生物素與抗生物素系統應用於膝關節軟骨細胞貼附與生長之研究

Evaluation of avidin-biotin binding system in chondrocyte adhesion and proliferation

王閔正(Min-Cheng Wang)

指導教授 ： 蔡偉博

國立臺灣大學/工學院/化學工程學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.02299

摘要

本研究為針對抗生物素與生物素的鍵結系統，應用於膝關節軟骨細胞在2-D表面(TCPS和可分解高分子)的貼附與生長之研究。在本研究中提供了一種有效且方便的系統，來增加軟骨細胞的貼附量。研究中利用3-sulfo-NHS-biotn與細胞膜上蛋白質的NH2官能基形成醯胺鍵(amide bond)，可於細胞膜表面接上生物素，形成生物素化細胞，再於料表面吸附抗生物素分子，兩者作結合就可使此系統運用於軟骨細胞的貼附。研究結果顯示，此系統可增加一開始軟骨細胞附著的數量，並在短時間內使細胞攤平，於基因表現上還具有軟骨細胞的特性。
本研究更進一步的對於PLGA材料表面，將此系統配合，幫助細胞附著的蛋白質fibronectin混和使用。結果顯示利用混和系統的生物素化細胞，不但有快速貼附與攤平的效果，而且於七天的培養下細胞數目明顯的比正常細胞只運用幫助細胞附著的蛋白質fibronectin來的多。同樣的在基因表現方面上還保有軟骨細胞的性質。本研究成功的將此鍵結系統，運用於增加關節軟骨細胞於TCPS表面和可分解高分子表面的貼附與生長。此結果可以進一步的運用於3-D生物可生解鷹架的培養上，對於軟骨組織工程將更有應用的潛力與價值。

關鍵字

膝關節軟骨細胞 ； 生物素與抗生物素系統

並列摘要

Cell adhesion to synthetic biomaterials is a prerequisite for anchorage cell culture and tissue engineering. The current study investigated the utilization of the avidin-biotin binding system in enhancing chondrocyte adhesion to tissue culture polystyrene (TCPS) and biodegradable polymer surface. Porcine chondrocytes were biotinylated by using 3-Sulfo-NHS-Biotin, and avidin was immobilized onto material surface by adsorption. In this study, we revealed that avidin-biotin system can enhance initial chondrocytes adhesion and spread out in a short time. The progeny of biotinylated chondrocytes still maintained the ability of expressing cartilage extracellular matrix proteins, such as type II collagen and aggrecan. Otherwise, we also used avidin-biotin system with fibronectin on PLGA surface. Contrast with using combine system and single system, combine system can enhance cell adhesion than avidin-biotin system and fibronectin system. After seven days culture, the progeny of these cell still maintain the ability of expressing cartilage extracellular matrix proteins. These results indicate that the avidin-biotin binding system could therefore help in the area of cell culture and tissue engineering.

並列關鍵字

avidin-biotin binding system ； chondrocyte

參考文獻

Balcells, M. & Edelman, E. R. 2002. Effect of pre-adsorbed proteins on attachment, proliferation, and function of endothelial cells. J Cell Physiol 191, 155-61.

Bhat, V. D., Truskey, G. A. & Reichert, W. M. 1998. Fibronectin and avidin-biotin as a heterogeneous ligand system for enhanced endothelial cell adhesion. J Biomed Mater Res 41, 377-85.

Bogdanov, A.A., Jr. Gordeeva, L. V. Baibakov, B. A. Margolis, L. B. & Torchilin, V. P. 1991. Restoration of adhesive potentials of Ehrlich ascites carcinoma cells by modification of plasma membrane. J Cell Physiol 147, 182-90.

Catherine, D. Reyes, A. J. G. 2003. A centrifugation cell adhesion assay for high-throughput screening of biomaterial surfaces. Journal of Biomedical Materials Research 67, 328-333.

Chilkoti, A. Tan, P. H. & Stayton, P. S. 1995. Site-directed mutagenesis studies of the high-affinity streptavidin-biotin complex: contributions of tryptophan residues 79, 108, and 120. Proc Natl Acad Sci U S A 92, 1754-8.