A Promoter-Specific TFO Prevents Liver Fibrosis

启动子特异性 TFO 可预防肝纤维化

• 批准号: 6728834
• 负责人: RAMAREDDY V GUNTAKA
• 金额: $ 28.91万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6728834
• 关键词: antifibrinolytic agents; collagen; deoxyribonucleotides; drug adverse effect; fibrosis; genetic promoter element; genetic transcription; histology; immunocytochemistry; in situ hybridization; inflammation; laboratory rat; liver cirrhosis; liver disorder; liver pharmacology; oligonucleotides; pharmacokinetics; western blottings

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.

描述（由申请人提供）：响应伤害，发生了进化保守的伤口愈合过程。如果出现问题，这种反应可能会导致一种称为纤维化的病理过程，这是由于异常细胞基质（ECM）的异常积累，并负责导致结构改变和相关器官的功能丧失。肝纤维化，如果未检查，可以发展为肝硬化并对肝脏造成不可逆转的损害。 ECM的主要成分是I型胶原蛋白，在肝纤维化的情况下，肝脏的活化星状细胞合成。激活后，星状细胞变得更加活跃，因为它们具有增殖，由于肌肉肌动蛋白 - 肌肉肌动蛋白 - 肌肉肌动蛋白的肌肉肌动蛋白增强，并使I型胶原蛋白的量增加。在我们的实验室中，我们开发了形成三氧化三纤维纤维纤维苷（TFO），该寡头纤维纤维苷（TFO）形成了三重螺旋结构，其A1（I）胶原基因启动子的C1区（-170至-141）形成了三重螺旋结构，并抑制了转录。此外，我们已经表明，该TFO抑制了大鼠的化学，二甲基氯糖胺（DMN）诱导的肝纤维化。现在，我们想开发这种抗基因TFO作为潜在的抗纤维化剂。我们假设该TFO选择性抑制活化星状细胞中的胶原蛋白合成。在此提案中，我们描述了实验，以解决TFO抑制胶原蛋白基因转录的机制。 TFO是否通过形成三环体或阻止导致炎症和激活星状细胞的事件来阻止转录。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，还将研究大鼠和不同细胞类型的不同组织的摄取，例如肝细胞，肝脏和库普弗细胞的肝脏。在这些研究中，我们将使用组织化学方法来监测纤维化，免疫组织化学方法检查毒性​​和炎症以及各种生化和核酸杂交技术，以定量肝组织中胶原蛋白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预防。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。