Construction of Hybrid Artificial Liver with Immortalized Human Hepatocytes

肝细胞原代培养在混合型人工肝开发中的应用是一个活跃的研究课题。然而，在正常培养条件下，肝细胞的原代培养仅能存活几周。利用腺病毒载体，将SV40原代基因导入大鼠和猴肝细胞原代培养中，成功地实现了转化，延长了培养周期，实现了永生化和规模化培养。方法与结果：1.将多个缺失SV40基因的腺病毒E1A、E1B基因重组到原代培养的人肝细胞中，加入到原代培养的人肝细胞中，在37ﾟC下培养2小时，48小时后固定在乙醇中，用SV40T抗体进行免疫染色。T抗原阳性率接近…MOI100最高可达20%，MOI10最高可达4%，MOI1最低可达0.5%，且这些结果依赖于MOI2。将腺病毒载体导入人肝细胞，接种于1×10~(-6)/瓶(25 cm~2)。3-4周后，从10个细胞中获得约100个转化克隆。转人SV40基因的人肝细胞批量培养12个月以上，细胞大多为SV40T抗原阳性，继续增殖，达到永生化。白蛋白免疫染色定位于细胞胞浆。结论：用腺病毒载体高效导入SV40初级基因并转化，可大量培养和永生化。讨论：在本次采用的单层培养方法下，通过测定尿素产量和酪氨酸氨基转移酶(TAT)活性，发现肝细胞功能随时间而恶化。我计划在未来用三级修养来改善这方面。较少

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