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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.