Construction of Hybrid Artificial Liver with Immortalized Human Hepatocytes

利用肝细胞原代培养培育杂交人工肝是目前研究的热点。然而，原代培养的肝细胞在正常培养条件下仅能存活数周。我利用腺病毒载体，将SV40原代基因导入大鼠和狨猴肝细胞原代培养中，成功实现了转化转化，延长了培养周期，实现了永生化，实现了大规模培养。利用同样的技术，我尝试用人类肝细胞制造出混合人工肝脏。方法与结果：将多缺失SV40原代基因的人腺病毒E1A和E1B基因重组得到的腺病毒载体，通过MI （multiplicity of infection）法加入人肝细胞原代培养中，在37℃下培养2 h，再在乙醇中固定48 h，用SV40T抗原抗体进行免疫染色。MOI 100的T抗原阳性率约为20%，MOI 10的阳性率为4%，MOI 1的阳性率小于0.5%，这些结果与MOI 2有关。将腺病毒载体引入人肝细胞moi100中，并镀于1x10^6/瓶（25cm^2）。3-4周后，10个细胞转化成约100个/菌落。引入原代SV40基因的人肝细胞作为一个整体培养超过12个月，大多数SV40T抗原阳性的细胞继续良好地繁殖并实现了永生。同时用白蛋白免疫染色法对这些细胞的细胞质进行定位。结论：利用腺病毒载体可以高效地导入SV40原代基因并进行转化转化，实现了SV40的大规模培养和永生化。讨论：在本次采用的单层培养法下，通过测定尿素产量和酪氨酸氨基转移酶（TAT）活性，发现肝细胞功能随着时间的推移而恶化。我计划在未来用三度培养来改善这方面。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