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

肝星状细胞（hepatic stellate cells，HSC）参与细胞外基质的合成和积累，导致肝纤维化.该项目的目的是开发一种可调节的腺病毒转导系统，以调节HSC活化，并最终消除其细胞外基质的产生或促进凋亡。为此，我们构建了两个重组腺病毒系统;一个在特异性启动子的控制下表达Cre基因，另一个包含有效的表达单位，该表达单位通过Cre重组酶介导的重组激活以去除上游LoxP侧翼的“填充物”序列，从而转录下游目的转基因（Cre-loxP系统）。该Cre-loxP系统被分析为实现HSC中特定基因的调控表达的潜在方法。当胶原1A 2基因的启动子驱动Cre重组酶在原代静止期大鼠HSC中表达时，观察到报告基因的表达。然而，在活化的HSC中，胶原启动子有效地驱动Cre重组酶活性，如通过表达评估的。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。HSC特异性表达Smad 7显著降低了培养物中I型胶原的表达，并减少了两种肝纤维化模型中的纤维化。这些结果证明了使用Cre/loxP系统的转录控制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.