人類軟骨細胞無血清培養基之開發與應用

Development of Serum-Free Medium for Human Chondrocytes

吳俊賢(Chun-Hsien Wu)

指導教授 ： 朱一民

國立清華大學/工學院/化學工程學系/碩士(2006年)

https://doi.org/10.6843/NTHU.2006.00092

摘要

此研究當中包含了三個部分包括：製造膠原蛋白二型載體、探討各種醣胺素對於人類軟骨細胞的影響以及人類軟骨細胞體外無血清培養基的開發。第一個部分當中，我們已經成功地從牛支氣管萃取出膠原蛋白第二型，再以genipin和膠原蛋白第二型載體交聯。交聯後確實提高了材料的強度以及物化性質，此仿效人類天然軟骨的載體將可供關節軟骨細胞正常地生長。第二部分主要是先篩選出醣胺素的種類及濃度對於軟骨細胞的影響，藉由添加在培養基當中觀察軟骨細胞的生化合成速率以及基因表現。實驗結果顯示添加10μg/ml CS-A、50μg/ml CS-B、50μg/ml CS-C、5μg/ml HS和500kDa Hyaluronan都能夠有效的增加胞外間質分泌與細胞增生速率，Aggrecan和Collagen type II這兩個基因表現也都有上昇的現象。雖然添加沒有裂解 (non-depolymerization) 的長碳鏈CS能夠使軟骨細胞正常分化及生長，但我們發現短碳鏈的CS-C6 (六個醣的CS-C) 對於軟骨影響更大，無論是調升應有的基因表現或者是抑制分解酵素基因的表現，都說明了添加短碳鏈的CS於培養基中的確有較佳的結果。最後一部份則是著重在無血清培養基的開發，先挑選七種軟骨細胞常用的生長因子，利用二階因子的實驗設計方法來篩選出人類軟骨細胞增殖及分化所需要的血清取代物組成，並搭配陡升路徑的實驗來找尋其適切的濃度，最終結果的配方：10.26 ng/ml FGF-2、10.30 ng/ml EGF、10.42 ng/ml TGF-β1、53.00 ng/ml BMP-2 和 11.01 ng/ml VEGF。此種經由實驗設計所開發出的無血清培養基，在軟骨細胞的主要基因表現上確實跟添加血清的表現量是雷同的，因此證明本研究所開發的無血清培養基是具有其應用價值。

關鍵字

膠原蛋白二型 ； 醣胺素 ； 無血清培養基 ； 軟骨細胞 ； 生長因子

並列摘要

In this study, there are there major parts including fabricating collagen type II scaffold, various glycosaminoglycans effect on chondrocytes, and serum-free media design for chondrocytes. In first part, we successfully isolated collagen type II from bovine trachea and crosslinked by genipin. Crosslinking by genipin indeed reinforced the scaffold intensity and physico-chemical characteristics. In second part, our aim is to investigate the effect of various glycosaminoglycans into medium cultured with genipin-crosslinked collagen type II scaffolds. Our results suggest that chondrocytes cultured within genipin-crosslinked scaffolds may show better tendency for differentiation by adding certain dose of GAGs (10μg/ml CS-A, 50μg/ml CS-B, 50μg/ml CS-C, 5μg/ml HS, and 500kDa Hyaluronan). On transcription level we show that treatment with CS-A, CS-C, and 500kDa HA lead to a significant up-regulation of collagen type II and aggrecan expression. These indicate that supplement with GAGs into medium are potent for promoting excreting ECM of normal cartilage. Although non-depolymerization CS-A and CS-C act as good addition of cultured medium, CS-C6 (hexasaccharides of CS-C) may be more powerful because it can inhibit further degradation due to catabolic activities, in the meantime heighten the anabolic ability. In the last part, seven kinds of growth factors which are most often used for chondrocytes growth and differentiation are selected. The 27-3 fractional factorial design is adopted here to determine what growth factors are required in the chondrogenic culture. There were five growth factors left to determine concentration in the serum-free medium along the steepest ascent path. Applicable serum-free and growth factor-containing medium for the chondrocytes is developed, which is composed of DMEM containing 10.26 ng/ml FGF-2, 10.30 ng/ml EGF, 10.42 ng/ml TGF-β1, 53.00 ng/ml BMP-2, and 11.01 ng/ml VEGF. Finally, we conclude that our serum-free medium including glycosaminoglycans and growth factors are functionally substituted for animal serum.

並列關鍵字

collagen II ； Glycosaminoglycans ； chondrocytes ； Growth Factors ； Serum-free medium

參考文獻

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