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Recombinant human fibroblast growth factor 7 obtained from stable Chinese hamster ovary cells enhances wound healing

  • 김영식 (Young Sik Kim, 아주대학교 일반대학원)

초록/요약

Wounds can occur due to various causes such as physical trauma or infection. To facilitate the healing of such wounds, it is necessary to apply active treatments utilizing growth factors like FGF7 or appropriate wound dressings combined with growth factors. FGF7, produced in large quantities from CHO cells, and animal models treated with FGF7 and collagen patches have showed effective promotion of wound healing. CHAPTER I: Fibroblast growth factor 7 (FGF7) is recognized as one of the growth factors promoting wound healing. However, study on its mass production remains scarce, particularly regarding its feasibility in Chinese hamster ovary (CHO) cells. Therefore, this study aimed to evaluate the potential of CHO cells for large-scale production of recombinant FGF7 and assess its efficacy in wound healing model. The FGF7 gene was stably transfected into CHO cells. A single clone was selected from the transfected CHO cell population using semi-solid method. The selected single clone achieved a productivity of 102.8 ± 6.8 mg/mL (9-day culture) through MTX amplification, screening of culture media, and optimization of culture temperature. The produced FGF7 had a molecular weight of 20-30 kDa due to oxidation, deamidation, and glycosylation, with an isoelectric point ranging from 4.61 to 7.34. The glycosylated FGF7 exhibited a complex N-glycan pattern with over 14 different types of glycans attached, and its biological activity showed a slightly higher activity of 7.49 ng/mL compared to the activity of E. coli-derived FGF7, which was 7.74 ng/mL. In conclusion, this study showed that the glycosylated FGF7 produced in CHO cells exhibits stable large-scale production. In summary, this study showed the capacity for FGF7 mass production in CHO cells and its potential as a biopharmaceutical. CHAPTER II: To facilitate rapid recovery of wound sites, the application of appropriate wound dressings and the supply of external growth factors such as FGF7, which is secreted during wound healing phase, can actively promote wound healing. The wound healing efficacy of produced N-glycosylated FGF7 was evaluated in animals on days 7 and 14 post-treatment using collagen patches (CP), FGF7 alone, and CP combined with FGF7 (CP+FGF7), with an untreated group serving as the control. Notably, wound closure was most effective in the CP+FGF7 group. Particularly, on day 7 post-treatment, both CP+FGF7 and FGF7-alone groups exhibited the highest expression of hydroxyproline, fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor β. Histological assessment by H&E staining revealed a parallel trend in epidermalization with hydroxyproline levels. Additionally, CP+FGF7 and FGF7-alone groups demonstrated notable vascularization on days 7 and 14. Therefore, this study has shown that using wound dressings in combination with FGF7 promotes wound healing more effectively than using wound dressings alone. In conclusion, FGF7 can be reliably mass-produced from CHO cells, and when used in combination with collagen patches, it significantly enhanced wound healing in animal models. Therefore, FGF7 production in CHO showed potential for commercialization due to its ability for large-scale production and its potential as a biopharmaceutical. Keywords: MTX amplification, culture media screening, recombinant FGF7, CHO, N- glycosylated, growth factors, wound healing, dressing, collagen patches

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목차

BACKGROUND 1
1. Skin function 1
2. Wound healing 3
2.1 Hemostasis and inflammation 3
2.2 Tissue regeneration 5
2.3 Tissue remodeling 5
3. Roles of growth factors in wound healing 8
3.1 Keratinocyte growth factor (KGF) 8
3.2 Fibroblast growth factors (FGFs) 9
3.3 Vascular endothelial growth factor (VEGF) 9
3.4 Transforming growth factor β (TGFβ) 10
3.5 Platelet derived growth factor (PDGF) 11
4. Wound dressing 13
5. The aim of this study 16
INTRODUCTION 17
CHAPTER I Stable cell line development of recombinant human fibroblast growth factor 7 in Chinese hamster ovary cells 20
ABSTRACT 21
I. INTRODUCTION 22
II. MATERIALS AND METHODS 24
II-1. Human FGF7 gene transfection 24
II-2. Preparation of FGF7 gene-transfected CHO 27
II-3. FGF7 gene amplification 27
II-4. Optimization of the culture conditions of FGF7 gene-transfected CHO cells 30
II-4-1. Media screening 30
II-4-2. Optimization of culture medium composition 30
II-5. Purification of FGF7 31
II-6. Characterization of FGF7 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting 31
II-7. Characterization of FGF7 by high-performance liquid chromatography (HPLC) 32
II-8. Determination of biological activity of FGF7 by BrdU assay 32
II-9. Glycosylation (N-Glycan) analysis of FGF7 33
II-10. Statistical analysis 34
III. RESULTS AND DISCUSSION 35
III-1. Preparation of FGF7 gene-transfected CHO cells 35
III-2. Clonal selection of FGF7 gene-transfected CHO cells 35
III-3. Optimization of FGF7 production from FGF7 gene-transfected CHO cells 38
III-4. Characterization of FGF7 produced from FGF7 gene-transfected CHO cells 43
III-5. Characterization of the glycosylation (N-Glycan) of FGF7 45
III-6. In vitro biological activity of FGF7 45
IV. CONCLUSION 50
CHAPTER II Efficacy assessment of the recombinant human fibroblast growth factor 7 with wound dressing (collagen patch) in wound healing animal model 51
ABSTRACT 52
I. INTRODUCTION 53
II. MATERIALS AND METHODS 55
II-1. Fabrication of FGF7-loaded collagen patches (CP+FGF7) 55
II-2. Release of FGF7 in collagen patch 55
II-3. Animal experiment 57
II-4. Determination of wound contraction 59
II-5. Determination of hydroxyproline in wound animal model 59
II-6. Determination of protein in wound animal model 60
II-7. Histological stain analysis of the wound animal model 60
II-8. Immunohistochemistry for angiogenesis (CD31 labeling) 61
II-9. Statistical analysis 62
III. RESULTS AND DISCUSSION 63
III-1. Fabrication and release of FGF7 in collagen patch 63
III-2. In vivo evaluation of wound contraction 63
III-3. Evaluation of inflammation and formation of hydroxyproline in the wound 67
III-4. In vivo assessment of wound healing 70
III-5. In vivo assessment of wound healing via histological stain 72
III-6. In vivo assessment of wound healing via histomorphometric analysis 75
IV. CONCLUSION 79
CONCLUSION 80
REFERENCE 81
국문요약 91

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