是否可以构建一个能够利用球体的肝脏特异性功能的模块。1)球体形成的分子通过对糖胺多聚糖和核心蛋白的分析，发现小软骨素/硫酸皮肤素蛋白多糖是负责球体形成的两种小分子。此外，似乎只有通过核心蛋白将核心蛋白和双聚糖固定在SURF…上时，它们才有效更多的王牌文化产品。利用合成的由不同类型的糖胺聚糖和磷脂酰乙醇胺组成的新蛋白多糖，分析了糖胺聚糖对球体形成的特异性。结果表明，硫酸软骨素包括硫酸皮肤素作为新蛋白多糖的糖链对球体的形成是有效的，但肝素和硫酸肝素的作用要小得多。2)球体中P450相关解毒能力的保存培养肝细胞在球体中的解毒能力：用特异性的单抗通过免疫印迹法检测P450蛋白，测量P450还原酶的转录信号，以及测定微粒体蛋白。结果表明，球形肝细胞比单层肝细胞具有更好的解毒能力。在P450蛋白中，苯巴比妥诱导型蛋白保存最好，球形肝细胞在第5天保留了初始水平的60%，而在单层肝细胞中仅保留了20%。3)利用肝特异性功能的模块为了利用肝细胞的特异性功能，用精氨酸钙凝胶液滴包裹球体，并将液滴包裹在中间回路中的生物反应室中。通过测定定期采集的培养液中的白蛋白和尿素来评价包裹的球体的肝特异性功能。白蛋白和尿素在循环中均呈线性积累；微囊化的产生率与未微囊化的球体相当。虽然我们的目标是利用生物反应器系统来评估药物代谢，但由于P450相关的解毒能力不能很好地保留在如上所述的球体中，药物被引入系统中。较少

We have previously found that isolated rat hepatocytes assembled into multicellular spheroids in the presence of liver-derived proteoglycans and various differentiated liver-specific functions were retained in the spheroids. The present research project aimed to solve the following questions, 1) What molecule in the liver-derived proteoglycans was responsible for the spheroid formation, 2) Whether the ability of p450-related detoxication was retained in the spheroid, and 3) Whether a module that can utilize liver specific functions of spheroids can be constituted.1) Molecules responsible for spheroid formationCharacterization of liver-derived proteoglycans by analyses of glycosaminoglycan and core proteins revealed that decorin (108kD) and biglycan (200kD), both small chondroitin/dermatan sulfate proteoglycans, were the molecules responsible for the spheroid formation. It also appeared that decorin and biglycan were effective only when they were immobilized via core protein to the surf … More ace of culture wares. Glycosaminoglycan specificity for the spheroid formation was analyzed by using synthetic neoproteoglycan which were constituted of various types of glycosaminoglycans and phosphatidyl ethanolamine. The result indicated that chondroitin sulfate including dermatan sulfate were effective as a sugar chain of neoproteoglycan for the spheroid formation, but heparin and heparan sulfate were much less.2) Preservation of p450-related detoxicating ability in spheroidDetoxication ability of cultured hepatocytes ability in spheroid the following procedures ; measuring p450 proteins by western blotting using specific monoclonal antibodies, measuring transcritional signal of p450 reductase, and measuring microsomal proteins. Results indicated that detoxication ability was better retained in spheroid hepatocytes than monolayer hepatocytes. Among p450 proteins, phenobarbital inducible type was best preserved ; 60% of the initial level was retained at day 5 in spheroid hepatocyte in contrast only 20% was in monolayer hepatocytes. However the total enzyme activity of p450 retained in spheroid was only a small percent of liver tissue in vivo.3) A module utilizing liver specific functions retained in spheroidsTo utilize liver specific functions of hepatocytes, the spheroids were encapsulated with calcium arginate gel droplet, and the droplets were entrapped in a bioreactor chamber which was inserted in the medium circuit. The liver specific functions of encapsulated spheroids were assessed by measuring the albumin and urea in medium samples periodically collected. Both albumin and urea accumulated in a linear fashion in the circulation ; the production rates obtained with encapsulated were equivalent to those with non-capsulated spheroids. Although we had aimed to utilize the bioreactor system for assessment of drug metabolism, drug was introduced in the system since p450-related detoxicating ability was not well retained in spheroids as described above. Less