博士論文代寫作品集案例：

荷蘭醫學博士開題報告 Research Project Proposal 

論文題目：Role of stromal cells in the progression of Inflammatory Bowel Disease

論文語言：英語 English

論文專業：醫學

字數：2000-3000

學校國家：荷蘭

是否有數據處理要求：否

您的學校：XXX大學 排名89

論文用于：Doctor Application 博士申請

截止日期：2021-01-09

補充要求和說明： 申請留學的RP，有摘要，論文參考格式不清楚

The Role of Stromal Cells in the Progression of Inflammatory Bowel Disease

Research Project Proposal

Name of the PhD Candidate: XXXX

Name of the Local Supervisor:Prof. dr. XXX

Dr. L. XXX

Dr. A. XXX

Date of Submission:                   19-01-2021

Table of Contents

Introduction 3

Background and Problem Statement 3

Purpose of the Study 4

A Preliminary Review of Literature 4

Introductions 4

Functions in the Gut 6

Stromal cells in IBD progression 6

Stromal cell subsets in IBD progression 6

Stromal Cells and Immune Regulation 8

Research Questions 9

Research Design, Methods and Procedures 10

Ethical Considerations 10

Institutional Framework 11

Time Table of the proposed PhD Project 11

Expected Research Budgeting 12

Expected Limitations 12

Significance of the Study 12

References 14

Introduction

Inflammatory Bowel Diseases (IBD), which comprise Crohn’s disease (CD) and ulcerative colitis (UC) are chronic and relapsing inflammatory diseases of unknown etiology (ref). The epidemiology of IBD is widely studied and internationally, the cases are approximately 0.5 to 24.5 for every 100,000 cases for ulcerative colitis and 0.1 to 16 cases for every 100,000 people with the Crohn disease (Ha & Khalil, 2015). Many of these patients either require medication or surgery or are often hospitalized with Click et al., (2015) noting that up to 80% of Crohn’s victims shall be hospitalized at some point in their disease ecosystem with a majority of others having either prolonged or active remission. In the United States, circa 1-2 million individuals have Crohn or ulcerative colitis (Kappelman et al., 2007). The incidence of both diseases in the US is estimated to be 75-150 cases per 100 000 people (Rowe, 2020). In Europe, epidemiological data by 2013 suggested that 2.5-3 million people were affected by IBD with a high economic burden on healthcare, a cost circa 4.6-5.6 Euros/year (Burisch et al., 2013). Internationally, the highest incidences of IBD are seen in developed countries as compared to developing countries. The pathogenesis of this condition including the UC and CD entail a variety of pathogenic elements such as the abnormal gut microbiota, environmental changes, immune response dysregulations as well as the variants within the genes (Lee, Eun & Cho, 2018). Treatment has always been to treat the inflammation directly using immunosuppressive medication and to prevent the flares. When anti-inflammatory drugs are insufficient, surgery is the only remaining option in these severe cases. 

Background and Problem Statement

 For a long time, treatment of IBD has focused on symptomatic and supportive care, focusing on targeting the inflammatory cells directly. Very recently evidence, however, has suggested a role of stromal cells (non-epithelial cells/fibroblast) in IBD progression (Kinchen et al., 2018). However very little is known of the role they play in disease progression, how they functionally interact with infiltrating immune cells, and how they affect/are affected by current therapies. Therefore, this research project focuses on the role of the stromal cells in the progression of IBD and response to current IBD medication. This knowledge is relevant both in the quest for new therapeutic options for IBD, assess parameters predicting he effectiveness of current IBD medication and increase basic knowledge on pathogenesis. 

Purpose of the Study

As defined by Nowarski, Jackson & Flavell (2017) stromal cells are non-epithelial, non-hematopoietic and non-endothelial cells. Besides playing a role in epithelial haemostasis, they also influence the haemostasis of the gut immune cells. The purpose of our project is to investigate how stromal cells affect, immune cell recruitment, activation and subsequently the progression of IBD and its complications such as stenosis and (perianal) fistulas. The first stage will involve an intensive literature review of credible journals and reliable sources in order to determine various hypothetical information available as well as gaps in the research topic. Further, we will analyze the stromal compartment in IBD patients using an imaging mass cytometry. Afterwards, the project shall entail a study of the interaction between immune and stroma cells in vitro using 3D coculture systems. The potential targets found in 2 different mouse models for colitis experiment shall be considered. 

A Preliminary Review of Literature

Introductions

This section of the research will handle relevant literature and past works of researchers in this field. It would also provide a basis of theoretical framework for the research project, analysing the various theories that attempt to explain the relationship between stromal cells and IBD progression.

