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。用腺病毒载体将SV40主要基因导入大鼠和绒猴原代肝细胞培养中。我成功地进行了转化，使修养周期更长、教化更长、修养更大。通过使用相同的技术，我尝试使用人类肝细胞来制造混合型人工肝。方法和结果：1.将E1a和E1b基因在多个缺失的SV40腺病毒中重组而成的重组腺病毒载体，经MOI(多重感染)加入人原代培养的肝细胞中，37℃培养2小时后，再培养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高于20%，MOI10大于4%，MOI1小于0.5%，这些结果与MOI有关。2.将腺病毒载体导入人肝细胞，接种于1×10~(-6)培养瓶(25 Cm)。3-4周后，从10个细胞中产生了大约100个转化克隆。3.将SV40原代基因导入人肝细胞，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中。结论：用腺病毒载体高效导入SV40主基因并转化为目的基因，可实现大批量培养和永生化。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。我计划在未来采用三度高密度栽培来改善这一方面。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 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