用于移植的非致瘤性大鼠肝细胞系的构建：通过SV40 T抗原的位点特异性切除逆转肝细胞永生化

Construction of a non-tumorigenic rat hepatocyte cell line for transplantation: reversal of hepatocyte immortalization by site-specific excision of the SV40 T antigen.

作者信息

Cai J, Ito M, Westerman K A, Kobayashi N, Leboulch P, Fox I J

机构信息

Department of Surgery, University of Nebraska Medical Center, Omaha, USA.

出版信息

J Hepatol. 2000 Nov;33(5):701-8. doi: 10.1016/s0168-8278(00)80299-8.

DOI:10.1016/s0168-8278(00)80299-8

PMID:11097476

Abstract

BACKGROUND/AIMS: Hepatocytes immortalized with a temperature-sensitive SV40 large T antigen (SV40Tag) function as well as primary hepatocytes following transplantation to reverse hepatic encephalopathy and improve survival in rodents with liver failure. The continued presence of SV40Tag in the conditionally immortalized hepatocytes may increase the risk of malignant tumor growth in transplant recipients.

METHODS

We immortalized hepatocytes using a recombinant retrovirus containing the gene encoding SV40Tag flanked by loxP recombination target sites. Excision of SV40Tag from immortalized cells could then be accomplished by site-specific recombination with Cre-recombinase.

RESULTS

Cells immortalized with this recombinant virus expressed SV40Tag and doubled in number every 48 h. After excision of the gene encoding SV40Tag with Cre-recombinase, cells stopped growing, DNA synthesis fell by 90%, and production of liver-specific mRNAs was either increased or became newly detectable. In addition, the morphology and epithelial cell polarity of the cells became more characteristic of differentiated hepatocytes. To determine their malignant potential, immortalized hepatocytes were transfected to express a second oncogene, activated H-ras. SV40Tag+/H-ras+-immortalized cells were capable of anchorage-independent growth and developed into tumors when injected in severe combined immunodeficiency mice. While Cre-recombinase delivery by recombinant adenovirus infection was not 100% efficient, when SV40Tag excision occurred anchorage-independent growth stopped and tumor formation in immunodeficient mice was abolished. Immortalized hepatocytes also contained the gene encoding herpes simplex virus thymidine kinase and treatment with ganciclovir produced complete regression of established tumors in mice.

CONCLUSIONS

These studies extend previous work that indicates that a transplantable hepatocyte cell line could be developed for clinical use.

摘要

背景/目的：用温度敏感型猿猴病毒40大T抗原（SV40Tag）永生化的肝细胞在移植后发挥的功能与原代肝细胞相似，可逆转肝性脑病并提高肝功能衰竭啮齿动物的存活率。条件永生化肝细胞中SV40Tag的持续存在可能会增加移植受者发生恶性肿瘤生长的风险。

方法

我们使用一种重组逆转录病毒使肝细胞永生化，该重组逆转录病毒含有编码两侧带有loxP重组靶位点的SV40Tag的基因。然后可通过与Cre重组酶进行位点特异性重组从永生化细胞中切除SV40Tag。

结果

用这种重组病毒永生化的细胞表达SV40Tag，每48小时数量翻倍。用Cre重组酶切除编码SV40Tag的基因后，细胞停止生长，DNA合成下降90%，肝脏特异性mRNA的产生增加或新可检测到。此外，细胞的形态和上皮细胞极性更具分化肝细胞的特征。为了确定其恶性潜能，将永生化肝细胞转染以表达第二个癌基因——激活的H-ras。SV40Tag+/H-ras+永生化细胞能够在无锚定条件下生长，注射到严重联合免疫缺陷小鼠体内时会发展成肿瘤。虽然通过重组腺病毒感染递送Cre重组酶的效率并非100%，但当发生SV40Tag切除时，无锚定生长停止，免疫缺陷小鼠中的肿瘤形成被消除。永生化肝细胞还含有编码单纯疱疹病毒胸苷激酶的基因，用更昔洛韦治疗可使小鼠体内已形成的肿瘤完全消退。