Chen Y, Li J, Liu X, Zhao W, Wang Y, Wang X
Institute of Infectious Diseases, Southwest Hospital, Third Military Medical University, Chongqing, China.
Transplant Proc. 2010 Jun;42(5):1907-14. doi: 10.1016/j.transproceed.2010.01.061.
The aim of this study was to investigate whether human fetal hepatocytes are amenable to simian virus 40 large T-antigen (SV40Tag) mediated immortalization and whether the immortalized cells rescue mice with acute liver failure induced by 90% hepatectomy.
We constructed a retroviral vector expressing a thermolabile mutant SV40Tag for transfer into primary human fetal hepatocytes. We quantitatively detected the synthetic ability for albumin and urea by the immortalized cells, which were subcutaneously inoculated into mice with severe combined immunodeficiency (SCID) to evaluate tumorigenzcity. The immortalized cells were also transplanted into the spleens of mice with acute liver failure.
One clone resulting after selection, referred to as HepCL, was highly differentiated, growing steadily in a chemically defined serum-free medium. HepCL cells were positive for albumin, cytokeratin 18, and cytokeratin 19 immunocytochemical staining. The average synthetic efficacies of HepCL cells for albumin and urea were comparable to that of unmodified primary human fetal hepatocytes. The population doubling time of HepCL cells in the logarithmic growth phase was approximately 17 hours. HepCL cells showed no oncogenicity in immunodeficient mice at 16 months. Mice receiving HepCL cells (G1) and primary human fetal hepatocytes (G2) showed significantly lower blood ammonia levels after 90% hepatectomy. Pairwise comparisons between the 4 groups showed that xenotransplantation of HepCL (G1) or primary fetal hepatocytes (G2) significantly improved survivals of recipient mice.
HepCL may be useful as a source of hepatic function for cell-based therapeutics in acute liver failure.
本研究旨在探究人胎儿肝细胞是否适合猿猴病毒40大T抗原（SV40Tag）介导的永生化，以及永生化细胞能否挽救90%肝切除诱导急性肝衰竭的小鼠。
构建表达温度敏感突变型SV40Tag的逆转录病毒载体，用于转染原代人胎儿肝细胞。定量检测永生化细胞白蛋白和尿素的合成能力，将其皮下接种到严重联合免疫缺陷（SCID）小鼠中评估致瘤性。永生化细胞也移植到急性肝衰竭小鼠的脾脏中。
筛选后得到的一个克隆，称为HepCL，高度分化，在化学成分明确的无血清培养基中稳定生长。HepCL细胞白蛋白、细胞角蛋白18和细胞角蛋白19免疫细胞化学染色呈阳性。HepCL细胞白蛋白和尿素的平均合成效率与未修饰的原代人胎儿肝细胞相当。HepCL细胞对数生长期的群体倍增时间约为17小时。HepCL细胞在免疫缺陷小鼠中16个月时无致瘤性。接受HepCL细胞（G1）和原代人胎儿肝细胞（G2）的小鼠在90%肝切除后血氨水平显著降低。4组之间的两两比较显示，HepCL（G1）或原代胎儿肝细胞（G2）的异种移植显著提高了受体小鼠的存活率。
HepCL可能作为急性肝衰竭基于细胞治疗的肝功能来源。
