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: 09671254
• 负责人: KATAYAMA Tokitaka
• 金额: $ 1.98万
• 依托单位: Tokai University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671254/
• 关键词: Primary culture; Human hepatocytes; Adenovirus vector; Immortalization; SV40T antigen; Hybrid artificial liver; 初代培養用細胞; 遺伝子導入; 形質転換コロニー; 初代培養肝細胞

Use of primary culture 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 succeeded in transformed conversion resulting in longer cultivation period, 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 vector 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℃ for two hours, then fixed in ethanol after 48 hours of additional cultivation, and immunostained by using antibody to SV40T antigen. Rate of T antigen positive was appr … More oximately 20% in MOI 100, 4% in MOI 10, less than 0.5% in MOI 1, and these results were depend on MOI.2. Adenovirus vector was introduced in human hepatocytes in MOI 100 and plated to 1xl0ィイD16ィエD1 flask(25 cmィイD13ィエD1). After 3-4 weeks, approximately 100/colonies undergoing transformed conversion were produced from 10 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 reproducefavorably 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 layer cultivation method employed this time, hepatocyte function was found to deterorated with time by measuring urea production and tyrosine amino transferase (TAT) activity. I plan to used third degree high dense cultivation to improve this aspect in the future. Less

利用原代肝细胞培养技术研制杂交型人工肝是目前研究的热点。然而，肝细胞的原代培养物在正常培养条件下仅能存活数周。利用腺病毒载体，将SV 40原代基因导入大鼠和绒猴原代培养的肝细胞中。我已经成功蜕变，修炼时间更长，长生化，大规模修炼。利用同样的技术，我尝试了利用人肝细胞制备杂交型人工肝。将E1 A和E1 B基因重组于人腺病毒中并与多个缺失的SV 40一级基因重组产生的腺病毒载体，通过MOI（感染复数）加入人肝细胞原代培养物中，在37℃下培养2小时，然后在额外培养48小时后在乙醇中固定，并通过使用抗SV 40 T抗原的抗体进行免疫染色。T抗原阳性率约为 ...更多信息 MOI 100时氧化苦参碱含量为20%，MOI 10时氧化苦参碱含量为4%，MOI 1时氧化苦参碱含量小于0.5%，且氧化苦参碱含量与MOI有关。将腺病毒载体以MOI 100引入人肝细胞中，并接种到1 × 105 × D16 × D13 × D1烧瓶（25 cm × D13 × D13 × D1）中。3-4周后，从10个细胞产生约100个/菌落进行转化转化。将原代导入SV 40基因的人肝细胞培养12个月以上，细胞大多呈SV 40 T抗原阳性，并可持续增殖，达到永生化。结论：利用腺病毒载体有效地导入SV 40原代基因并转化，可实现大规模培养和永生化，并通过测定尿素生成和酪氨酸氨基转移酶（达特）活性，发现在单层培养方法下，肝细胞功能随时间的推移而下降。我打算以后用三度高密度培养来提高这方面。少