Multiple etiologic factors have been proposed as contributors to the pathogenesis of IBD; however, no element if dominant. From the definition of IBD, inflammation as a component of pathogenesis has been the most accepted scientific basis. Environmental causes, gene variants, microbiota, immune system dysregulation and other forms of pathogenesis have drawn a wide range of debate and form a controversial research subject with no certainty.  However, in recent research developments, the concept of stromal cell contributing to IBD pathogenesis is emerging but many things remain unknown nevertheless (McDowell et al., 2020). To lay the foundation, it is crucial to understand the differences between Crohn’s disease and ulcerative colitis. While Crohn’s disease affects the entire gastrointestinal tract, from the mouth to the anus, ulcerative colitis is limited to the colon (Chang et al., 2020). Crohn’s disease exhibits a trans mural granulomatous inflammation in a skip lesion pattern (normal areas interspersed with diseased regions). Furthermore, due to the trans mural involvement, inflammation in Crohn’s disease has a tendency to form deep ulcers which can develop into complications such as fistulas to loops of bowel, vagina or perianal skin (Ramos & Papadakis, 2019). Contrarily, ulcerative colitis begins in the rectum and extends proximally, without skip lesions and the inflammation is limited to the mucosa and submucosa (Ramos & Papadakis, 2019). Different immune cells are found in the two forms of IBD (lymphocytes, cytokines, and macrophages) and also different stromal cell subsets (fibroblasts, mesenchymal cells, pericytes) seem to play a role (Kinchen et al., 2018).

There is no consensus on the exact definition of stromal cells, fibroblast and related cells due to the absence of specific and selective markers. We will, according to the most accepted form, define stromal cells as non-hematopoietic, non-endothelial and non-epithelial cells (Barnhoorn, 2020). Examples of stromal cells include fibroblasts, myofibroblasts, smooth muscle cells, mesenchymal stromal cells and pericytes. Stromal cells are present in all the layers of the gastrointestinal wall in human beings. Serosal mesothelium which stems from mesoderm is the origin the majority of the stromal cells in the gastrointestinal wall (Ramos & Papadakis, 2019). However, stromal cells can also develop from other mechanisms including endothelial to mesenchymal or epithelial to mesenchymal transmission (Ramos & Papadakis, 2019). Other stromal cells also migrate to the intestines from the bones. In this research our primary cell of interest are the local fibroblasts.

Functions in the Gut

Stromal cells and especially fibroblasts play an important role in intestinal homeostasis. Understanding their role in a healthy gut, might aid in the understanding their role in the disease bowel. The stromal cells are responsible for the structure and the form of the gut. Fibroblasts play a role in production of extracellular matrix (ECM). In a diseased gut, particularly in Crohn’s disease, stenosis of the gut lumen occurs as a result of excessive production of ECM by fibroblasts (Ramos & Papadakis, 2019)  

The inflammatory mediators play a role in pathologic and clinical manifestations of IBD. Cytokines, macrophages and T-helper cells are widely implicated in the inflammation cascades (Nowarski, Jackson & Flavell, 2017)

Stromal cells in IBD progression

Just like stromal cells contribute to the pathogenesis of other diseases such as rheumatoid arthritis (Bustamante et al., 2017), it is believed that they also have important roles in the IBD. A study by (Moniuszko et al., 2018) justifies the same by burnishing the immunoregulatory and the hypo-immunogenic properties of mesenchymal stem cells in management of severe repercussions of Crohn’s disease such as fistulas.

Stromal cell subsets in IBD progression

Although the research on stromal cells is still a novelty, few studies support the role of stromal cells in wound healing and immune cell modulation in the pathogenesis of IBD. Kinchen et al. (2018) contributed hugely to the understanding of the stromal cells in IBD. In their research, stromal cell subsets were isolated from the colon of IBD patients and analysed using RNA sequencing and mass cytometry time-of-flight. Kinchen and colleagues found 12-different non-hematopoietic and non-epithelial cells in the colon of patients with ulcerative colitis. Other than myofibroblasts, there were other four clusters of fibroblast-like cells defined named cluster S1, cluster S2, cluster S3, and cluster S4. In a different study by Smillie et al. (2019), eight fibroblast like-cells were isolated from ulcerative colitis tissue. In addition, the study by Martin et al. (2019) shows robust evidence in the implication of stromal cells in IBD pathogenesis. Four clusters of stromal cells were identified from the lamina propria from ileal tissues of patients with Crohn's disease. The cells included fibroblasts, pericytes, smooth muscle cells, and activated fibroblasts (Martin et al., 2019). The studies above show that even though inflammatory cells and other factors are implicated in the pathogenesis of IBD, stromal cells play a crucial function in the human body.

