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。

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02807074/
• 关键词: proteoglycans; glycosaminoglycans; multicellular spheroids; p450; detoxication; primary culture; hepatocyte; グリコサシノグリカン; p450; 年代培養肝細胞; 三次元培養; 解毒能; 人工肝; 初代培養肝細胞

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

我们以前已经发现，分离的大鼠肝细胞组装成多细胞球体的存在下，肝源性蛋白聚糖和各种分化的肝脏特异性功能保留在球体中。本研究旨在解决以下问题：1）肝源性蛋白聚糖中的什么分子负责球状体的形成，2）球状体中是否保留了p450相关的解毒能力，以及3）是否可以构成能够利用球状体的肝脏特异性功能的模块。1）负责球状体形成的分子通过分析糖胺聚糖和核心蛋白衍生的蛋白聚糖揭示了核心蛋白聚糖（108 kD）和双糖蛋白聚糖（200 kD），两者都是小的软骨素/硫酸皮肤素蛋白聚糖，是负责球状体形成的分子。此外，核心蛋白聚糖和双糖蛋白聚糖似乎只有在通过核心蛋白固定在冲浪时才有效 ...更多信息 文化用品的王牌。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来分析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状体的形成是有效的，而肝素和硫酸乙酰肝素则相对较少。2）球状体中p450相关的解毒能力的保存培养肝细胞的解毒能力球状体中的解毒能力如下：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还原酶的转录信号，以及测量微粒体蛋白。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，苯巴比妥诱导型保存得最好;在第5天，球形肝细胞中保留了60%的初始水平，而单层肝细胞中仅保留了20%。然而，保留在球体中的p450的总酶活性仅占体内肝组织的一小部分。3）利用保留在球体中的肝特异性功能的模块为了利用肝细胞的肝特异性功能，将球体用精氨酸钙凝胶液滴包封，并将液滴包封在插入培养基回路的生物反应器室中。通过定期收集的培养基样品中的白蛋白和尿素来评估包封的球状体的肝脏特异性功能。白蛋白和尿素在循环中均以线性方式积累;用包封的球状体获得的生产率与用非包封的球状体获得的生产率相当。尽管我们的目的是利用生物反应器系统来评估药物代谢，但由于如上所述p450相关的解毒能力不能很好地保留在球状体中，因此将药物引入系统中。少

项目成果

K Sakaguchi,N Koide,et al.: "Promotion of spheroid asembly of adult rat hepatocytes by some factor(s)present in the initial 6h conditioned medium of the primary culture." Pathobiology. 59. 351-356 (1991)

K Sakaguchi、N Koide 等人：“原代培养物初始 6 小时条件培养基中存在的一些因子促进成年大鼠肝细胞的球体组装。”

H Takabatake,他: "Encapsulated multicellular pheroids of rat hepatocytes produce albumin and urea in a spouted bed circulating culture system." Artificial Organs. 15. 474-480 (1991)

H Takabatake 等人：“大鼠肝细胞的封装多细胞球体在喷射床循环培养系统中产生白蛋白和尿素。”15. 474-480 (1991)。

H Matsushima,N Koide,et al.: "Electron microscopic localization of alkaline phosphatase activity in he patocyte spheroids." J clin.Electron Microscopy.23. 660-661 (1991)

H Matsushima、N Koide 等人：“肝细胞球体中碱性磷酸酶活性的电子显微镜定位”。

H Hada,N Koide,T Tsuji: "Sequence variations in the envelope protein of the variant virus obtained by the polymerase chain reaction." Acta Med Okayama.45. 347-355 (1991)

H Hada、N Koide、T Tsuji：“通过聚合酶链式反应获得的变异病毒包膜蛋白的序列变异。”

小出 典男、他: "肝細胞spheroidをバイオリアクターとする人工肝機能補助装置のプロトタイプ開発" 組織培養、. 18. 418-423 (1992)

Norio Koide 等人：“使用肝细胞球体作为生物反应器的人工肝功能支持装置的原型开发”《组织培养》，18. 418-423 (1992)