Weber-Benarous A, Decaux J F, Bennoun M, Allemand I, Briand P, Kahn A
INSERM U. 129, Institut Cochin de Génétique Moléculaire, Paris, France.
Exp Cell Res. 1993 Mar;205(1):91-100. doi: 10.1006/excr.1993.1062.
Cultured adult rodent hepatocytes are extensively used as a model system for gene transfer in vitro. In the present study, we examined the influence differentiation status and growth capacity of the hepatocytes on their infectivity in vitro by a retroviral vector. These parameters were initially studied in primary cultures of rat hepatocytes transduced with an ecotropic retroviral vector containing Escherichia coli beta-galactosidase. However, significant differences observed in the infectivity of hepatocytes from 12-day-old and adult rats led us to also examine hepatocytes from a transgenic mouse strain in which the SV40 large T antigen is fused to the regulatory sequences of the human anti-thrombin III gene. The large T antigen is expressed in the liver and these mice develop hepatoma within 7 months. A comparison of infectivity of hepatocytes from normal and transgenic mice of different ages indicated that in contrast to previous reports, hepatocytes which express differentiated functions during the first week of culture can still be efficiently infected by retroviral vectors. Optimal infection was observed between the second and fourth day of culture and does not appear to be due to transient cell dedifferentiation, but is more likely due to transient mitotic activity of mice cells since the role of growth factors seems crucial for infection. The peak of infection did not appear to correspond to transient cell dedifferentiation. We also found differences of infectivity between hepatocytes from normal and transgenic mice of different ages. Such differences are correlated with differences in in vitro BrdU incorporation, which was used to determine the proportion of dividing hepatocytes. These results indicate that the efficiency of infectivity of hepatocytes by recombinant retrovirus is probably related to their normal proliferative potential and not to some dedifferentiated stage. Hence these findings provide a model for efficient gene transfer in differentiated cells and suggest an approach for studies of liver-specific gene regulation and for somatic gene therapy of metabolic diseases as well.
培养的成年啮齿动物肝细胞被广泛用作体外基因转移的模型系统。在本研究中，我们通过逆转录病毒载体研究了肝细胞的分化状态和生长能力对其体外感染性的影响。这些参数最初是在转导了含大肠杆菌β-半乳糖苷酶的嗜亲性逆转录病毒载体的大鼠肝细胞原代培养物中进行研究的。然而，在12日龄大鼠和成年大鼠肝细胞的感染性中观察到的显著差异，促使我们也研究了一种转基因小鼠品系的肝细胞，在该品系中，SV40大T抗原与人抗凝血酶III基因的调控序列融合。大T抗原在肝脏中表达，这些小鼠在7个月内会发生肝癌。对不同年龄的正常小鼠和转基因小鼠肝细胞感染性的比较表明，与先前的报道相反，在培养第一周表达分化功能的肝细胞仍可被逆转录病毒载体有效感染。在培养的第二天至第四天观察到最佳感染，这似乎不是由于短暂的细胞去分化，而更可能是由于小鼠细胞的短暂有丝分裂活性，因为生长因子的作用似乎对感染至关重要。感染高峰似乎与短暂的细胞去分化不对应。我们还发现不同年龄的正常小鼠和转基因小鼠肝细胞之间存在感染性差异。这种差异与体外BrdU掺入的差异相关，BrdU掺入用于确定分裂肝细胞的比例。这些结果表明，重组逆转录病毒对肝细胞的感染效率可能与其正常增殖潜力有关，而不是与某些去分化阶段有关。因此，这些发现为分化细胞中的有效基因转移提供了一个模型，并为肝脏特异性基因调控研究以及代谢疾病的体细胞基因治疗也提供了一种方法。
