Molecular and Cellular Changes in the Peritoneal Surface Mesothelium during Adhesion Formation

粘连形成过程中腹膜表面间皮的分子和细胞变化

基本信息

批准号：
9051598
负责人：
Jonathan Michael Tsai
金额：
$ 3.55万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-01-01 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9051598
关键词：
Abdomen Actins Address Adhesions Aftercare Antibodies Area Blocking Antibodies CD44 Antigens Cells Clustered Regularly Interspaced Short Palindromic Repeats Coagulation Process Color Complementary DNA Complication Data Disease Elements Etiology Female infertility Fibrin Fluorescence-Activated Cell Sorting Gene Targeting Genes Goals Guide RNA Hour Hypertrophy Image Immunofluorescence Immunologic Immunohistochemistry Inflammatory Injury Interferon Type II Intervention Intestinal Obstruction Ischemia Knock-in Mouse Knock-out Label Lead Mediator of activation protein Medical Mesothelium Modeling Molecular Molecular Target Monoclonal Antibodies Morbidity - disease rate Mus Nature Operative Surgical Procedures Organ PECAM1 gene PTPRC gene Pathogenesis Pathway interactions Patients Peritoneal Plasmids Population Postoperative Period Prevalence Prevention RNA Interference RNA Sequence Analysis RNA Sequences Reporter Resolution Role Small Intestines Solutions Sorting - Cell Movement Staging Staining method Stains Surface System Techniques Testing Therapeutic Time Transfection Up-Regulation base chronic pain cytokine follow-up improved in vivo injured insight intraperitoneal knock-down lymphatic vessel operation podoplanin prevent promoter public health relevance receptor research study response small hairpin RNA therapeutic target transcription factor transcriptome sequencing

项目摘要

DESCRIPTION (provided by applicant): Intraperitoneal adhesions have been a major and expensive complication of abdominal surgeries and lead to a significant fraction of postsurgical morbidities, small bowel obstructions, female infertility, and chronic pain. Despite its prevalence not much is known about the molecular and cellular mechanisms that underlie adhesion formation. Studies have implicated the surface mesothelium, and suggest either its interaction with fibrin matrices or its denudation as causes for adhesion formation. Because much is still unknown about adhesion pathogenesis, few effective preventative or postoperative treatments exist. We have shown that the peritoneal surface mesothelium activates and reacts to local insults such as abrasion and ischemia, leading to hypertrophy and proliferation, culminating in adhesion formation. We have begun to investigate this response for its role as a mediator of adhesion pathogenesis. Our initial experiments showed we can selectively label and purify mouse mesothelium using surface marker stains PDPN+LYVE-1-CD31-CD45-. Immunofluorescence imaging showed substantial mesothelial thickening in mice ~2h after initial insult and adhesion formation as early as 72h after. We purified surface mesothelium using FACS and performed RNA sequencing on mesothelium with no injury and 6h, 12h, and 24h after injury. Aim 1 proposes to a) investigate the cellular dynamics of adhesion formation using a Cre dependent multi-color fluorescent reporter, and b) to perform parallel sorting and RNA sequencing analysis on other adhesion forming models. Our multi-color reporter will be crossed with a global Cre under the actin promoter, and a mesothelium specific Cre allowing us to address two fundamental questions regarding adhesion formation: 1) does only the mesothelium give rise to peritoneal adhesions? 2) is the surface mesothelium homogenous, or do there exist subsets within the mesothelium that specifically give rise to adhesions? Though we have a list of targets we believe are key mediators of adhesion formation, further analysis of our RNA seq data in conjunction with new analyses of other adhesion forming models will refine this list and provide insight towards a global mechanism. Aim 2 proposes to use an in vivo system we have recently raised interrogate our molecular targets. We have developed a technique in which we electroporate the peritoneal wall of mice and observe robust transfection of plasmid containing a cDNA cassette or an shRNA system. We propose to use this system to provide transient upregulation or knock down of genes we find to be highly involved in adhesion formation. We expect that electroporating in these genes after initial adhesion-forming insults will prevent its pathogenesis. We are pursuing a parallel approach in which we will electroporate in plasmids containing single guide RNAs into a Cas9 knock-in mouse to achieve permanent knockout and expect similar results. Finally, we have accumulated a list of surface markers highly upregulated following adhesion induction, and are testing blocking antibodies and expect to prevent adhesion formation. These studies have the potential to establish a new understanding of peritoneal adhesions and a mechanism to facilitate discovery of new therapies.

描述（由申请人提供）：腹膜内粘连一直是腹部手术的主要且昂贵的并发症，并导致很大一部分术后发病、小肠梗阻、女性不孕和慢性疼痛，尽管人们对其普遍性知之甚少。研究表明，表面间皮细胞与纤维蛋白基质的相互作用或其剥脱是导致粘附形成的原因。由于粘连的发病机制尚不清楚，因此很少有有效的预防或术后治疗方法，我们已经证明腹膜表面间皮会激活并对局部损伤（如磨损和缺血）做出反应，导致肥大和增殖，最终导致粘连形成。我们已经开始研究这种反应作为粘附发病机制介质的作用。我们的初步实验表明，我们可以使用表面选择性地标记和纯化小鼠间皮。标记染色 PDPN+LYVE-1-CD31-CD45- 免疫荧光成像显示小鼠在最初损伤后约 2 小时出现显着的间皮增厚，并且早在 72 小时后我们就使用 FACS 纯化了表面间皮，并对间皮进行了 RNA 测序，没有损伤。目标 1 建议 a) 使用损伤后 6 小时、12 小时和 24 小时研究粘附形成的细胞动力学。 a Cre 依赖性多色荧光报告基因，b) 对其他粘附形成模型进行平行分选和 RNA 测序分析。我们的多色报告基因将与肌动蛋白启动子下的全局 Cre 和间皮特异性 Cre 杂交。解决有关粘连形成的两个基本问题：1）只有间皮会引起腹膜粘连吗？2）表面间皮是同质的，还是内部存在子集？尽管我们有一系列我们认为是粘连形成关键介质的靶标，但对我们的 RNA seq 数据的进一步分析以及对其他粘连形成模型的新分析将完善该列表并提供对全球粘连的见解。目标 2 提出使用我们最近提出的体内系统来询问我们的分子靶标。我们开发了一种技术，可以对小鼠腹膜壁进行电穿孔并观察质粒的稳健转染。我们建议使用该系统来提供与粘附形成高度相关的基因的瞬时上调或敲低，我们预计在最初的粘附形成损伤后对这些基因进行电穿孔将阻止其发生。我们正在寻求一种平行的方法，将含有单向导RNA的质粒电穿孔到Cas9敲入小鼠中以实现永久敲除，并期望获得类似的结果。粘连诱导后上调，并且正在测试阻断抗体并期望防止粘连形成。这些研究有可能建立对腹膜粘连的新认识和促进新疗法发现的机制。