2005 Fiscal Year Final Research Report Summary
Functional analyses of novel collagen formed network-matrix and its application for cell culture
| Project/Area Number |
16500307
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biomedical engineering/Biological material science
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| Research Institution | Kinki University |
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Principal Investigator |
MORIMOTO Koichi Kinki University, Faculty of Biology-Oriented Science and Technology, Assistant Professor, 生物理工学部, 講師 (10319741)
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| Co-Investigator(Kenkyū-buntansha) |
SAITO Takuya Kinki University, Faculty of Biology-Oriented Science and Technology, Associate Professor, 生物理工学部, 助教授 (30101398)
YOSHIKAWA Takafumi Nara Medical University, Department of Orthopaedic Surgery, Assistant Professor, 整形外科, 講師 (90275347)
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| Project Period (FY) |
2004 – 2005
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| Keywords | Collagen / Cell culture / Cell adhesion / Neutrophil / Fibroblast 3T3-L1 / Immunoelectron microscopy / Fluorescence microscopy / Scanning electron microscopy |
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| Research Abstract |
Type I collagen is the most abundant component in tissues such as skin and assembles spontaneously to fibrils in vitro in physiological conditions. It has been known that collagen has biological functions as well as mechanical functions.
Methods : Atelocollagen and AP-collagen were purified from chicken skin by pepsin and actinidain limited digestion, respectively. The purified collagen species were identified by 5% SDS-polyacrylamide gel electrophoresis. Morphological difference between two collagen matrices was observed by an S-900 (Hitachi) ultra-high resolution scanning electron micrograph (SEM). By using anti-collagen monoclonal antibody (COL-1), we compared immunoreactivities against two collagen matrices. We examined each polypeptide fragment digested by collagenase to obtain further evidence for the ultra-structural difference. In addition, to evaluate whether two collagen matrices may lead to recruitment of neutrophils, we observed biological behaviors of mouse neutrophil on the collagen matrices by SEM.
Results : The SDS-PAGE pattern showed that the atelocollagen consists of at least four components : α1, α2, β, and γ chain. In contrast, the AP-collagen gave two bands corresponding to α1 and α2 chain. The immunoreactivities of COL-1 to two collagen matrices were different from at pH 4.0 and at pH 6.5 conditions. Digested fragments of two collagen matrices showed different pattern in SDS-PAGE. It seemed that the adhesive and the migration activity of neutrophil were clearly different between two collagen matrices.
Conclusion : The AP-collagen formed the unique matrix such as three dimensional meshwork layers. Additional lines of evidence of the recognition sites of COL-1 antibody and collagenase against two collagen matrices strongly indicated difference in the ultra-structure. We demonstrated that the AP-collagen matrix clearly enhances the neutrophil adhesion and migration activity.
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