DEVELOPMENT OF BIOARTIFICIAL LIVER

为了开发生物人工肝(BAL)支持装置，建立高密度培养的高分化肝细胞至关重要。研制了一种混合型肝支持系统，该系统通过多孔中空纤维组件进行血浆灌流，将100亿个猪肝细胞接种在Engelbreth-Holm-Sarm(EHS)凝胶中。在建立门腔分流术和肝脏断流术后8小时，该系统被应用于缺血性肝功能衰竭猪。在接受BAL支持系统治疗的动物中，血液碳酸氢盐水平在治疗后立即增加，血流动力学稳定性得到改善。另一方面，在对照组中，血液中的碳酸氢盐水平和血压保持在低水平。使用BAL装置治疗的猪的血氨和乳酸水平降低，但对照组动物没有。我们构建了携带大鼠人神经营养因子-4α基因的腺病毒载体，并将其导入肝癌细胞，以增强外源人神经营养因子-4α基因的表达。用Norhim blotting和Western blotting分析表达hNf-4α的腺病毒载体感染细胞后hnf-4、hnf-1和肝脏特异性基因的表达。腺病毒介导的hNF4α基因转移导致转化的肝癌细胞hNF4、hNFI和肝脏特异性基因如αL抗胰蛋白酶、载脂蛋白和谷氨酰胺合成酶的表达增加。与感染对照载体的细胞相比，高表达hnf-4α的细胞从添加NH4CL的培养基中去除氨的程度更大。这些结果表明，BAL支持装置结合中空纤维模块和EHS凝胶包裹的肝细胞在临床上具有潜在的优势，使用表达HNF-4a的腺病毒感染的肝细胞对生物人工肝的发展具有潜在的优势。

For the development of a bioartificial liver (BAL) support device, it is most important to establish highly differentiated liver cells cultured at high density. A hybrid liver support system was developed, and consisted of plasma perfusion through porous hollow fiber modules inoculated with 10 billion porcine hepatocytes entrapped in Engelbreth-Holm-Swarm (EHS) gel. This system was applied to pigs with ischemic liver failure 8 hours after creation of a portocaval shunt and hepatic devascularization. In animals treated with the BAL support system, blood bicarbonate levels were incrcased immediately after treatment, and hemodynamic stability was improved. In control pigs, on the other hand, blood bicarbonate levels and blood pressure remained low. Plasma levels of ammonia and lactate decreased in pigs treated with the BAL device, but not in control animals. We constructed adenovirus vector carring rat HNF-4α cDNA, and transfected the adenovirus vector to hepatoma cells to enforce expression of the exogenous HNF-4α gene. We analyzed expression of HNF-4, HNF-1, and liver specific genes in cells infected by the adenovirus vector expressing HNF-4α by Northem blotting and Western blotting analysis. Adenovirus-mediated HNF-4α gene transfer resulted in the increases of HNF-4, HNF-I, and liver specific genes such as αl-antitrypsin, apolipoproteins, and glutamine synthetase by transformed hepatoma cells. Cells overexpressing HNF-4α removed ammonia from medium supplemented with NH_4Cl to greater extent than cells infected control vector. These results suggest that the use of the BAL support device in combination with a hollow fiber module and hepatocytes entrapped in EHS gel has potential advantages for clinical use in patients with fulminant hepatic failure and that the use of liver cells infected by adenovirus expressing HNF-4a has potential advantages for the development of bioartificial liver.