Construction of Hybrid Artificial Liver with Immortalized Human Hepatocytes.

永生化人肝细胞混合人工肝的构建。

• 批准号: 05807109
• 负责人: KATAYAMA Tokitaka
• 金额: $ 1.09万
• 依托单位: Tokai University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05807109/
• 关键词: Primary culture; Human hepatocytes; Adenovirus vector; Immortalization; SV40 T antigen; Hybrid artificial liver; ハイブリッド型人工肝臓; 初代培養肝細胞; 不死化ヒト肝細胞

Use of primary cultures of hepatocytes in developing hybrid artificial liver is a topic of active research. However, primary cultures of hepatocytes remain viable for only a few weeks under normal cultivation conditions. By using adenovirus vector and introducing SV40 primary gene into rat and marmoset primary cultures of hepatocytes, I have suceeded in transformed conversion resulting in longer cultivation peroid, immortalization, and massive cultivation. By using the same technique, I have attempted to produce hybrid artificial liver by using human hepatocytes.Methods and Results : 1.Adenovirus vecotr produced by recombination of E1A and E1B genes in human adenovirus with multiple deletion SV40 primary genes, was added to human primary cultures of hepatocytes by MOI (multiplicity of infection) and cultivated under 37ﾟC for two hours, then fixed in ethanol after 48 hours og additional cultivation, and immunostained by using antibody to SV40T antigen. Rate of T antigen positive was app … More roximately 20% in MOI 100, 4% in MOI 10, less than 0.5% in MOI1 ; and these results were dependent on MOI.2.Adenovirus vector was introduced in human hepatocytes in MOI 100 and plated to 1x10^6/flask (25cm^2). After 3-4 weeks, approximately 100/colonies undergoing transformed conversion were produced from 10^6 cells. 3.Human hepatocytes introducing with primary SV40 genes were cultivated as a bulk for longer than 12 months and the cells mostly SV40T antigen positive, continued to reproduce favorably and attained immortalization. Also immunostaining with albumin was localization in cytosol of these cells.Conclusion : By using adenovirus vector, efficient introduction of SV40 primary gene and transformed conversion was possible, resulting in massive cultivation and immortalization.Discussion : Under the single layr cultivation method employed this time, hepatocyte function was found to deteriorated with time, by mesuring urea production and tyrosine amino transferase (TAT) activity. I plan to use third degree cultivation to improve this aspect in the future. Less

利用肝细胞原代培养物培育杂交人工肝是一个活跃的研究课题。然而，原代培养的肝细胞在正常培养条件下仅能存活数周。我利用腺病毒载体，将SV40原代基因导入大鼠和狨猴肝细胞原代培养中，成功实现了转化转化，使培养周期延长，永生化，大规模培养。通过同样的技术，我尝试用人类肝细胞制造混合型人工肝脏。方法与结果：将多缺失SV40原代基因的人腺病毒E1A和E1B基因重组制备腺病毒载体，经MOI （multiplicity of infection）法加入人肝细胞原代培养物中，在37 C条件下培养2 h，再在乙醇中固定48 h，用SV40T抗原抗体进行免疫染色。MOI 100组T抗原阳性率约为20%，MOI 10组为4%，MOI1组小于0.5%；这些结果依赖于moi。将腺病毒载体引入人肝细胞moi100中，并镀于1x10^6/瓶（25cm^2）。3-4周后，10^6个细胞进行转化转化，产生约100/菌落。3.引入原代SV40基因的人肝细胞作为一个整体培养超过12个月，大多数SV40T抗原阳性的细胞继续良好地繁殖并实现了永生。同时用白蛋白免疫染色法对这些细胞的细胞质进行定位。结论：利用腺病毒载体可以高效地导入SV40原代基因并进行转化转化，实现了SV40的大规模培养和永生化。讨论：本实验采用单层培养法，通过测定尿素产量和酪氨酸氨基转移酶（TAT）活性，发现肝细胞功能随着时间的推移而恶化。我计划在未来用三度培养来改善这方面。少