The recent studies have established a probable existence of stromal cells in IBD tissues. This factor rubberstamps the essence of understanding the actual mechanism in which stromal cells function. In a study by Karpus et al. (2019), stromal cells play a role in wound healing in IBD. In a diseased state, IBD disrupts the integrity of the epithelial cell layer of the gut lumen therefore requiring restoration. Restoration of the damaged epithelium in a normal gut is achieved via migration of fibroblast, deposition of collagen, and rebuilding of epithelial layer (Karpus et al., 2019; Kinchen et al., 2018). However, in a diseased gut (IBD), the migratory capacity of the fibroblasts is impaired therefore impairing healing of damaged gut wall. In an in vitro setting, studies have shown that the migration of fibroblasts from lamina propria from IBD patients is greatly decreased as compared to a control population without a diseased gut (Kinchen et al., 2018). Further, the studies show even a further decrease in the migratory capacity of fibroblasts derived from Crohn’s disease fistula patients (Kinchen et al., 2018). Moreover, the proliferation rate of fibroblasts derived from Crohn's disease or ulcerative colitis tissues is higher with an increased amount of collagen deposition as compared to normal tissues (Karpus et al., 2019). This, therefore, explains the high risk of fibrosis in patients with IBD which ultimately causes gut lumen stenosis, and burnishes the previous studies on the stromal cell involvement in IBD pathogenesis. 

Stromal Cells and Immune Regulation

Other than the role in wound healing, stromal cells are actively involved in immune-regulation. As aforementioned, the aetiology of IBD involves a complex interaction between genetic factors, dysregulated immune response, and environmental factors. In the study by Shi et al. (2018), stromal cells can produce or mount a response to cytokines and chemokines. In a recent sc RNA sequencing study of IBD tissue, it was revealed that fibroblasts produce monocyte chemoattractant factors, neutrophil attractants, T-cell recruitment factors, and factors involved in fibrosis (Martin et al., 2019). To support the statement, in vitro studies have shown that in an inflamed murine colon, fibroblasts produce a neutrophil attractant, CX CL2 in response to the damaged epithelium. The CX CL2, therefore, attracts lymphocytes to the damaged mucosa.

Other than the role of stromal cells in immune regulation, it also influences gut response to microbiota. In a non-intact epithelial barrier, fibroblasts directly respond to microbial antigens such as lipopolysaccharides (Barnhoorn et al., 2020). This is achieved through the expression of toll-like receptors (TLRs). Other than the TLRs, fibroblasts have expressed on them the nucleotide-binding oligomerization domain-containing protein 2 [NOD2] which enables them to recognize bacterial antigens (Barnhoorn et al., 2020). In the presence of a loss-of-function mutation in NOD 2, the individuals have high-risk factors for ileal Crohn's disease. This indicates the role of stromal cells in the progression of IBD. 

The evidence described above vividly illustrates the active involvement of stromal cells in the pathogenesis of IBD. The three main mechanisms in which they are involved include wound healing, immune regulation, and response to microbiota. Even though the topic and the evidence provided above are correlated, the information does not exhaust the significance of stromal cells in the progression of IBD. Further, the use of the information available in clinical applications and laboratory experiments is still lacking. This project is aimed not only at exploring the role of stromal cells in the progression of IBD but also targets to apply the knowledge in vitro to investigate the immunoregulatory role of fibroblast, and to investigate fibroblast-Regulatory T cell interactions. Further, the information could be applied in the search for alternative pharmacologic therapies to IBD that target the stromal cells.

Having discussed more on the role of stroma cells on IBD, this section examines the connection between efficacy of the mesenchymal stroma cell (MSC) therapy to this condition. The MSC therpies are significantly becoming a novel regenerative as well as immunodulatory manner to prevent or treat diseases. The MSC, as indicated in REF posses tropism in regards to inflamed and damanged tissues. Therefore, determining the routes of therapeutic cell delivery is an important aspect of this clinical practice. 

Research Questions

1. What is the role of stromal cell subsets in the progression of IBD and response to therapy?

2. How can the changes in the relative abundance of stromal cell subsets affect immune cell infiltration and activation and subsequently IBD disease progression?

