Transplantation of Immortalized Human Fetal Hepatocytes Prevents Acute Liver Failure in 90% Hepatectomized Mice
Transplantation of Immortalized Human Fetal Hepatocytes Prevents Acute Liver Failure in 90% Hepatectomized Mice
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DOI：
10.1016/j.transproceed.2010.01.061
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发表时间：
2010-06-01
影响因子：
0.9
通讯作者：
Wang, X.
中科院分区：
文献类型：
--
作者：
Chen, Y.;Li, J.;Wang, X.
瞄准。本研究旨在探讨人胎肝细胞是否对猴病毒40大T抗原(SV40Tag)介导的永生化具有适应性，以及永生化细胞能否挽救90%肝切除所致的急性肝衰竭小鼠。我们构建了表达不耐热突变SV40Tag的逆转录病毒载体，并将其转移到原代人胎肝细胞中。我们定量检测了永生化细胞对白蛋白和尿素的合成能力，并将其接种到严重联合免疫缺陷(SCID)小鼠的皮下，以评估其致瘤性。永生化细胞也被移植到急性肝功能衰竭小鼠的脾内。经过选择后得到的一个克隆，称为HepCL，是高度分化的，在化学定义的无血清培养基中稳定生长。细胞白蛋白、细胞角蛋白18和细胞角蛋白19免疫细胞化学染色阳性。HepCL细胞对白蛋白和尿素的平均合成效率与未修饰的原代人胎肝细胞相当。HepCL细胞对数生长期的群体倍增时间约为17小时。免疫缺陷小鼠16个月时，HepCL细胞无致瘤性。接受HepCL细胞(G1)和原代人胎肝细胞(G2)的小鼠在90%肝切除后血氨水平显著降低。4组间的配对比较显示，异种移植HepCL(G1)或原代胎肝细胞(G2)显著提高了受体小鼠的存活率。在急性肝功能衰竭的细胞治疗中，HepCL可能是有用的肝功能来源。
Aim. 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.Methods. 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.Results. 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.Conclusions. HepCL may be useful as a source of hepatic function for cell-based therapeutics in acute liver failure.