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The Effects of Sea Cucumber Extracts (Holothuria Scabra) on Human Placenta- Derived Mesenchymal Stem Cells

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The Effects of Sea Cucumber Extracts (Holothuria Scabra) on Human Placenta- Derived Mesenchymal Stem Cells THE EFFECTS OF SEA CUCUMBER EXTRACTS (HOLOTHURIA SCABRA) ON HUMAN PLACENTA- DERIVED MESENCHYMAL STEM CELLS BY MISS JUTARAT SAENGSUWAN A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE (BIOCLINICAL SCIENCES) CHULABHORN INTERNATIONAL COLLEGE OF MEDICINE THAMMASAT UNIVERSITY ACADEMIC YEAR 2017 COPYRIGHT OF THAMMASAT UNIVERSITY Ref. code: 25605729040302HAO THE EFFECTS OF SEA CUCUMBER EXTRACTS (HOLOTHURIA SCABRA) ON HUMAN PLACENTA- DERIVED MESENCHYMAL STEM CELLS BY MISS JUTARAT SAENGSUWAN A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE (BIOCLINICAL SCIENCES) CHULABHORN INTERNATIONAL COLLEGE OF MEDICINE THAMMASAT UNIVERSITY ACADEMIC YEAR 2017 COPYRIGHT OF THAMMASAT UNIVERSITY Ref. code: 25605729040302HAO Thesis Title The effects of sea cucumber extracts (Holothuria scabra) on human placenta mesenchymal stem cells Author Miss Jutarat Saengsuwan Degree Master of Science in Bioclinical Sciences Major Field/Faculty/University Stem cell and Regenerative Medicine Chulabhorn International College of Medicine Thammasat University Thesis Advisor Assistant Professor Napamaee Kornthong, Ph.D. Thesis Co-Advisor Associate Professor Sirikul Manochantr, Ph.D. Associate Professor Chairat Tantrawatpan, Ph.D. Academic Years 2017 Ref. code: 25605729040302HAO (1) ABSTRACT Sea cucumber, Holothuria scabra, has been emphasized on their ability in the regeneration of their own body. They may provide new pathways to target the treatment of degenerating diseases in humans, due to their regeneration ability. The thesis was focused on the effects of H. scabra extracts on mesenchymal stem cells (MSCs) derived from human placenta, including MSCs proliferation and neuronal differentiation. The H. scabra crude protein extracts were prepared from the body wall (BW), viscera (VI), radial nerve cord (RN) and nerve ring (NR) by using different extraction buffers, including 0.1M phosphate buffer saline (PBS) and 0.1M acetic acid buffers. The SDS- PAGE showed protein abundance, with various molecular mass, within the BW and VI extracts using 0.1M PBS buffer. Less protein abundance was observed for all organ extracts using 0.1M acetic acid buffer (AA). The western blot analysis demonstrated that nerve growth factor (NGF) was expressed in VI-PBS at 13 kDa. On the other hand, epidermal growth factor (EGF) also showed in BW-PBS, BW-AA, VI-PBS, RN-PBS and NR-PBS at 160 kDa. The MSCs were then treated with different doses of H. scabra extracts using MTT assay for cytotoxicity and cell proliferation. We found that the treatment of 0.1 and 1 µg/ml of H. scabra extracts increased the proliferative rate of MSCs when compared with the sham. In addition, real-time PCR of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) transcripts was performed and shown that both genes shown higher expression level than control on day10 at 1 µg/ml of BW-PBS. After neural induction, the MSCs was detected MAP2, Nestin and β-tubulin III by immunocytochemistry. MSCs expressed β tubulin III and Nestin positive signals in the cytoplasm, although MAP2 signal is very limited signal. Moreover, real-time PCR of MAP2, Nestin and β-tubulin III transcripts were carried out and shown that β-tubullin III mRNA showed very high level at RNL2-0.1 neural MSCs group, comparing to sham control medium group. MAP2 mRNA expression showed high level at BWL1-0.1, BWL1-1, BWL2-0.1, BWL2-1 and RNL2-0.1 groups. Interestingly, Nestin mRNA expressed within the similar level between the groups. In this study, it was the first report of the ability of H. scabra crude extracts on MSCs proliferation and differentiation. While the further studies are necessary in order to conclude the regulation and mechanism on the effects of sea cucumber on MSCs. Ref. code: 25605729040302HAO (2) Keywords: Holothuria scabra, Mesenchymal stem cells, Sea cucumber Ref. code: 25605729040302HAO (3) ACKNOWLEDGEMENTS I would like to express my gratitude to all people who have contributed to improve this thesis and also develop me as a good scientist during these years. Without all peoples, this work could not be succeeded. First and foremost, I would like to express my deeply grateful acknowledgement to my major advisor, Assist. Prof. Dr. Napamanee Kornthong for supervising this project and always encourage and support during these years. I also would like to express my gratefulness to my co-advisors, Assoc. Prof. Dr. Sirikul Manochantr and Assoc. Prof. Dr. Chairat Tantrawatpan for providing scientific thinking, support and technical advice during the study. My grateful appreciation also extended Prof. Dr. Prasert Sobhon for kindness suggestion and valuable recommendations in this work. Moreover, my sincere thanks also extend to all members of the Center of excellence in stem cell research, Thammasat University (TCSR), Miss Supawadee Duangprom, Miss Supawadee Kheowkae, Miss Jutaporn Pollawat and Miss Wilailuk Ampansri, staffs