3. How do stromal cells subsets affect IBD complications like fistulas?

Research Design, Methods and Procedures 

The research design will entail a combination of mostly quantitative approach and a few qualitative elements. The initial dominant approach shall be both descriptive, correlational and entailing experimental research based on a chose study population and research techniques relevant. Sampling techniques shall adopt four common probability sampling methods including random sampling, systematic, stratified and cluster sampling approaches. The data collection methods shall also be done in accordance with the medical requirements including making use of the patient reported data, proxy data well as ambulatory medical records. The main activities in the research are as follows

1.  We will perform an intensive literature review in order to understand the role of stromal cells in IBD pathogenesis and fuel an experiment in vitro, regarding the immune-regulatory role of fibroblasts

2. We will analyse the stromal compartment in IBD patients using imaging mass cytometry

3. We will study the interaction between immune and stromal cells in vitro using 3D co culture systems

4. We will target potential targets found in 2 in different mouse models for experimental colitis

Ethical Considerations

The following are the ethical considerations considered before the initiation and during the progress of the project.

1. The research participants will not be subjected to any form of harm 

2. Full consent will be obtained before participation in the project

3. The privacy and confidentiality of the participants will be given key priority

4. Participants will remain anonymous

5. Communication concerning the project will be done with honesty and transparency upon completion of the research plan

Institutional Framework

The framework of the study will be based on the university and affiliated resources. I, as the PhD applicant will ensure that I adhere to the framework provided by the university in collaboration with all relevant research bodies. The people necessary in this project shall include my local supervisor, as well as every participant within the study (participants and research assistants). 

Time Table of the proposed PhD Project

The estimated time duration is presented in the chart below. 

Expected Research Budgeting 

The expected budget accruing from the research shall be detailed and examined in the course of the proposal output including the various angles that financial implications shall be involved. Most of the income shall be covered by the participant in collaboration with a few research financing institutions that the researcher is in the process of identifying and approaching. 

Expected Limitations

Up to now most research on stromal cells in IBD in the Leiden University Medical Centre has been focused on murine samples. The findings in mouse models will therefore be extrapolated to human beings. Despite the similarities between the two species, they have diverse differences. The major problem is translating the data from such studies into a pragmatic experience with human beings. Therefore, the pieces of evidence from literature and previous research might be inconclusive about the role of stromal cells on human IBD. Thus, the animal models will always be a model while the mouse element is nased. The focus will be to complement these models with human models thus posing research limitations. 

Additionally, despite the studies on the role of stromal cells on IBD, discussed above, there is lacking evidence concerning the efficacy of mesenchymal stromal cell therapy. Therefore, even with the hope that the findings in this study will help fuel the development of MSC-therapy, there is still doubt whether the patients with IBD will find relief in mesenchymal stromal cell therapy.

Significance of the Study

The goal of this research project is to define new targets for IBD on stromal cells and how medication affects current IBD medication and effectiveness. The current treatment guidelines of IBD recommend the use of antibiotics, immune-suppressants, corticosteroids, and aminoglycosides (Hidalgo-Garcia et al., 2018). Even though their efficacy has been proven in patients with IBD, there are various side effects associated with the chronic use of drugs. Furthermore, these drugs are not capable of healing the disease. Due to that, there is a need to develop new treatments that are safe and efficacious. The knowledge of the use of stromal cells in the progression of IBD therefore proves pertinent. The knowledge is applied in vitro to investigate the immune-regulatory role of fibroblasts and possibly, to develop new treatment options for IBD.

References

Ananthakrishnan, A. N., Kaplan, G. G., & Ng, S. C. (2020). Changing global epidemiology of inflammatory bowel diseases: Sustaining health care delivery into the 21st century. Clinical Gastroenterology and Hepatology: The Official Clinical Practice Journal of the American Gastroenterological Association, 18(6), 1252–1260. https://doi.org/10.1016/j.cgh.2020.01.028

Binienda, A., Ziolkowska, S., Hauge, I., & Salaga, M. (2020). The Role of Immune and Epithelial Stem Cells in Inflammatory Bowel Disease Therapy. Current Drug Targets, 21(14), 1405-1416. https://doi.org/10.2174/1389450121666200504074922

Burisch, J., Jess, T., Martinato, M., & Lakatos, P. (2021). The burden of inflammatory bowel disease in Europe.

