角蛋白添加幾丁聚醣對於豬脂肪幹細胞生長及分化之影響

The proliferation and differentiation of porcine adipose-derived stem cells by keratins incorporated with chitosan-Az

黃耀樟(Yao-Chang Huang)

指導教授 ： 游佳欣

國立臺灣大學/工學院/化學工程學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.01691

摘要

隨著萃取、純化技術的進步，以及不論是從頭髮、羊毛等來源取得的角蛋白的特性有更多的認識，有關角蛋白的研究與應用也前仆後繼地不斷有人去鑽研探討。角蛋白是個具有特有的生物活性、生物降解性及良好的生物相容性材料，而且由於它取得來源簡單、便宜，因此也是個實用方便的生醫材料。另外，硬骨修復一直是臨床上很熱門的課題，特別是在大型骨頭修復再生上，所以使用有再生能力的生醫材料在硬骨組織工程也有相當廣泛的研究。在本研究中，我們希望以角蛋白作為生醫材料的基底，並加入幾丁聚醣以期改善角蛋白本身極為脆弱的機械性質，之後在將細胞培養於這樣的基材上觀察其促進細胞的貼附及增生的能力，然後會利用誘導因子促進分化成硬骨細胞，並且對於在硬骨組織工程的應用上是否有良好的幫助。
本研究中，我們先從頭髮中萃取出角蛋白，並利用SDS－PAGE確認此蛋白質是否為我們要的角蛋白，之後加入經改質過後的幾丁聚醣加強其機械性質，並將此混合後的材料做成膜跟支架，將細胞養在這兩種方式上，分別觀察幹細胞在上面的貼附及增生的情況，並進一步的加入硬骨細胞的誘導因子，觀察幹細胞在這材料上的分化情形。經由測試證實，我們能夠成功地從頭髮中萃取出我們所需的角蛋白，並且此角蛋白能夠提供良好的環境給細胞貼附並且生長；而在加入幾丁聚醣後，也大大的提升了角蛋白本身較缺乏的機械性質。在誘導分化方面，在替換含誘導因子的培養液後，可以發現細胞型態有所改變，並在後續的測試中證實幹細胞確實有成功的分化成硬骨幹細胞，並且有持續的增長。
本研究中，我們成功的證實角蛋白是個具有良好生物活性及生物相容性的材料，並能順利促進幹細胞的貼附，增生及分化。因此，角蛋白未來在組織工程應用中還有極大的發展潛力。

關鍵字

角蛋白 ； 幾丁聚醣 ； 膜 ； 支架 ； 硬骨組織工程 ； 脂肪幹細胞

並列摘要

Advances in the extraction, purification, and understanding of the characterization of keratin proteins from hair and wool let researchers continuously study the application of keratin-based biomaterials. Keratins have intrinsic bioactivity, biodegradability, and biocompatibility materials. Keratins are also convenient, natural biomaterials due to the source of keratins is convenient and cheap. In the other hand, issue of bone repair clinically is very popular, especially regeneration of large traumatic bone defects. Therefore, using of regenerative biomaterials has been extensively studied for bone tissue engineering.
In this study, we hope to use keratins as biomaterials base, and mix with chitosan which is modified to enhance the mechanical property of keratins. Then we cultured cell to the keratin-chitosan substrates, and observe the ability of cell adhesion and proliferation. Furthermore, we added differentiation-inducing factors to induce stem cells to become osteoblasts and observe it, and we wish to support some contribution to bone tissue engineering.
First, we extracted keratins from human hair, and used SDS-PAGE to confirm the protein whether the keratins which we wanted to get. Then we mixed with chitosan which is crosslinked by photochemical to strengthen the mechanical property of keratins, and fabricated the films and scaffolds with keratin-chitosan composite materials. After seeding cell on the films or scaffolds, we observe the situation of cell adhesion and proliferation on the films and scaffolds, respectively. We further induced and observed the stem cells to differentiate osteoblasts. Through our research, we succeeded to obtain the keratins from human hair, and these keratins could enable to provide suitable environment for cell attachment and proliferation. We also improved the keratin weak mechanical properties after mixing with chitosan-az. In the differentiation aspect, we could find the morphology of cells that is different with original cells, and we showed the stem cells to convert to osteoblasts, and still keeping growing up.
Our study successfully demonstrates keratins that have wonderful bioactive and biocompatibility materials. Keratins promote cell attachment, proliferation and differentiation. Hence, keratins may still have huge potential of development in tissue engineering in the future.

並列關鍵字

keratin ； chitosan ； film ； scaffold ； bone tissue engineering ； adipose-derived stem cells

參考文獻

1. Lysaght, M.J. and A.L. Hazlehurst, Tissue engineering: The end of the beginning. Tissue Engineering, 2004. 10(1-2): p. 309-320.

2. Jaklenec, A., et al., Progress in the Tissue Engineering and Stem Cell Industry "Are we there yet?". Tissue Engineering Part B-Reviews, 2012. 18(3): p. 155-166.

3. R Langer , J.V., Tissue engineering. Science, 1993. 260(5110): p. 920-926.

4. Hashin Z, S.S., A variational approach to the theory of the elastic behavior of multiphase materials. J. Mech. Phys. Solids 1963(11): p. 127-40.

5. Burg KJ, P.S., Kellam JF, Biomaterial developments for bone tissue engineering. Biomaterials, 2000. 21(23): p. 2347-59.