CELL-SELECTIVE EXPRESSION OF FIBROTIC GENE PATHWAYS

纤维化基因途径的细胞选择性表达

基本信息

批准号：
7315442
负责人：
Nestor F Gonzalez-Cadavid
金额：
$ 20.81万
依托单位：
LUNDQUIST INSTITUTE FOR BIOMEDICAL INNOVATION AT HARBOR-UCLA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-07 至 2009-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7315442
关键词：
Abbreviations Actins Age Aging Animal Model Arterial Medias Arteries Arteriosclerosis Biological Assay Blood Vessels Body of uterus Cell Communication Cell Nucleus Cells Collagen Collagen Fiber Condition Confocal Microscopy Control Groups Corpora Cavernosa Cultured Cells DAPI DNA Microarray Chip DNA Microarray format Data Deposition Detection Development Diabetes Mellitus Disease Dorsal End Point Erectile dysfunction Fibroblasts Fibrosis Fluorescence Functional disorder Gene Expression Gene Expression Alteration Genes Goals Green Fluorescent Proteins Hydroxyproline Image Analysis Immunofluorescence Immunologic Immunohistochemistry Implant Injury Label Lead Microspheres Molecular Molecular Profiling Molecular Target Myofibroblast Names Nitric Oxide Synthase Numbers Organ Pathway interactions Pattern Peyronie Disease Polymerase Chain Reaction Process Public Health RNA Rattus Reactive Oxygen Species Role Saline Smooth Muscle Myocytes Specificity Stem cells Time Tissues Transforming Growth Factors Transplantation Tunica Adventitia Tunica Albuginea Vascular Diseases Vascular System Veno-occlusive Vimentin Week Western Blotting adult stem cell age related aged arterial stiffness cell type day implantation in vivo laser capture microdissection male novel paracrine penis promoter research study selective expression

项目摘要

DESCRIPTION (provided by applicant): This project aims to clarify some aspects that would help to define a common molecular profile of vascular fibrosis, that in the penis causes Peyronie's disease (PD) and aging-related vasculogenic erectile dysfunction, and that in the arteries leads to aging-related arteriosclerosis and arterial stiffness, in order to detect cellular and molecular targets of antifibrotic therapy. We will investigate: a) the cell specificity of the alterations of gene expression occurring in penile and vascular fibrosis, clarifying the role of smooth muscle cells (SMC) and myofibroblasts in these processes; b) whether the SMC in the penile trabecular tissue are responsible for excessive collagen synthesis; c) the ontogenic relationship of myofibroblasts and SMC, and d) the effect of cell to cell interactions on their differentiation leading to excessive collagen deposition. In Aim 1, we will determine gene expression patterns in SMC and myofibroblasts in penile and vascular fibrosis. Male rats in groups: 1) "Young"; 2) "Aged"; 3) "PD-like plaque"; and 4) "Normal tunica", will receive in the corpora cavernosa or tunica albuginea an adenoviral collagen I promoter-green fluorescent protein (ColP-gfp) construct. Gfp be detected by dual fluorescence at 6 days in: a) SMC, and possibly myofibroblasts in the corpora and PDA, and: b) fibroblasts and myofibroblasts in the PDA adventitia or the tunica. Some of these cells will be dissected by laser capture microdissection (LCM). In Aim 2 we will determine the in vivo differentiation of penile fibroblasts and stem cells into SMC and myofibroblasts, and of myofibroblasts into SMC, active in collagen synthesis. Penile cultures from the rat tunica (fibroblasts) and the PD plaque (myofibroblasts) will be used as such, or after Sca1+ selection for stem cells, transfected with the ColP-gfp construct, and labeled with DAPI. Implantation will be for 1 week into: A: corpora cavernosa of old rats, with tunical: 1) fibroblasts; or 2) stem cells; and B: TGF¿1-induced PD-like plaque in the tunica, with: 3) tunical fibroblasts; 4) PD-myofibroblasts; or 5) tunical stem cells. End-points will be: A) LCM in tissue sections, with immunofluorescence for gfp, fibroblasts (vimentin), myofibroblasts/SMC (ASMA) and SMC (smoothelin), followed by DNA microarrays for gene expression profiles; B) fibrosis by immunohistochemistry/QIA (quantitative image analysis), RT/real time PCR, and western blots, and assays for hydroxyproline; B) for implanted DAPI+ cells: by QIA fluorescence/dual confocal microscopy for cell markers. Tissue fibrosis is an excessive deposition of collagen fibers, often accompanied by the loss of cellular mass, that occurs in most organs with aging, diabetes, injury, toxic insults, and other conditions, and that severely impairs the function of those organs and is a key factor in various diseases. Using rat animal models and cell cultures, we aim to clarify aspects that would help to define whether there is a common molecular and cellular profile of fibrosis, that in penile tissues causes Peyronie's disease (PD) and erectile dysfunction, and that in the arterial wall leads to arteriosclerosis. We will focus on studying the role of cells named fibroblasts, smooth muscle cells, and myofibroblasts, as well as adult stem cells, in these processes, and whether some of these cells can interconvert into each other. We expect that our studies would lead to the detection of novel targets of antifibrotic therapy for penile and vascular diseases of considerable public health impact.

