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IL-lp, IL-4, IL-5 AND IL-13 CYTOKINES THROUGH THE APPLICATION OF cDNA

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UNIVERSITITEKNOLOGI MARA

GENE EXPRESSION PROFILING OF HUMAN BRONCHIAL SMOOTH MUSCLE AND LUNG FIBROBLASTS CELLS STIMULATED WITH

IL-lp, IL-4, IL-5 AND IL-13 CYTOKINES THROUGH THE APPLICATION OF cDNA

MICROARRAY.

FAIZAH BINTIJAAFAR

Thesis submitted in fulfilment of the requirements for the degree of

Master of Science

Faculty of Medicine

November 2011

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AUTHOR'S DECLARATION

I declare that the work in this thesis was carried out in accordance with the regulations of Universiti Teknologi MARA. It is original and is the result of my own work, unless otherwise indicated or acknowledged as reference work. This thesis has not been submitted to any other academic institution or non-academic institution for any degree or qualification.

I, hereby, acknowledge that I have been supplied with the Academic Rules and Regulations for Post Graduate, Universiti Teknologi MARA, regulating the conduct of my study and research.

Name of Student : Faizah binti Jaafar

Student I.D. No. : 2006666496

Programme : Master of Science (Molecular Medicine) Faculty : Medicine

Thesis Title

Signature of Student Date

Gene Expression Profiling Of Human Bronchial Smooth Muscle And Lung Fibroblasts Cells Stimulated With IL-lp, I L ^ , IL-5 And IL-13 Cytokines Through The Application Of cDNA Microarray.

November 2011

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ABSTRACT

Both Thl and Th2 cytokines are known to be potent stimulants acting on resident airway cells by inducing inflammatory response. Thl cytokines were known as proinflammatory mediators while Th2 cytokines, in particular play critical role in eliciting the activation and recruitment of the IgE antibody producing B cells, mast cells and eosinophils which are the key features of allergic asthma. However, the molecular mechanisms involved are still poorly understood. The aim of this study is to investigate the molecular mechanism underlying the mechanism of the cytokines in inducing asthma. In this study, cultured normal human bronchial smooth muscle cells (BSMC)'and normal human lung fibroblasts (NHLF) were stimulated with cytokines namely IL-IB, IL-4, IL-5, IL-13 and combination of all four cytokines at two timepoint (1 and 24 hours) which represent the early and late onset of inflammatory response . The application of cDNA microarray in this study has generated a gene expression database for both BSMC and NHLF. Some of the key- genes involved in the stimulatory response were selected for further validation by real-time PCR and protein assays. Microarray data was generated by using third party software. Two algorithms were chosen to summarize the data; RMA and PLIER. Multi-way ANOVA test was conducted at p value= 0.01 with >2 fold change. Results clearly showed that in NHLF stimulation, IL-IB one hour showed high number of expressed genes for 89 and 67 genes for both PLIER and RMA analysis. In IL-IB 24 hours stimulation, the expressed genes are 313 and 246 for both algorithm respectively. In contrast, Th2 cytokines of I L ^ , IL-13 and IL-5 merely generating non-significant genes compared to IL-IB and combination of all four cytokines. The combination stimulation generated more to IL-IB like expression. In BSMC stimulation, IL-5 surprisingly showed a similar pattern of expression to that of IL-IB in both 1 and 24 hours stimulation. There are about 199 genes expressed common to both IL-5 and IL-IB stimulation. RMA analysis proved to be more stringent in data generation compared to PLIER. Despite activating in a common signaling pathway, IL-5 when compared to other Th2 cytokine type like I L ^ and IL- 13 induced dramatically different patterns of gene expression in primary cultures of airway smooth muscle cells, and lung fibroblasts, with little overlap among cell types. The most prominent effects of IL-IB and IL-5 were observed on the airway smooth muscle stimulation. A better understanding on the relationship of IL-5 and IL-IB was further investigated and found to be that IL-5 is acting through the same pathway as in the IL-IB cytokines which is responsible for the inflammatory response in modulation of the smooth muscle expression. However, the stimulation of IL-4 and IL-13 on the other hand did not yield to a significance result. This might be due to the low concentration of cytokine used in the stimulation. IL-IB on the other hand is taken as positive control due to its ability as potent stimulant in both NHLF and BSMC. On the overall, effects of cytokine on the airway resident cells in this study are able to provide molecular insight especially during inflammatory response.

iii

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ACKNOWLEDGEMENTS

In the name of Allah, the All-merciful, the All-compassionate.

All my praise goes to Allah, whom have given me strength and only with His blessings that made this master thesis possible. First and foremost, I would like to express my gratitude to my supervisor Assoc. Prof. Dr. Gabriele Anisah whom has effortlessly guided me throughout the completion of my thesis with her patience and knowledge and tonnes of advice. My sincere thanks also go to my co-supervisor Assoc. Prof. Dr. Tengku Saifuddin whom without his valuable knowledge and assistance this study would not have been successful. Special thanks to Assoc. Prof.

Dr. Saifulaman whom has introduced me to this institution and open the door to my Master Degree and for bringing me into his project. I would like to express my thanks to my best friend Norita Salim whom has given me a great deal of help doing the laborious tissue culture work and provide me with the comfort of friendship and support in many ways. Above all, I am really indebted to my dearest father Jaafar b.

Haji Mansor whom has never fails to believe in me and provides me with an incredible strength for me to endure the painstaking years in doing my masters with his endless love, prayers and encouragement. He, in the first place is the person who put the fundamental of my learning character by showing me the joy of intellectual pursuit ever since I was a child and that there is nothing impossible to be achieve in life as long as there is God to guide the way. I am dedicating this thesis whole heartedly to my father whom deserve the appreciation more than myself for his unconditional love and dedication that makes me who I am now. To my younger brother Firdaus Jaafar, thank you for your love and support. Not forgetting my dear husband, Suhaimi Alias for giving me an endless support and taken the load off my shoulder by keeping me company till late at night while I am editing the thesis, I do appreciate your assistance and thank you very much.

Faizah Jaafar November 2011

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TABLE OF CONTENTS

TITLE PAGE i AUTHOR'S DECLARATION ii

ABSTRACT iii ACKNOWLEDGEMENTS iv

TABLE OF CONTENTS v LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATIONS xii

CHAPTER ONEt.INTRODUCTION

1.1 Background 1 1.2 Problem statement 3

1.3 Hypothesis 3 1.4 Significant contribution to new knowledge 3

1.5 Objectives 4 CHAPTER TWO : LITERATURE REVIEW

2.1 Cytokines 5 2.2 Cytokines in inflammatory responses 6

2.2.1 IL-lp 8 2.2.2 IL^t 9 2.2.3 IL-13 11 2.2.4 IL^5 13

v

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