Construction of Hybrid Artificial Liver with Immortalized Human Hepatocytes.

利用原代培养的肝细胞研制杂交型人工肝是一个活跃的研究课题。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。通过使用腺病毒载体，将SV 40原代基因导入大鼠和绒猴原代培养的肝细胞中，获得了转化转化，从而延长了培养周期，实现了永生化和大量培养。通过使用相同的技术，我尝试了使用人肝细胞生产杂交型人工肝。方法和结果：1.将E1 A和E1 B基因重组于人腺病毒SV 40多基因缺失的腺病毒载体中，以MOI法加入人原代肝细胞培养物中，（感染复数），并在37 ℃下培养2小时，然后在额外培养48小时后在乙醇中固定，并通过使用针对SV 40 T抗原的抗体进行免疫染色。T抗原阳性率 ...更多信息 在MOI 100时，病毒滴度为20%，MOI 10时为4%，MOI 1时小于0.5%，且这些结果依赖于MOI。2.将腺病毒载体以MOI 100导入人肝细胞中，并以1 × 10^6/瓶（25 cm ^2）接种。3-4周后，从10^6个细胞中产生了约100个进行转化转化的集落。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，可以有效地将SV 40原代基因导入细胞，并转化转化，实现细胞的大量培养和永生化.讨论：在单层培养方法下，通过测定尿素生成和酪氨酸氨基转移酶（达特）活性，发现肝细胞功能随时间的推移而恶化.我打算以后用三度修炼来提高这方面。少

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