A Promoter-Specific TFO Prevents Liver Fibrosis

描述（由申请人提供）：响应损伤，发生进化保守的伤口愈合过程。这种反应，如果出了差错，可能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，并负责引起所涉及器官的结构改变和功能丧失。肝纤维化，如果不检查，可以进展为肝硬化，并对肝脏造成不可逆的损害。ECM的主要成分是I型胶原，在肝纤维化的情况下，其由肝的活化星状细胞合成。在活化时，星状细胞变得更加活跃，因为它们是增殖的，由于α-平滑肌肌动蛋白而收缩，并且合成增加量的I型胶原。本实验室开发了一种三链形成寡脱氧核糖核苷酸（triple-forming oligodeoxyribonucleotide，TFO），它与α 1（I）胶原基因启动子的C1区（-170 ~-141）形成三螺旋结构，抑制转录。此外，我们已经表明，这种TFO抑制大鼠中由施用化学物质二甲基硝基胺（DMN）诱导的肝纤维化。现在，我们希望开发这种抗原TFO作为一种潜在的抗纤维化剂。我们推测，这种TFO选择性地抑制活化的星状细胞中的胶原合成。在这个提议中，我们描述了实验来解决TFO抑制胶原基因转录的机制。TFO是否通过形成三链体或通过阻断导致炎症和星状细胞活化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、肝星状细胞和枯否细胞）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定量肝组织中胶原mRNA的水平。此外，我们将测定肝功能，以评估DMN对肝脏的损害程度和TFO的预防作用。该项目产生的数据可能会导致人类治疗肝纤维化的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.