運用雙光子和二倍頻顯微術來研究人類骨髓間葉幹細胞在幾丁聚醣鷹架上的組織形成

Utilizing two-photon fluorescence and second harmonic generation microscopy to study human bone marrow mesenchymal stem cell morphogenesis in Chitosan scaffold

黃啟修(Chi-Hsiu Huang)

指導教授 ： 董成淵

國立臺灣大學/理學院/物理研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.02859

摘要

雙光子顯微術由於使用波長較長的近紅外光作為激發光源，所以具有穿透深度深、較少的光破壞，且可以激發所有可見光波段的自發螢光和二倍頻訊號，使得我們可以直接透過自發螢光和二倍頻訊號來觀測樣品，而不需要將樣品切片和染色。因此，我希望能將這種非線性光學顯微術技術來應用於組織工程上的觀測與分析。

在組織工程裡，培養體外軟骨組織用來移植一直是組織工程極欲努力研究的目標。最常用的方式就是將間葉幹細胞種入三維可分解和可降解的生物相容性鷹架材料，然後再加入適當的訊息因子-TGF-β3來誘導細胞產生膠原蛋白，進而長成軟骨組織。在這個研究裡，我們將間葉幹細胞種入幾丁聚醣鷹架中，加入訊息因子TGF-β3來誘發軟骨組織的產生，並且利用雙光子螢光和二倍頻顯微術來取得這個過程中的影像。

過程中會發現，藉由加入TGF-β3刺激所產生的膠原蛋白，在四個禮拜左右的期間就可以被觀察到。膠原蛋白的產生與時間並非成線性的關係，大約到了第二十一天的時候就達到飽和了，且膠原蛋白產生會依據鷹架孔洞的形狀而生長。

關鍵字

雙光子 ； 二倍頻 ； 組織工程 ； 間葉幹細胞 ； 幾丁聚醣

並列摘要

Multiphoton microscopy ,using longer wavelength of near infrared light as excitation light source has deeper penetration and less photodamage. Besides,it can capable all visible autofluorescence and second harmonic generation,making us observe specimens through autofluorescence and second harmonic generation directly without slicing and dying. Hence,I hope to apply this technology of nonlinear optical microscopy to observe and analysize in tissue engineering.

One important goal in tissue engineering is cultiviating cartilage tissue for transplantation.The most common method to cultivate cartilage is to seed human bone marrow mesenchymal stem cells on three dimensional biocompatiable and biodegradable scaffold materials,then add the signal factor TGF-β3 to induce the cells to produce collagen that would grow into cartilage tissue.In this study,we seeded mesenchymal stem cells on a chitosan scaffold and added TGF-β3 to induce cartilage tissue producing and image the process using two-photon fluorescence and second harmonic generation microscopy.

In the course of imaging,we found that the production of collagen by the addition of TGF-β3 can be followed over a four-week period. The growth of collagen is not linear with time,and it approaches saturation at around 21th day.And the collagen fibers have a tendency to grow following the shape of scaffold pores.

並列關鍵字

Two photon ； Second harmonic generation ； Tissue Engineering ； Mesenchymal stem cell ； Chitosan

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