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

我们建立了大鼠肝星状细胞（维生素A储存细胞）和肝实质细胞的分离方法。将这些细胞培养在补充有10%胎牛血清的Dulbecco改良Eagle培养基或William培养基E中。当在补充有长效维生素C衍生物（L-抗坏血酸2-磷酸盐; Asc 2-P）的培养基中共培养时，分离的大鼠星状细胞和人肝癌HepG 2细胞直接接触并形成三维结构。与星状细胞接触后，HepG 2细胞内磷酸化蛋白的表达和三维空间分布发生了显著变化，Asc 2-P通过促进细胞外基质的产生来调节细胞功能。细胞外基质的三维结构对星状细胞的形态、增殖和功能具有可逆性调节作用。肽DGEA和整合素α 2 β 1是必要的直接粘附的星状细胞和I型胶原。我们目前的研究表明，信号从整合素刺激磷酸化的酪氨酸和磷脂酰肌醇，组装的微管和纤维状肌动蛋白，和调节的形态的星状细胞。Asc 2-P刺激肝星状细胞产生的天然I型胶原对肝星状细胞的形态和功能的调节具有重要作用。本研究所获得的数据将促进三维人工肝的发展。

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.