DEVELOPMENT OF THE THREE-DIMENSIONAL ARTIFICIAL LIVER USING A LONG-ACTING VITAMIN C DERIVATIVE

使用长效维生素 C 衍生物开发三维人工肝

• 批准号: 08557001
• 负责人: SENOO Haruki
• 金额: $ 4.1万
• 依托单位: AKITA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08557001/
• 关键词: artificial liver; three-dimensional structure; L-ascorbic acid 2-phosphate; hepatic stellate cells; hepatic parenchymal cells; collagen; extracellular matrix; integrin; 細胞外マトリックス; 人工肝臓(三次元); ビタミンC; ビタミンA肝蔵細胞

We established the procedure for isolation of hepatic stellate cells (vitamin A-storing cells) and parenchymal cells by perfusion of collagenase through the hepatic portal vein of rat. These cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum or William's medium E.Isolated rat stellate cells and human hepatoma HepG2 cells contacted directly and formed a three-dimensional structure when co-cultured in the medium supplemented with a long-acting vitamin C derivative (L-ascorbic acid 2-phosphate ; Asc 2-P). Expression and three-dimensional distribution of phosphorylated proteins in HepG2 cells changed dramatically by contact with the stellate cells.Asc 2-P regulates cellular functions through promotion of the production of extracellular matrix. A kind and three-dimensional structure of extracellular matrix was found to regulate reversibly morphology, proliferation, and functions of the stellate cells. A peptide DGEA and integrin alpha2beta1 was essential for the direct adhesion of the stellate cells and type I collagen. Our present study demonstrated that signals from integrin stimulated phosphorylation of tyrosine and phosphatidylinositol, assembly of microtubules and fibrillar actin, and regulation of the morphology of the stellate cells. The native form of type I collagen which production is stimulated by Asc 2-P was important for the regulation of morphology and function of the hepatic stellate cells. The data obtained in our present study will promote the development of the three-dimensional artificial liver.

我们建立了通过大鼠肝门静脉灌注胶原酶来分离肝星状细胞（维生素A储存细胞）和实质细胞的程序。这些细胞在添加了10%胎牛血清的Dulbecco改良Eagle培养基或William培养基E中培养。分离的大鼠星状细胞和人肝癌HepG2细胞在添加了长效维生素C衍生物（L-抗坏血酸2-磷酸；Asc 2-P）的培养基中共培养时直接接触并形成三维结构。 HepG2细胞中磷酸化蛋白的表达和三维分布因与星状细胞的接触而发生巨大变化。Asc 2-P通过促进细胞外基质的产生来调节细胞功能。发现一种三维的细胞外基质结构可逆地调节星状细胞的形态、增殖和功能。肽 DGEA 和整合素 α2β1 对于星状细胞和 I 型胶原的直接粘附至关重要。我们目前的研究表明，整合素发出的信号刺激酪氨酸和磷脂酰肌醇的磷酸化、微管和纤维状肌动蛋白的组装以及星状细胞形态的调节。由 Asc 2-P 刺激产生的天然形式的 I 型胶原蛋白对于肝星状细胞的形态和功能的调节非常重要。我们本研究获得的数据将促进三维人工肝的发展。

项目成果

Senoo H.: "Three-dimensional structure of extracellular matrix regulates morphology,proliferation,and collagen metabolism of perisinusoidal stellate cells(vitamin A-storing cells)" Cell Biol.Int.20. 501-512 (1996)

Senoo H.：“细胞外基质的三维结构调节窦周星状细胞（维生素 A 储存细胞）的形态、增殖和胶原代谢”Cell Biol.Int.20。

Imai K: "Morphology of sites adhesion between hepatic sttellate cells(vitamin A-storing sells)and a three-dimensional structure of extracellular matrix" Anat.Rec.250(in press). (1998)

Imai K：“肝星状细胞（维生素 A 存储出售）与细胞外基质三维结构之间粘附位点的形态”Anat.Rec.250（印刷中）。

Senoo H: "Hepatic stellate cells-From the viewpoint of vitamin A handling and functions of extracellular matrix-" Acta Anat.Nippon.72・2(in press). (1997)

Senoo H：“肝星状细胞-从维生素A处理和细胞外基质功能的角度来看-”Acta Anat.Nippon.72·2（出版中）（1997年）。

Senoo H.: "Hepatic stellare sells-from the viewpoint of retioid handling and funetion of extracecllular matrix" Acta Anat.Nippon.72. 79-94 (1997)

Senoo H.：“从维甲酸处理和细胞外基质功能的角度来看，肝星状细胞的销售”Acta Anat.Nippon.72。

Imai K,Senoo H.: "Morphology of sites of adhesion between hepatic stellate cells (vitamin A-storing cells) and a three-dimensional extracellularmatrix." Anat Rec. 250 (in press). (1998)

Imai K, Senoo H.：“肝星状细胞（维生素 A 储存细胞）和三维细胞外基质之间粘附位点的形态学。”