from Chulabhorn International College of Medicine for guidance, suggestion, support and shared the knowledges. They didn’t not only teach and advice of techniques, procedures and methods for laboratories but also making my life more enjoyable over the past few years. Finally, I would like to thank my dearest family, my parents, my older sister, my family, my friends and others person for all their support and constant encouragement throughout the period of this thesis and the research grant from Thammasat University, Chulabhorn International College of Medicine and the Center of excellence in stem cell research, Thammasat University (TCSR). Miss Jutarat Saengsuwan Ref. code: 25605729040302HAO (4) TABLE OF CONTENTS Page ABSTRACT (1) ACKNOWLEDGEMENTS (3) LIST OF TABLES (8) LIST OF FIGURES (9) LIST OF ABBREVIATIONS (13) CHAPTER 1 INTRODUCTION 1 1.1 Introduction 1 1.2 Objective 2 1.2.1 Overall Objective 2 1.2.2 Specific objectives 2 CHAPTER 2 REVIEW OF LITERATURE 3 2.1 Sea cucumbers 3 2.1.1 Biology of sea cucumber 3 2.1.2 Concept of regeneration of sea cucumber 3 2.1.3 Visceral regeneration of sea cucumber 7 2.2 Mesenchymal stem cells 16 2.2.1 Morphology of MSCs 16 2.2.2 Differentiation potential of MSCs 17 2.3 The effect of sea cucumber extracts condition on proliferation 17 CHAPTER 3 RESEARCH METHODOLOGY 20 Ref. code: 25605729040302HAO (5) 3.1 Sea cucumber extraction 20 3.1.1 Tissue collection 20 3.1.2 Protein preparation 20 3.1.3 Measuring concentration of protein 20 3.1.4 SDS-PAGE 21 3.1.5 Western blot analysis 21 3.2 Cell preparation 21 3.2.1 Mesenchymal stem cell derived from human placenta 21 (PL-MSCs). 3.3 Cell culture 22 3.4 Characterization of MSCs 22 3.4.1 Multi-linage cell differentiation 22 3.4.1.1 Osteogenic differentiation 22 3.4.1.2 Adipogenic differentiation 22 3.4.2 Immunophenotypical 23 3.5 Effect of H. scabra on MSCs proliferation 23 3.5.1 Cytotoxicity test 23 3.5.2 Growth kinetic curve 23 3.5.3 Proliferative gene expression analysis by qRT-PCR 24 3.6 Treat sea cucumber crude extract 26 3.6.1 Neural differentiation 26 3.6.1.1 Commercial neural differentiation medium 26 3.6.1.2 In-House neural differentiation medium 26 3.6.2 Immunofluorescence staining of neurogenic-MSCs 27 3.7 RNA extraction 27 3.8 First-strand cDNA synthesis 28 3.9 Quantitative real-time polymerase chain reaction (qRT-PCR) 28 CHAPTER 4 REVIEW OF LITERATURE 30 Ref. code: 25605729040302HAO (6) Page 4.1 Isolation and characterization of PL-MSCs from placenta 30 4.1.1 Isolation PL-MSCs from placenta 30 4.1.2 Characterization of PL-MSCs 32 4.1.2.1 Adipogenic differentiation potential of PL-MSCs 32 4.1.2.2 Osteogenic differentiation potential of PL-MSCs 34 4.1.2.3 Immunophenotype of MSCs derived from placenta 36 4.2 Proliferative effect of H. scabra on PL-MSCs 39 4.2.1 Protein profile of H. scabra using SDS-PAGE 40 4.2.2 Protein identification of H. scabra growth factor using 41 Western blot analysis 4.2.3 Cell proliferation and cell cytotoxicity determined by MTT 44 assay 4.2.4 Cell growth determined by direct cell counting 47 4.2.5 Proliferative genes expression of PL-MSCs by qRT-PCR 50 4.3 Neural differentiation potential of MSCs derived from placenta 57 with sea cucumber extracts 4.3.1 Commercial neural differentiation medium 57 4.3.2 In-House neural differentiation medium 64 4.4 Neural genes expression by RT-PCR 68 CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS 72 REFERENCES 77 APPENDICES 80 APPENDIX A 81 APPENDIX B 86 Ref. code: 25605729040302HAO (7) Page APPENDIX C 91 BIOGRAPHY 100 Ref. code: 25605729040302HAO (8) LIST OF TABLES Tables Page 3.1 The sequence of primers for proliferative genes and 25 house-keeping gene 3.2 The sequence of primers for neurogenic genes and 29 house-keeping gene 4.1 The surface marker expression profiles of MSCs from placenta 37 Ref. code: 25605729040302HAO (9) LIST OF FIGURES Figures Page 1.1 Regeneration of various organism 5 1.2 Principle of basic mechanism of regeneration in vertebrates and 6 invertebrates. 1.3 Schematic diagram shows the tissue layers of the holothurians 8 digestive tract 1.4 Diagram showing the nervous system of A.japonicus 9 1.5 Anatomical diagram showing organization of the regenerating 11 digestive tube in a holothurian, at different times points after evisceration 1.6 Histological sections of normal and regenerating intestine during 12 the different regeneration stages in the sea cucumber A.japonicus 1.7 Uninjured and regenerating stages of the radial nerve cord 13 (RNC) of H. glaberrima 1.8 KEGG pathway and putative transcripts in regenerative 14 A. japonicus transcriptome 1.9 Bioactive components of sea cucumber extracts and their 15 biological effects on various human cancer cells and cancer animal models 1.10 The interactions between a sulfated polysaccharide from Haishen 18 (HS) and EGF (20 ng/ml) or FGF-2 (20ng/ml) on the cell viability of rat NSPCs in vitro 1.11 The effect of Stichopus variegatus water extract (SVWE) and 19 EGF treatments on spinal astrocytes culture in vitro 4.1 Morphology of mesenchymal stromal cells 31 4.2 Adipogenic differentiation of PL-MSCs 33 4.3 Osteogenic differentiation of PL-MSCs 35 Ref.
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