Construction of Hybrid Artificial Liver with Immortalized Human Hepatocytes

永生化人肝细胞混合人工肝的构建

基本信息

批准号：
12671175
负责人：
INOKUCHI Sadaki
金额：
$ 2.3万
依托单位：
Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

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

使用肝细胞原代培养物开发混合型人工肝脏是一个活跃的研究课题，然而，通过使用腺病毒载体并将SV40原代基因引入大鼠和狨猴原代中，肝细胞的原代培养物只能存活几周。我已经成功地进行了转化，从而延长了培养周期，并进行了大规模培养。方法和结果：1。将多重缺失SV40原代基因的人腺病毒E1A和E1B基因重组产生的腺病毒载体，按MOI（感染复数）添加到人肝细胞原代培养物中，37℃培养2小时，48℃后乙醇固定。额外培养数小时，并使用 SV40T 抗原抗体进行免疫染色，T 抗原阳性率在 MOI 中约为 20%。 100，MOI 10 中为 4%，MOI1 中小于 0.5%，这些结果取决于 MOI。 2. 将腺病毒载体以 MOI 100 引入人肝细胞中，并在 3- 后铺板至 1x10^6 烧瓶（25 cm）。 4周，从10个细胞产生约100个/集落进行转化。 3.引入初级SV40基因的人肝细胞。大量培养超过12个月，细胞大多呈SV40T抗原阳性，继续良好繁殖并实现永生化，并且白蛋白免疫染色定位于这些细胞的细胞质中。结论：通过使用腺病毒载体有效导入SV40初级基因。讨论：在这次采用的单层培养方法下，通过测量尿素产量和肝细胞功能，发现肝细胞功能随着时间的推移而恶化。今后我计划采用三级高密栽培来改善这方面。