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

我们之前已经发现，分离的大鼠肝细胞在肝源性蛋白聚糖存在的情况下组装成多细胞球体，并且在球体中保留了各种分化的肝脏特异性功能。本课题旨在解决以下问题：1)肝源性蛋白聚糖中哪些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)是否可以构建一个利用球体肝脏特异性功能的模块。通过对糖胺聚糖和核心蛋白的分析，对肝脏来源的蛋白聚糖进行了表征，结果表明，decorin （108kD）和biglycan （200kD）都是小的软骨素/硫酸皮肤聚糖蛋白聚糖，是导致球体形成的分子。此外，decorin和biglycan只有通过核心蛋白固定在表面时才有效。采用不同类型的糖胺聚糖和磷脂酰乙醇胺组成的合成新蛋白聚糖，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（包括硫酸皮素）作为新蛋白多糖的糖链对球的形成是有效的，而肝素和硫酸肝素的作用要小得多。2)保存p450相关的球形解毒能力培养肝细胞的球形解毒能力使用特异性单克隆抗体，通过western blotting测定p450蛋白，测定p450还原酶的转录信号，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。p450蛋白中，苯巴比妥诱导型保存最好；球状肝细胞在第5天保留了初始水平的60%，而单层肝细胞仅保留了20%。然而，p450酶的总酶活性在体内仅在肝组织中占很小的比例。为了利用肝细胞的肝脏特异性功能，将精氨酸钙凝胶液滴包裹在球体上，并将液滴包裹在生物反应器腔中，该生物反应器腔插入介质回路中。通过定期采集的培养基样品中白蛋白和尿素的测定来评价包封球体的肝脏特异性功能。白蛋白和尿素在血液循环中呈线性积累；包被球体的产率与未包被球体的产率相当。虽然我们的目的是利用生物反应器系统来评估药物代谢，但由于p450相关的解毒能力在球体中没有很好地保留，因此我们将药物引入了该系统。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