描述（由申请人提供）：该项目旨在澄清一些方面，有助于定义血管纤维化的常见分子特征，阴茎中的血管纤维化会导致佩罗尼氏病（PD）和与衰老相关的血管源性勃起功能障碍，以及动脉中的血管纤维化导致与衰老相关的动脉硬化和动脉硬化，为了检测抗纤维化治疗的细胞和分子靶点，我们将研究：a）改变的细胞特异性。阴茎和血管纤维化中发生的基因表达，阐明平滑肌细胞 (SMC) 和肌成纤维细胞在这些过程中的作用；b) 阴茎小梁组织中的 SMC 是否负责过度胶原蛋白合成；c) 肌成纤维细胞和肌成纤维细胞的个体关系； SMC，d) 细胞间相互作用对其分化的影响，导致胶原蛋白过度沉积。在目标 1 中，我们将确定 SMC 和肌成纤维细胞中的基因表达模式。阴茎和血管纤维化组：1)“年轻”；2)“老年”；3)“PD样斑块”；和4)“正常被膜”，将在海绵体或白膜中接受腺病毒。胶原蛋白 I 启动子-绿色荧光蛋白 (ColP-gfp) 构建体可在 6 天时通过双荧光检测：a) SMC，并且可能还可以检测到 Gfp。体和 PDA 中的肌成纤维细胞，以及： b) PDA 外膜或膜中的成纤维细胞和肌成纤维细胞其中一些细胞将通过激光捕获显微切割 (LCM) 进行解剖。在目标 2 中，我们将确定阴茎成纤维细胞的体内分化。和干细胞转化为 SMC 和肌成纤维细胞，以及将肌成纤维细胞转化为 SMC，在阴茎培养物中具有活性。来自大鼠被膜（成纤维细胞）和 PD 斑块（肌成纤维细胞）的细胞将直接使用，或者在选择 Sca1+ 干细胞后，用 ColP-gfp 构建体转染，并用 DAPI 标记，植入：A 中 1 周。：老年大鼠海绵体，带外膜：1) 成纤维细胞；或 2) 干细胞；B：TGF¿ 1) 皮膜中诱导的 PD 样斑块，其中：3) 皮膜成纤维细胞；4) PD 肌成纤维细胞；或 5) 皮膜干细胞：A) 组织切片中的 LCM，对 gfp、成纤维细胞进行免疫荧光检测。 (vimentin)、肌成纤维细胞/SMC (ASMA) 和 SMC (smoothelin)，然后是 DNA 微阵列基因表达谱；B) 通过免疫组织化学/QIA（定量图像分析）、RT/实时 PCR 和蛋白质印迹以及羟脯氨酸测定来检测纤维化；B) 对于植入的 DAPI+ 细胞：通过 QIA 荧光/双共聚焦显微镜检查细胞标记。组织纤维化是胶原纤维的过度沉积，通常伴随着细胞质量的损失，发生在大多数因衰老、糖尿病、损伤、中毒性损伤和其他原因而导致的器官中。条件，并严重损害这些器官的功能，是各种疾病的关键因素，我们的目的是利用大鼠动物模型和细胞培养物来阐明有助于确定纤维化是否存在共同分子和细胞特征的方面。阴茎组织中的细胞会导致佩罗尼氏病 (PD) 和勃起功能障碍，动脉壁中的细胞会导致动脉硬化。我们将重点研究成纤维细胞、平滑肌细胞和动脉硬化细胞的作用。肌成纤维细胞以及成体干细胞在这些过程中的作用，以及这些细胞中的一些是否可以相互转化，我们希望我们的研究能够发现抗纤维化治疗的新靶点，以治疗影响公共健康的阴茎和血管疾病。影响。