DEVELOPMENT OF BIOARTIFICIAL LIVER

生物人工肝的开发

• 批准号: 10670462
• 负责人: NAGAKI Masahito
• 金额: $ 1.98万
• 依托单位: Gifu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670462/
• 关键词: BAIOARTIFICIAL LIVER; ACUTE HEPATIC FAILURE; EXTRACELLULAR MATRIX; GENE THERAPY; TRANSCRIPTION FACTOR

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.

高密度培养的高分化肝细胞是研制生物人工肝支持装置的关键。开发了一种混合型肝支持系统，该系统由通过多孔中空纤维模块的血浆灌注组成，该模块接种有包埋在Engelbreth-Holm-Swarm（EHS）凝胶中的100亿个猪肝细胞。在建立门腔静脉分流和肝血管离断术后8小时，将该系统应用于缺血性肝功能衰竭的猪。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升高，血流动力学稳定性得到改善。另一方面，在对照组猪中，血液碳酸氢盐水平和血压保持较低。在接受BAL装置治疗的猪中，氨和乳酸盐的血浆水平降低，但在对照动物中未降低。我们构建了携带大鼠HNF-4α cDNA的腺病毒载体，并将其转染肝癌细胞，以促进外源性HNF-4α基因的表达。我们通过Northem 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和肝特异性基因的表达。腺病毒介导的HNF-4α基因转移导致转化的肝癌细胞中HNF-4、HNF-Ⅰ和肝特异性基因如α l-抗胰蛋白酶、载脂蛋白和谷氨酰胺合成酶的表达增加。过表达HNF-4α的细胞比感染对照载体的细胞更大程度地去除NH_4Cl培养基中的氨。这些结果表明，BAL支持装置与中空纤维模块和包埋在EHS凝胶中的肝细胞的组合使用在暴发性肝衰竭患者的临床应用中具有潜在的优势，并且使用表达HNF-4a的腺病毒感染的肝细胞对于生物人工肝的开发具有潜在的优势。

项目成果

Nagaki M.: "Tumor necrosis factor a prevents tumor necrosis factor. receptormediated mouse hepatocyte apoptosis but not Fas-mediated apoptosis : role of NF-kB"Hepatology. 32. 1272-1279 (2000)

Nagaki M.：“肿瘤坏死因子 a 可以预防肿瘤坏死因子。受体介导的小鼠肝细胞凋亡，但不是 Fas 介导的凋亡：NF-kB 的作用”肝病学。

Nagaki M, Miki K, Kim Y-I, Ishiyama H, Hirahara I, Takahashi H, Sugiyama A, Muto Y, Moriwaki H: "Development and characterization of a hybrid bioartificial liver using primary hepatocytes entrapped in a basement menbrane matrix."Digest Dis Sci. (in press)

Nagaki M、Miki K、Kim Y-I、Ishiyama H、Hirahara I、Takahashi H、Sugiyama A、Muto Y、Moriwaki H：“利用基底膜基质中的原代肝细胞开发和表征混合生物人工肝。”Digest Dis Sci

Sano K, Nagaki M, Sugiyama A, Hatakeyama H, Ohnishi H, Muto Y, Moriwaki H.: "Effects of cytokines on the binding of leukocytes to cultured rat hepatocytes and on the expression of ICAM-1 by hepatocytes."Digest Dis Sci. 44. 796-805 (1999)

Sano K、Nagaki M、Sugiyama A、Hatakeyama H、Ohnishi H、Muto Y、Moriwaki H.：“细胞因子对白细胞与培养的大鼠肝细胞结合以及肝细胞表达 ICAM-1 的影响。”Digest Dis Sci

Nagaki M, Kim YI, Miki K, Ishiyama H, Hirahara I, Iwai H, Noda N, Sugiyama A, Ohnishi H, Moriwaki H, Muto Y.: "Clinical and experimental studies on apheresis and development of a bioartificial liver in fulminant hepatic failure."Jpn J Apheresis. 16. 23-24

Nagaki M、Kim YI、Miki K、Ishiyama H、Hirahara I、Iwai H、Noda N、Sugiyama A、Ohnishi H、Moriwaki H、Muto Y.：“暴发性肝病中单采术和生物人工肝发育的临床和实验研究

Nagaki M.: "Regulation of hepatic genes and liver transcription factors in rat hepatocytes by extracellular matrix" Biochem Biophys Res Commun. 210. 38-43 (1995)

Nagaki M.：“细胞外基质对大鼠肝细胞中肝基因和肝转录因子的调节”Biochem Biophys Res Commun。