博碩士論文 104881603 詳細資訊



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姓名 維加亞(Gudivada Vijaya Narasimha)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 環狀BMP-2肽對肌原細胞中BMP-2蛋白信號的上調
(Upregulation of BMP-2 protein signaling in myogenic cell lines by cyclic BMP-2 peptide)
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摘要(中) 在2007年，骨型態蛋白-2被廣泛應用在臨床研究，然而根據FDA資料顯示BMP-2蛋白經常誘導如頸部腫脹和相應需要插管的呼吸及吞嚥困難等併發症，其中20%-70%的併發症對生命是具威脅性的，FDA在2008年發布了關於BMP-2蛋白的安全和有效性警告，但BMP-2 仍然是骨科創傷的首選，許多研究人員正在尋找 BMP-2 的替代品。基本上，BMP-2 蛋白通過與BMP的I型和II型受體形成異四聚體複合物進而發揮作用。許多II型受體已被揭示它會增加某些成骨信號的傳導，例如骨形態發生蛋白受體-II (BMPRII) 和活化素受體 (ACVR2A/2B)。藉由抗體、小分子、適體和胜肽的使用其作為潛在目標藉由阻斷BMPR2去限制BMP-2的活性，其中相較於抗體、小分子和適體，胜肽被認為是穩定且更好的選擇。胜肽具有易合成、處理、具生物降解性以及低免疫原性。研究中，我們設計了一種具有BMP-2線性關節抗原決定區的BMP-2衍生肽，通過將兩個半胱氨酸殘基摻入不同序列環化線性胜肽，並製備了四種環狀胜肽且其C端的活性區可以分別在環狀結構的內部或外部。
表面電漿共振(SPR)用於檢測BMPRII阻斷作用，SPR結果顯示在環狀結構內具有活性區域的胜肽與其它胜肽相比具有更高的鍵結親和力。此外，BMPR2阻斷所引起的體外活性由肌源性細胞C2C12檢測。在與不同肽共培養 C2C12 後，通過RUNX2的西方墨點法和鹼性磷酸酶(ALP) 染色驗證BMP-2信號傳導和骨分化。
基於親和力和體外實驗結果，與線性肽相比，胜肽P-05是最適合成骨的選擇，其具有更高的鍵結親和力、RUNX2、ALP的表現增加。。
摘要(英) In 2007, Bone morphogenetic protein -2 (BMP-2) protein has been used extensively in clinic. However, some information from Food and drug administration (FDA) data had revealed that BMP-2 protein could induce the complications frequently, such as cervical swelling and corresponding dyspnea and dysphagia requiring intubation. Among these, more than 20 to 70 % of cases were life threatening. Although FDA issued a warning in 2008 about the safety and efficacy of BMP-2 protein, BMP-2 remains the preferred option for orthopedic trauma. Many researchers are searching an alternative of BMP-2. Basically, BMP-2 protein functions through formation of hetero-tetrameric complex with type I and type II receptors of BMP. Many type II receptors has been revealed their increasing osteogenic signaling, such as bone morphogenetic protein receptor-II (BMPRII) and activin receptor (ACVR2A/2B). It became a potential target to limit the activity of BMP-2 through blocking BMPR2 by using antibodies, small molecules, aptamers and peptides. In spite of antibodies, small molecules and aptamers with their unstable drawbacks, peptides could be considered as a better alternative. Comparing with others, peptides was with their ease of synthesis, handling, biodegradability as well as low immunogenic activity. In this study, we designed a BMP-2-derived peptide with BMP-2 linear knuckle epitope (73-92). The linear peptide was cyclized by incorporating two cysteine residues into different sequence. Four cyclic peptides were prepared respectively. The active region in C-terminal of the peptide could be either inside or outside the cyclic structure. Then, BMPRII-blocking activity was performed by surface plasmon resonance (SPR). The SPR study showed that the peptides with an active region inside the cyclic ring had a higher binding affinity in comparison to the other peptides. In addition, the in-vitro activity caused by BMPR2 blocking was carried on myogenic C2C12 cell lines. After co-culture C2C12 with different peptides, BMP-2 signaling and osteo-differentiation were performed by western blotting of RUNX2 and staining of alkaline phosphatase (ALP). Based on the affinity data and the in vitro experiments, peptide P-05 could be a suitable candidate for osteogenesis, with higher binding affinity and increased RUNX2 and ALP expression in comparison to the linear peptides.
