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A Promoter-Specific TFO Prevents Liver Fibrosis

启动子特异性 TFO 可预防肝纤维化

基本信息

批准号：
7336384
负责人：
RAMAREDDY V GUNTAKA
金额：
$ 26.86万
依托单位：
UNIVERSITY OF TENNESSEE HEALTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-01-01 至 2009-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7336384
关键词：
Abbreviations Address Amphibia Antisense Oligodeoxyribonucleotides Biochemical Biodistribution Biological Assay Cell Nucleus Cells Chemicals Chronic Cicatrix Cirrhosis Collagen Collagen Gene Collagen Type I Complement component C1s Condition Cytoplasm Cytoskeleton Data Deposition Dose EMSA Electrophoretic Mobility Shift Assay Event Extracellular Matrix Failure Fibroblasts Fibrosis Frequencies Gene Expression Genetic Transcription Granulation Tissue Green Fluorescent Proteins Hepatic Hepatocyte Human Hyperplasia Inflammation Injury KLK3 gene Kupffer Cells Laboratories Lead Liver Liver Fibrosis Mammals Mesenchymal Messenger RNA Methods Modeling Monitor Myofibroblast Natural regeneration Nitrosamines Nucleic Acid Hybridization Nucleotides Oligonucleotides Organ Pathologic Processes Pathway interactions Phase I Clinical Trials Physiological Play Prevention Process Production Rattus Reactive Oxygen Species Relative (related person)Rodent Model Role Route Smooth Muscle Actin Staining Method Stretching Structure Techniques Therapeutic Tissues Toxic effect Transcription Initiation Site Vertebral column Vertebrates Wound Healing antigene cell type in vivo liver function loss of function metabolic abnormality assessment mutant phosphorothioate prevent promoter research study response response to injury stellate cell transcription factor triple helix uptake

项目摘要

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）：响应损伤，发生进化上保守的伤口愈合过程。如果这种反应出现问题，可能会导致称为纤维化的病理过程，这是由于细胞外基质 (ECM) 的异常积累造成的，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控制，可能会发展为肝硬化，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，它是由肝脏激活的星状细胞合成的。激活后，星状细胞变得更加活跃，因为它们因平滑肌肌动蛋白而增殖和收缩，并合成更多量的 I 型胶原蛋白。在我们的实验室中，我们开发了一种三链体寡脱氧核糖核苷酸 (TFO)，它与 a1(I) 胶原蛋白基因启动子的 C1 区域（-170 至 -141）形成三螺旋结构并抑制转录。此外，我们还表明，这种 TFO 可以抑制大鼠体内由化学物质二甲基亚硝胺 (DMN) 诱导的肝纤维化。现在我们希望开发这种反基因 TFO 作为潜在的抗纤维化药物。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。在本提案中，我们描述了一些实验来解决 TFO 抑制胶原蛋白基因转录的机制。将研究 TFO 是否通过形成三链体或通过阻断导致炎症和星状细胞激活的事件来阻断转录。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，还将研究大鼠不同组织和不同细胞类型（例如肝脏的肝细胞、星状细胞和库普弗细胞）的摄取。在这些研究中，我们将使用组织化学方法来监测纤维化，使用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蛋白 mRNA 的水平。此外，我们将检测肝功能，以评估 DMN 对肝脏的损害程度以及 TFO 的预防程度。该项目产生的数据可能会导致治疗肝纤维化的人体一期试验。