乙二醇幾丁聚醣結合明膠之水凝膠上形成的人類脂肪幹細胞球其軟骨分化傾向

Chondrogenic Differentiation Potential of Human Adipose Stem Cell Spheroid Forming on Glycol Chitosan and Gelatin Hydrogel

陳信宇(Hsin-Yu Chen)

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(2016年)

https://doi.org/10.6342/NTU201602735

摘要

軟骨組織由於缺乏血管和神經系統，遭受損傷後極難修復，且由於其多於關節等骨頭相鄰處作為緩衝，受損未經修復容易惡化致使整個關節失去功能，因此修復軟骨組織為組織工程中一重要課題。人類脂肪幹細胞可發現於成人脂肪組織中，具有多種諸如脂肪、硬骨和軟骨等分化潛力，易於取得且可用於自體移植避免免疫反應之發生，已成為組織工程研究中一重要之細胞來源。乙二醇幾丁聚醣擁有和幾丁聚醣相似的特性且可溶於中性的水中，其結構和軟骨組織中的糖胺聚醣相似。此研究將其和明膠混合形成之水凝膠作為人類脂肪幹細胞之載體，觀察細胞生長情形和型態，並分析其軟骨分化之潛力。
明膠的存在可增進水凝膠的細胞貼附性質，然而明膠會隨著時間從膠中釋出。經觀察，幹細胞會逐漸在水凝膠表面聚集最後形成球狀，推測其原因可能為水凝膠之貼附性質降低，且幾丁聚醣的表面有助於幹細胞球的生成，即有研究是以幾丁聚醣形成之薄膜使幹細胞於其表面成球。細胞球的結構已被證實可以增進幹細胞的分化能力，傳統形成細胞球的技術諸如液滴、模具和幾丁聚醣膜等，其中細胞的聚集和避免細胞的攤平貼附為重要之因素。
細胞球之外觀和性質由顯微鏡和螢光染色等觀察之。而後以軟骨分化培養液對細胞球進行軟骨分化的刺激，在一定的天數以螢光染色和測量基因表現來分析軟骨分化的傾向。在水凝膠上以一定的細胞密度培養，可在一至兩天形成細胞聚集進而成球，然而由於明膠的脫離，水凝膠難以支持長時間的培養。
在軟骨分化方面，細胞的聚集成球對於幹細胞在SOX9和COL II兩者的基因表現有所助益，顯示對於軟骨分化的潛力。

關鍵字

細胞球 ； 乙二醇幾丁聚醣 ； 水凝膠 ； 軟骨分化

並列摘要

The repair of damaged cartilage is a challenge due to its hard self-repair in cartilage tissue engineering. Human adipose stem cells are proved that have the ability to differentiate into different linage including adipogenic, osteogenic and chondrogenic lineage. They are considered to be the cell sources in cartilage tissue engineering.
Glycol chitosan, which is a soluble derivative of chitosan, is studied for cartilage repairing due to its similar structure to glycosaminoglycans in cartilage and keeps the properties of biocompatibility and degradability as chitosan. In this study, the hydrogel was combined by glycol chitosan and gelatin which made cells able to attach on and its performance in cell culture was researched. This hydrogel could make cells adhere on its surface and make the hASCs form a sphere structure after culturing for several days. The formation of spheroids was attributed to the releasing of gelatin and the main material, glycol chitosan. These spheroids were observed by SEM and fluorescence and tested their properties and gene expression of differentiation.
There were studies showing that the stem cells in spheroids were viable mostly and had better differentiation ability. Moreover, the material we used was usually studied for cartilage tissue engineering. Therefor the chondrogenic differentiation potential of cells in spheroids was researched. We hope the GAGs-like glycol chitosan in hydrogel and formation of spheroids are able to promote the chondrogenic differentiation.
The results showed the chondrogenic differentiation performance appeared in spheroids. Moreover, the spheroids formed with induction medium were larger and had more complete shape of sphere. They also had higher performance in chondrogenic differentiation.

並列關鍵字

Spheroid ； Glycol chitosan ； Hydrogel ； Chondrogenic differentiation

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