Conditionally immortalized mouse hepatocytes for use in liver gene therapy
Conditionally immortalized mouse hepatocytes for use in liver gene therapy
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DOI：
10.1046/j.1440-1746.2000.02328.x
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发表时间：
2000-11-01
影响因子：
4.1
通讯作者：
Whitehead, RH
中科院分区：
文献类型：
--
作者：
Allen, KJ;Reyes, R;Whitehead, RH
背景和目的：肝细胞用于基因治疗的用途由于难以维持和改变培养物中的原代肝细胞而受到限制。已开发出一种条件永生化小鼠肝细胞系，该细胞系可以在允许的温度（33°C）下无限期传代，但在体外非允许的温度（39°C）下无法增殖并死亡。方法：使用改良的两步胶原酶灌注，从携带耐热 SV40 Large T 基因的 6 周龄雄性转基因小鼠（“immortomouse”）中收获肝细胞方法，33℃连续传代1年以上。为了评估永生肝细胞移植和填充小鼠肝脏的能力，将细胞通过门静脉（n = 10）或经过（n = 2）和未（n = 2）部分肝切除的脾脏注入部分肝切除的同系小鼠中。使用报告基因增强型绿色荧光蛋白（EGFP）评估转染永生肝细胞的能力。结果：培养物中的所有永生肝细胞的肝细胞标记物白蛋白、AFP、CK8和CK18的免疫组织化学染色呈阳性。在早期培养物中，一部分细胞的胆道上皮标记物 CK7 和 CK19 也呈强烈染色。晚期细胞培养物 M2PK 和 CK7 呈阴性，并用抗 CK19 抗体进行不同染色。细胞转移至不允许的温度39℃，在体外1周内停止增殖并死亡。通过 PCR 和 Southern blot 分析，在肝细胞移植后 2 周内，在所有术后小鼠的肝脏中检测到大 T DNA。永生肝细胞很容易转染报告基因。结论：永生肝细胞转移到肝脏后可以在体内存活，可作为肝脏基因治疗的模型。 (C) 2000 布莱克韦尔科学亚洲有限公司
Background and aims: The use of hepatocytes for gene therapy is limited by the difficulty of maintaining and altering primary liver cells in culture. A conditionally immortalized mouse hepatocyte cell line has been developed which can be passaged indefinitely at the permissive temperature (33 degreesC), but fails to proliferate and dies at the non-permissive temperature (39 degreesC) in vitro.Methods: Hepatocytes were harvested from a 6 week-old male transgenic mouse ('immortomouse') carrying a thermolabile SV40 Large T gene, using a modified two-step collagenase perfusion method, and serially passaged at 33 degreesC for more than 1 year. To assess the ability of immortohepatocytes to engraft and populate mouse liver, cells were infused into partially hepatectomized congenic mice via the portal vein (n = 10) or the spleen with (n = 2) and without (n = 2) partial hepatectomy. The ability to transfect immortohepatocytes was assessed using the reporter gene enhanced green fluorescent protein (EGFP).Results: All immortohepatocytes in culture stained positive by immunohistochemistry for the hepatocyte markers albumin, AFP, CK8 and CK18. In early cultures a proportion of cells also stained strongly for the biliary epithelial markers CK7 and CK19. Late cell cultures were negative for M2PK and CK7 and stained variably with anti-CK19 antibodies. Cells transferred to the non-permissive temperature of 39 degreesC ceased proliferation and died within 1 week in vitro. Large T DNA was detected in the liver of all postoperative mice up to 2 weeks post-hepatocellular transplantation via PCR and Southern blot analysis. The immortohepatocytes were easily transfected with a reporter gene.Conclusions: Immortohepatocytes can survive in vivo after transfer to liver, and will be useful as a model for hepatic gene therapy. (C) 2000 Blackwell Science Asia Pty Ltd.