Bustamante, M. F., Garcia-Carbonell, R., Whisenant, K. D., & Guma, M. (2017). Fibroblast-like synoviocyte metabolism in the pathogenesis of rheumatoid arthritis. Arthritis Research & Therapy, 19(1). https://doi.org/10.1186/s13075-017-1303-3

Click, B., Vargas, E. J., Anderson, A. M., Proksell, S., Koutroubakis, I. E., Ramos Rivers, C., ... & Binion, D. G. (2015). Silent Crohn's disease: asymptomatic patients with elevated C-reactive protein are at risk for subsequent hospitalization. Inflammatory bowel diseases, 21(10), 2254-2261. Retrieved from https://www.academia.edu/download/39398548/silentCD.pdf

Ha, F., & Khalil, H. (2015). Crohn’s disease: a clinical update. Therapeutic advances in gastroenterology, 8(6), 352-359. Retrieved from https://scholar.google.com/scholar?output=instlink&q=info:8bDyQTLMEuUJ:scholar.google.com/&hl=en&as_sdt=0,5&as_vis=1&scillfp=1989048096759038866&oi=lle

Hidalgo-Garcia, L., Galvez, J., Rodriguez-Cabezas, M., & Anderson, P. (2018). Can a Conversation Between Mesenchymal Stromal Cells and Macrophages Solve the Crisis in the Inflamed Intestine?. Frontiers In Pharmacology, 9. https://doi.org/10.3389/fphar.2018.00179

Kappelman, M. D., Rifas–Shiman, S. L., Kleinman, K., Ollendorf, D., Bousvaros, A., Grand, R. J., & Finkelstein, J. A. (2007). The prevalence and geographic distribution of Crohn’s disease and ulcerative colitis in the United States. Clinical Gastroenterology and Hepatology, 5(12), 1424-1429. Retrieved from https://www.cghjournal.org/article/S1542-3565(07)00715-X/fulltext

Karpus, O. N., Westendorp, B. F., Vermeulen, J. L. M., Meisner, S., Koster, J., Muncan, V., Wildenberg, M. E., & van den Brink, G. R. (2019). Colonic CD90+ crypt fibroblasts secrete semaphorins to support epithelial growth. Cell Reports, 26(13), 3698-3708.e5. https://doi.org/10.1016/j.celrep.2019.02.101

Kinchen, J., Chen, H., Parikh, K., Antanaviciute, A., Jagielowicz, M., & Fawkner-Corbett, D. et al. (2018). Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease. Cell, 175(2), 372-386.e17. https://doi.org/10.1016/j.cell.2018.08.067 Retrived 11 Febuary 2000, from https://www.020lunwen.com/overseas/

Lee, S. H., eun Kwon, J., & Cho, M. L. (2018). Immunological pathogenesis of inflammatory bowel disease. Intestinal research, 16(1), 26. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797268/

Martin, J., Chang, C., Boschetti, G., Ungaro, R., Giri, M., & Grout, J. et al. (2019). Single-Cell Analysis of Crohn’s Disease Lesions Identifies a Pathogenic Cellular Module Associated with Resistance to Anti-TNF Therapy. Cell, 178(6), 1493-1508.e20. https://doi.org/10.1016/j.cell.2019.08.008

McDowell, C., Farooq, U., & Haseeb, M. (2021). Inflammatory Bowel Disease. Ncbi.nlm.nih.gov. Retrieved 12 January 2021, from https://www.ukassignment.org/ResearchProposal.

Moniuszko, A., Sarnowska, A., Rogowski, W., Durlik, M., Wluka, A., & Rydzewska, G. (2018). Successful treatment of an enterovesical fistula due to Crohn’s disease with stem cell transplantation: a case report. Przeglad Gastroenterologiczny, 13(4), 332–336. https://doi.org/10.5114/pg.2018.79814

Nowarski, R., Jackson, R., & Flavell, R. A. (2017). The stromal intervention: Regulation of immunity and inflammation at the epithelial-mesenchymal barrier. Cell, 168(3), 362–375. https://doi.org/10.1016/j.cell.2016.11.040

Ramos, G. P., & Papadakis, K. A. (2019). Mechanisms of disease: Inflammatory bowel diseases. Mayo Clinic Proceedings. Mayo Clinic, 94(1), 155–165. https://doi.org/10.1016/j.mayocp.2018.09.013

Shi, Y., Wang, Y., Li, Q., Liu, K., Hou, J., Shao, C., & Wang, Y. (2018). Immunoregulatory mechanisms of mesenchymal stem and stromal cells in inflammatory diseases. Nature Reviews. Nephrology, 14(8), 493–507. https://doi.org/10.1038/s41581-018-0023-5

Smillie, C., Biton, M., Ordovas-Montanes, J., Sullivan, K., Burgin, G., & Graham, D. et al. (2019). Intra- and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis. Cell, 178(3), 714-730.e22. https://doi.org/10.1016/j.cell.2019.06.029