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: 12671175
• 负责人: INOKUCHI Sadaki
• 金额: $ 2.3万
• 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: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671175/
• 关键词: Primary culture; Adenovirus vector; Gene transfer; SV40Tantigen; Transformation colony; Immortalization; Hybrid artificial liver; 初代培養ヒト肝細胞

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 adenovims 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, moralization, 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 approxim … More ately 20% in MOI 100, 4% in MOI 10, less than 0.5% in MOI1, and these results were depend on MOI. 2. Adenovirus vector was introduced in human hepatocytes in MOI 100 and plated to 1x10^6 flask(25 cm). 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 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 layer cultivation method employed this time, hepatocyte function was found to deteriorated 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。我已经成功的转化，导致更长的修炼时间，道德化，大量的修炼。通过使用相同的技术，我已经尝试通过使用人肝细胞来生产杂交人工肝。方法与结果：1.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100%， ...更多信息 MOI 100时，接种率为20%，MOI 10时为4%，MOI 1时小于0.5%，且接种率与MOI有关。2.将腺病毒载体以MOI 100引入人肝细胞中，并接种到1 × 10^6烧瓶（25 cm）中。3-4周后，从10个细胞产生约100个/菌落进行转化转化。3.将导入原代SV 40基因的人肝细胞作为一个整体培养超过12个月，并且细胞大多数为SV 40 T抗原阳性，继续有利地繁殖并实现永生化。结论：利用腺病毒载体可有效地将SV 40原代基因导入细胞并转化，实现细胞的大量培养和永生化。讨论内容：在单层培养方法下，通过测量尿素产生和酪氨酸氨基转移酶（达特）活性，发现肝细胞功能随时间而恶化。我打算以后用三度高密度培养来提高这方面。少