Construction of Hybrid Artificial Liver with Immortalized Human Hepatocytes

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。通过使用腺病毒载体并将SV 40原代基因导入大鼠和绒猴的原代肝细胞培养物中，我成功地转化了转化，从而导致了更长的培养期、永生化和大量培养。利用同样的技术，我尝试了以人肝细胞为材料制备人工肝。通过MI（感染复数）将在具有多个缺失的SV 40初级基因的人腺病毒中重组E1 A和E1 B基因产生的腺病毒载体加入到人肝细胞的原代培养物中，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抗原阳性率约为 关于我们 MOI 100时氧化苦参碱含量为20%，MOI 10时为4%，MOI 1时小于0.5%，这些结果与MOI有关。将腺病毒载体以MOI 100引入人肝细胞中，并铺板至1 × 10^6/烧瓶（25 cm ^2）。3-4周后，从10个细胞产生约100个/菌落进行转化转化。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，实现细胞的大规模培养和永生化.讨论：采用单层培养方法，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 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 hydrid 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 culture of hepatocytes by MI (multiplicity of infection) and cultivated under 37ﾟC 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 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 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 mesureing urea production and tyrosine amino transferase (TAT) asctivity. I plan to use third degree cultivation to improve this aspect in the future. Less