利用二倍頻顯微術觀察膠原蛋白的自我聚集過程

Abstract

二倍頻顯微術用來對膠原蛋白鷹架成像已經被確立為一個可以實行且有效的技術；對於描述膠原蛋白的結構特性，這個顯微技術也同時兼具了低侵入、低光學破壞的特性。由於膠原蛋白是人體中含量最多的蛋白質之一（特別是第一類的膠原蛋白），其自我聚集過程而使其成為具有功能性的單元是許多組織中一個維持平衡的重要過程。可是膠原蛋白自我聚集過程的詳細動力機制卻仍不清楚。因此，在這個研究中，我們利用以脈衝式雷射及掃描系統作為基礎的二倍頻顯微術，「即時的」觀察膠原蛋白的自我聚集過程。況且利用二倍頻顯微術監測膠原蛋白的自我聚集過程來得到這個過程中的結構性資訊，對於理解膠原蛋白生成纖維的過程是相當重要的。 我們實驗中所用的膠體，在過去，已被廣泛的透過其他不同的技術所研究。這些技術像是電子顯微鏡，反射式共軛焦顯微鏡以及光譜光度分析儀都曾用來研究膠原蛋白以及其自我聚集的過程。然而，只有需要非中心對稱結構的二倍頻能夠讓我們明確的研究形成膠原蛋白纖維的過程，並且不需要做額外的樣本處理。此外，前向對後向的二倍頻比值更可以得到膠原蛋白纖維直徑的資訊。 我們的結果證明了利用二倍頻顯微術可以對活體外的膠原蛋白自我聚集過程進行研究；配合更多的發展，這個方法更可以延伸至活體內的膠原蛋白纖維生成過程的研究

Second harmonic generation (SHG) microscopy has already established as a viable and useful technics in imaging collagen scaffold; it is also holds promise as a noninvasive, less photodamage imaging technics for characterizing collagen structure. Since collagen, especially type-I collagen, is one of the most abundant protein in human body, its assembly into functioning units is an important process in maintaining homeostasis of many tissue types. However, the kinetic details of collagen self-assembly process remains unclear. Therefore, in this study, we used SHG microscopy based on a pulsed laser scanning system to monitor the collagen self-assembly process in real-time. Using SHG microscopy to monitor collagen self-assembly process can provide structural informationsI that is important for understanding the fibrillogenesis process. We used self-assembly in collagen hydrogel as our model as this system has already been investigated extensively using other many different techniqus. Electron microscopy, reflected confocal microscopy, and spectrophotometry had been widely used to investigate the process of collagen self-assembly. However, only the non-centrosymmetric requirement of SHG allows the assembly into collagen fibrils to be studied unequivocably and without additional specimen processing. Moreover, the informations of forwad and backward SHG ratio provide information of the fibril diameter. Our results demonstrate that the collagen self assembly process can be studied by SHG microscopy in vitro, with additional development; our approach can be extended to in vivo investigations of the fibrillogenesis process.