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

原代培养中肝细胞p450相关解毒的研究。

基本信息

批准号：
02807074
负责人：
KOIDE Norio
金额：
$ 1.02万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

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

我们之前发现，在肝源性蛋白聚糖存在的情况下，分离的大鼠肝细胞组装成多细胞球体，并且球体中保留了各种分化的肝脏特异性功能，本研究项目旨在解决以下问题：1）肝源性蛋白聚糖中存在哪些分子。肝源性蛋白聚糖负责球体的形成，2) p450 相关解毒能力是否保留在球体中，以及 3) 是否有一个模块1) 负责球体形成的分子通过糖胺聚糖和核心蛋白的分析对肝源性蛋白聚糖进行表征，发现核心蛋白聚糖 (108kD) 和双糖链蛋白聚糖 (200kD) 都是小软骨素/硫酸皮肤素蛋白聚糖，是负责球体形成的分子。核心蛋白聚糖和通过使用由各种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，只有当它们通过核心蛋白固定到表面时才有效。包括硫酸皮肤素在内的硫酸盐作为新蛋白聚糖的糖链对于2)球状体中p450相关解毒能力的保存培养肝细胞球状体中的解毒能力按照以下步骤使用特异性单克隆抗体通过蛋白质印迹法测量p450蛋白，测量p450的转录信号。还原酶，并测量微粒体蛋白，结果表明解毒能力得到了更好的保留。在 p450 蛋白中，苯巴比妥诱导型保留得最好；第 5 天，球状肝细胞中保留了 60%，而单层肝细胞中仅保留了 20%。球体仅占体内肝组织的一小部分。3）利用球体中保留的肝脏特定功能的模块利用肝脏特定功能肝细胞中，用精氨酸钙凝胶液滴封装球体，并将液滴捕获在插入培养基回路的生物反应器室中，通过测量定期收集的培养基样品中的白蛋白和尿素来评估封装球体的肝脏特异性功能。白蛋白和尿素在循环中以线性方式积累；尽管我们的目标是利用封装球体，但封装球体获得的生产率相同。用于评估药物代谢的生物反应器系统，由于 p450 相关的解毒能力在球体中没有得到很好的保留，因此将药物引入系统中。