Targeted and regulable expression of transgenes in hepatic stellate cells using an adenoviral Cre-loxP system

肝星状细胞(HSCs)负责细胞外基质合成的积聚，导致肝纤维化。该项目的目的是开发一种可调控的腺病毒转导系统，以调节HSC的激活，最终消除HSC产生细胞外基质或促进细胞凋亡。为此，我们构建了两个重组腺病毒系统；一个在特定启动子的控制下表达Cre基因，另一个包含一个由Cre重组酶介导的重组激活的有效表达单位，以去除上游的loxP侧翼的‘Stuffier’序列，从而转录下游的目的基因(Cre-loxP系统)。分析了该Cre-loxP系统作为一种潜在的方法来实现特定基因在HSCs中的表达调控。当胶原1A2基因启动子驱动Cre重组酶在原代静止大鼠肝星状细胞中表达时，可观察到报告基因的表达。然而，在激活的HSC中，胶原启动子有效地推动了Cre重组酶的活性，根据表达情况进行了评估。相反，当胶原启动子在肝细胞中表达时，几乎没有观察到表达。在两种肝纤维化模型中，HSC特异性表达Smad7可显著降低培养上清中I型胶原的表达，减轻肝纤维化程度。这些结果证明了使用Cre/loxP系统进行转录控制的基因治疗在体内改善肝纤维化的潜在效用。

Hepatic stellate cells (HSCs) are responsible for extracellular matrix synthesis accumulation, leading to liver fibrosis. The aim of this project was to develop a regulable adenoviral transduction system in order to modulate HSC activation and ultimately to either abrogate their generation of extracellular matrix or promote apoptosis. For this purpose, we constructed two recombinant adenoviral systems ; one expressing the Cre gene under the control of specific promoters and the other containing a potent expression unit that was activated by Cre recombinase-mediated recombination to remove an upstream LoxP-flanked ‘stuffier' sequence, thereby transcribing the downstream transgene of interest (Cre-loxP system). This Cre-loxP system was analyzed as a potential method to achieve the regulated expression of specific genes in HSCs. When promoter for the collagen 1A2 gene drove Cre recombinase expression in primary quiescent rat HSC, expression of reporter gene was observed. However, in activated HSC, the collagen promoter effectively drove Cre recombinase activity, as assessed by the expression. In contrast, the expression was barely observed when the collagen promoter was expressed in hepatocytes. HSC-specific expression of Smad7 significantly reduced the expression of type I collagen in culture and decreased fibrosis in two liver fibrosis models. These results demonstrate the potential utility of transcriptionally controlled gene therapy using a Cre/loxP system to ameliorate hepatic fibrosis in vivo.