A Promoter-Specific TFO Prevents Liver Fibrosis

启动子特异性 TFO 可预防肝纤维化

• 批准号: 7169847
• 负责人: RAMAREDDY V GUNTAKA
• 金额: $ 27.41万
• 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/7169847
• 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

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。这种反应如果出​​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星状细胞炎症和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监测纤维化，使用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。