關鍵字(中) ★ 骨型態發生蛋白
★ 環形胜肽
★ 骨型態發生蛋白關節表位
★ 雙硫鍵
★ 肌原細胞		 關鍵字(英) ★ Bone morphogenetic protein (BMP-2)
★ Myogenic cells
★ BMP-2 knuckle epitope
★ Disulphide bridging
★ Cyclic peptide
論文目次 Chinese abstract ……………………………………………………………… i
English abstract ……………………………………………………………… ii
Acknowledgments ……………………………………………………………… iv
Declaration ……………………………………………………………… v
Table of contents ……………………………………………………………… vi
List of figures ……………………………………………………………… xi
List of tables ……………………………………………………………… xiv
Abbreviation ……………………………………………………………… xv
CHAPTER I
I. Introduction .………………………………………………….. 1
CHAPTER II
II. Literature Review, Background and Hypothesis
2.1. Literature review and Background 3
2.1.1. BMP-2 protein binds with multiple receptor complex……………….. 3
2.1.2. ACVR2A/ACVR2B functions as BMPRII………………………… 4
2.2. Hypothesis…………………………………………………….. 6
2.3. Molecules to modulate activation of receptors.………………… 7
2.3.1. Antibody as drug molecules.…………………………………… 8
2.3.1.1. Higher costs of antibodies production…………………………….. 8
2.3.2. Small molecules.……………………………………………….. 9
2.3.3. Aptamers………………………………………………………. 9
2.3.4. Peptides……………………………………………………… 10
2.3.4.1. Peptides in clinic for bone related disorders……………………… 11
2.4. BMP-2 based peptides and their applications.………………… 11
2.4.1. BMP-2 peptide immobilized on alginate hydrogel.…………………. 11
2.4.2. Fragments of BMP-2 protein knuckle epitope and its activity……… 13
2.4.3. BMP-2 peptide with rat calf muscle and its dosage.………………… 14
2.4.4. BMP-2 peptide in combination with Bone marrow stromal cells.…….. 15
2.4.5 Repair of 20 mm radial fractures in Rabbit with 73-92 knuckle epitope…………………………………………………………
15
2.5. Different ways to increase the activity of BMP-2 peptide.………………………………………………………..
15
2.5.1. Bicyclic analogue of somatostatin peptide.………………………… 16
2.5.2. Conformational constraint in RGD peptide with organic moiety………………………………………………………… 17
2.5.3. Types of cyclization……………………………………………. 18
2.6. Cyclic disulphide peptides and their properties…………………….. 19
2.6.1. Structure of Nesiritide…………………………………………… 21
2.6.2. Disulphide bonds as redox switches………………………………. 22
2.6.3. Disulphide bonds enhances pharmacological properties……………... 22
2.6.4. Proteolytic stability of Disulphide bonds…………………………… 22
2.6.5. Disulphide bonds stabilizes the secondary structures………………… 23
CHAPTER III Materials and Methods
III. Materials
3.1.1. Chemical reagents for peptide synthesis…………………………… 24
3.1.2. Biological reagents and proteins………………………………….. 26
3.1.3. Equipment’s and glassware……………………………………… 28
3.2. Methodology
3.2.1. Peptide Design.………………………………………………… 30
3.2.2. Study of molecular events for peptide evaluation…………………… 31
3.3. Experimental section…………………………………………….. 33
3.3.1. Peptide synthesis……………………………………………… 33
3.4. Interaction of peptides with BMPRII……………………………… 38
3.5. Cell Culture…………………………………………………… 39
3.5.1. Staining to study cell differentiation………………………………. 39
3.5.2. Expression of cell signaling marker……………………………… 40
3.5.3. Quantification of cell differentiation (ALP assay).…………………. 40
3.5.4. Application of peptide………………………………………… 41
3.5.5. Thermal stability of the linear (P-01) and cyclic peptide (P-05)……… 42
3.5.6. pH stability of linear (P-01) and cyclic peptide (P-05)………………. 43
3.5.7. Preparation of GHG (Gelatin-Hydroxyapatite and Glutaraldehyde) scaffold…………………………………………………………
43
CHAPTER IV

IV. Results
4.1. Analysis of peptides
4.1.1. HPLC analysis………………………………………………….. 45
4.1.2. Mass Spectroscopic analysis……………………………………… 47
4.2. Interaction of the peptides with BMPRII……………………… 50
4.3. Differentiation of C2C12 cells to osteogenesis……………………. 53
4.3.1 Dose dependent curve…………………………………………… 53
4.3.2. Cell differentiation……………………………………………… 54
4.3.3. Cell signaling marker (Anti RUNX2 antibody)..…………………… 55
4.3.4. Quantification of osteogenic differentiation (ALP assay).…………… 56
4.3.5. Stability of peptides at higher temperatures………………………… 57
4.3.6. Cell differentiation of heat-treated peptides………………………… 59
4.3.7. Cell differentiation (ALP staining of pH-treated peptides)…………… 61
4.3.8. Application of linear and cyclic peptides in the scaffold…………… 63
CHAPTER V
V. Discussion……………………………………………………65
CHAPTER VI
VI Conclusion…………………………………………………….. 71
CHAPTER VII
VII. Future Work………………………………………………… 72
CHAPTER VII
VIII. References…………………………………………………….. 76
CHAPTER IX.
IX Appendix
9.1. Conferences…………………………………………………… 81
9.2. Publications…………………………………………………… 82
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指導教授 董國忠 羅月霞(Guo-Chung Dong Yueh-Hsia Luo) 審核日期 2022-1-22
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