雙性離子材料結合胜肽製成水凝膠對人類脂肪組織幹細胞 培養與分化的影響

The Influence of pSBMA Hydrogels Incorporated with RGD/QK Peptides on The Human Adipose-derived Stem Cells Proliferation and Differentiation

王思文(Shih-Wen Wang)

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(2015年)

https://doi.org/10.6342/NTU.2015.02292

摘要

抗結垢表面(anti-fouling surface)為許多生醫材料之重要特性之一。兩性離子材料憑藉著結構中同時帶有部分正負電誘導出高水合的特性，此高水合特性使得以此做為基材的生醫材料表面，亦能擁有良好的抗結垢性質。因此，兩性離子材料在在組織工程上為目前一廣為研究的主題。在本研究中，我們致力於發展生醫材料結合人類脂肪組織幹細胞應用於組織工程。
二維水凝膠表面研究除了材料機械強度測試外，實驗架構主要探討人類脂肪組織幹細胞(hASCs)在胜肽改質的磺基甜菜鹼單體(sulfobetaine mathacrylate) 雙性離子材料水凝膠表面上生長情形，其中包含細胞貼附測試、細胞增生測試以及促進分化後其相關基因表現的定量。並分別討論輔以接枝不同功能之胜肽鏈，比較出人類脂肪組織幹細胞在不同組別的磺基甜菜鹼基雙性離子水凝膠表面上表現關係。首先，利用RGD胜肽鏈修飾以標定細胞吸附能力，確立人類脂肪組織幹細胞的最佳化培養條件外並比較在不同濃度的RGD修飾下對脂肪分化及硬骨分化的表現影響。再者，加入QK胜肽鍵並觀察此胜肽鍵對人類脂肪幹細胞分化的影響。
主要研究在探討將人類脂肪組織幹細胞培養在不同濃度的RGD-modified 磺基甜菜鹼基雙性離子水凝膠上並觀察細胞貼附情形及誘導脂肪組織幹細胞其脂肪分化及硬骨分化的效果。研究結果發現：(1) 接枝RGD序列能有效增進細胞貼附性質；(2) 接枝QK序列對於人類脂肪組織幹細胞並無貼附效果；(3) 接枝QK序列對於人類脂肪組織幹細胞除了改變機械性質而影響脂肪分化與硬骨分化效果外，仍在探討其對內皮分化的影響；(4) 雖然接枝不同功能之胜肽序列對於細胞在二維材料平面上有顯著的影響，但相對的添加如上述的序列會降低水凝膠的機械強度而影響人類脂肪組織幹細胞的分化表現。

關鍵字

雙性離子材料水凝膠 ； 人類脂肪組織幹細胞 ； 脂肪分化 ； 硬骨分化 ； 胜肽鍵

並列摘要

Zwitterionic compounds are electrically neutral ionic compounds, which have partially positive and negative charges simultaneously. Nonfouling surface has been widely used in biomaterials due to its potential resistance in non-specific adsorption. The distinctive highly hydrated and non-fouling features are the most important properties of zwitterionic compounds. Zwitterionic poly-sulfobetaine methacrylate (pSBMA) has been well studied for its superhydrophilic and ultralow biofouling properties, making it to be a promising material for high biocompatibility.
Adipose-tissue derived stem cells (ASCs), which are isolated from fat tissues, are easily obtainable and also can differentiate to multiple lineages, including adipogenesis, osteoblast and chondrocytes. ASCs have valuable applications for reconstruction and surgery to restore injury-related soft tissue loss within the subcutaneous layer.
Although pSBMA hydrogel being an ultralow biofouling material, it can be modified with peptides containing the amino acid sequence arginine-glycine-aspartic acid (Arg-Gly-Asp, RGD) to promote cell adhesion ability. In addition, the mechanical property, which plays a crucial role directing the cellular functions and supporting the structures, decreased when peptides graft onto hydrogels. Manipulating the mechanical property was thus necessary and the most related factor was the monomer concentration.
A chemical crosslinking poly-sulfobetaine methacrylate hydrogel was employed to form an in situ gel that was embedded with two formulations of peptide containing surface-modified and human adipose-derived stem cells. In this study, seeded human adipose-tissue derived stem cells both on different concentration RGD-modified and RGD/QK peptides-modified pSBMA hydrogel, through in vitro cell culture studies to observe hASCs cell adhesion ability and differential potential of adipogenic and osteogenic lineage by analyzing gene expression and intracellular lipid accumulation. And compared the differential potential via RT-PCR and staining quantification.Though no significant difference showed in gene expression, staining quantification indicated that cells cultured in higher RGD concentiraion condition might have better differential potential at day 7 in adipogenic lineage. For the differential potential comparison of RGD/QK groups, even though positive gene expression showed up at day 10 in both culture environment. The osteogenic differentiation showed no significant difference in RGD and RGD/QK groups.

並列關鍵字

Zwitterionic hydrogel ； human adipose-tissue derived stem cells ； adipogenesis ； osteoblast ； peptide-grafted

參考文獻

[1] Jiang S, Cao Z. Ultralow-fouling, functionalizable, and hydrolyzable zwitterionic materials and their derivatives for biological applications. Advanced materials. 2010;22:920-32.

[2] Mi L, Jiang S. Integrated antimicrobial and nonfouling zwitterionic polymers. Angew Chem Int Ed Engl. 2014;53:1746-54.

[3] El-Sherbiny IM, Yacoub MH. Hydrogel scaffolds for tissue engineering: Progress and challenges. Global cardiology science & practice. 2013;2013:316-42.

[4] Vats K, Benoit DS. Dynamic manipulation of hydrogels to control cell behavior: a review. Tissue Eng Part B Rev. 2013;19:455-69.

[5] Hwang YS, Zhang C, Varghese S. Poly(ethylene glycol) cryogels as potential cell scaffolds: effect of polymerization conditions on cryogel microstructure and properties. J Mater Chem. 2010;20:345-51.