A Promoter-Specific TFO Prevents Liver Fibrosis

描述（由申请人提供）：在对损伤的反应中，发生了一个进化保守的伤口愈合过程。这种反应，如果出错，会导致一种被称为纤维化的病理过程，这是由于细胞外基质（ECM）的异常积累，并导致相关器官的结构改变和功能丧失。肝纤维化如果不加以检查，就会发展成肝硬化，对肝脏造成不可逆转的损害。ECM的主要成分是I型胶原，在肝纤维化的情况下，它是由活化的肝星状细胞合成的。激活后，星状细胞变得更加活跃，因为它们是增殖的，由于平滑肌肌动蛋白而收缩，并合成更多的I型胶原蛋白。在我们的实验室中，我们开发了一种三联体形成的寡脱氧核糖核苷酸（TFO），它与a1(I)胶原基因启动子的C1区（-170至-141）形成三螺旋结构，并抑制转录。此外，我们已经证明，这种TFO抑制大鼠肝纤维化，由化学物质二甲基-硝基氨基（DMN）的管理诱导。现在我们想开发这种抗原TFO作为一种潜在的抗纤维化药物。我们假设这种TFO选择性地抑制活化星状细胞中的胶原合成。在本提案中，我们描述了解决TFO抑制胶原基因转录机制的实验。TFO是否通过形成三联体或通过阻断导致星状细胞炎症和激活的事件来阻断转录将被研究。已经提出了开发最有效的TFO并研究其毒性、稳定性和生物分布的实验。此外，还将研究大鼠不同组织和不同细胞类型（如肝细胞、星状细胞和库普弗细胞）的摄取情况。在这些研究中，我们将使用组织化学方法来监测纤维化，免疫组织化学方法来检查毒性和炎症，以及各种生化和核酸杂交技术来量化肝组织中胶原mRNA的水平。此外，我们将检测肝功能，以评估DMN对肝脏的损害程度和TFO的预防程度。该项目产生的数据可能会导致用于治疗肝纤维化的人体I期试验。

DESCRIPTION (provided by applicant): In response to injury, an evolutionarily conserved wound healing process occurs. This response, if gone awry, can result in a pathologic process known as fibrosis, which is due to abnormal accumulation of extra cellular matrix (ECM) and is responsible for causing structural alterations and loss of function of the involved organ. Hepatic fibrosis, if not checked, can progress into cirrhosis and cause irreversible damage to the liver. The main component of the ECM is type I collagen, which, in the case of hepatic fibrosis, is synthesized by the activated stellate cells of the liver. Upon activation, stellate cells become more active in that they are proliferative, contractile due to a -smooth muscle actin and synthesize increased amounts of type I collagen. In our laboratory we have developed a triplex-forming oligodeoxyribonueleotide (TFO), which forms a triple helix structure with the C1 region (-170 to -141) of the a1(I) collagen gene promoter and inhibits transcription. Further we have shown that this TFO inhibits liver fibrosis, induced by administration of the chemical, dimethyl-nitorosamine (DMN), in rats. Now we would like to develop this antigene TFO as a potential antifibrotic agent. We hypothesize that this TFO selectively inhibits collagen synthesis in activated stellate cells. In this proposal, we describe experiments to address the mechanism by which the TFO inhibits collagen gene transcription. Whether the TFO blocks transcription by forming triplexes or by blocking events that lead to inflammation and activation of stellate cells will be studied. Experiments to develop a most efficacious TFO and to study its toxicity, stability, and biodistribution have been proposed. Further, the uptake by different tissues in rats and by different cell types, such as hepatocytes, stellate and Kupffer cells of the liver will also be studied. In these studies we will be using histochemical methods to monitor fibrosis, immunohistochemical methods to examine toxicity and inflammation and various biochemical and nucleic acid hybridization techniques to quantitate the levels of collagen mRNA in the liver tissues. In addition, we will assay for liver function to assess the extent of damage to the liver by the DMN and prevention by the TFO. The data generated from this project may lead to Phase I trials in humans for the treatment of liver fibrosis.