開發活體多光子的眼表影像平台

Abstract

透明的角膜是保護眼睛的第一屏障並避免外部感染。輪部上皮和角膜上皮的分層結構有助於角膜維持生理平衡與再生。儘管在體外實驗中研究過各種信息路徑，但開發活體小鼠的眼表影像是必要的。在本研究，我們開發了一個活體影像系統，其使用多光子顯微鏡與和帶有標記螢光特定細胞群的基因轉殖小鼠進行不同細胞種類和各種細胞結構的成像和追蹤。藉由我們的影像平台以及螢光蛋白的表現，我們可以獲取從整個角膜從上皮到內皮甚至橫跨到結膜的三維活體細胞級解析度的影像。在K5-H2B-EGFP小鼠的EGFP有助於辨別角膜上皮細胞和輪部上皮細胞，而R26R-GR小鼠的廣泛細胞核螢光蛋白讓我們可以觀察到所有種類細胞的細胞核。mT-mG小鼠所有細胞膜上的螢光蛋白可以界定出全部細胞的邊界、神經纖維和血管。同步收集來充滿角膜膠原間質層的二倍頻訊號提供結構上的位置對照。縮時攝影紀錄也觀察到角膜上皮和輪部上皮細胞的細胞分裂行為。透過二維影像手動選取細胞核的邊界進而重組三維細胞切割，發現基底細胞的三維細胞核體積從中央角膜顯著性增加到周邊角膜。有趣的是，周邊角膜的基底細胞密度與中央角膜的密度相近。透過FUCCI mice發現位於周邊的角膜上皮細胞進入S/G2/M期之數量多於在中央角膜和輪部上皮細胞。因此我們提出在細胞增殖和細胞損失之間達成平衡才能維持角膜上皮的基底細胞密度。利用K5creER; mT-mG mice發現基底細胞會在24小時內向上脫離基底層。根據周邊角膜的高增殖率，我們進一步探討哺乳動物標靶雷帕黴素複合物1(mTORC1)在角膜上皮中的角色。在生理平衡時，mTORC1僅表達在角膜中的翼細胞和表層細胞而機械清創後在輪部上皮的mTORC1會被快速活化。儘管剃除raptor基因的小鼠在角膜上皮中仍保有屏障功能，但p63表現減少導致角膜上皮的厚度下降。因此，mTORC1信號被認為藉由受損的細胞增殖來減少角膜上皮的厚度。在這項研究中，我們開發了一個活體影像平台來追踪單個細胞，其是一個潛在的眼科藥物篩選系統。我們還證實mTORC1信號在角膜上皮的角色，這對於眼科研究至關重要。

The transparent cornea acts as a first ocular barrier and protects against external infections. The hierarchical organization of limbal epithelium and corneal epithelium contribute to homeostasis and regeneration. Despite the in vitro signals pathways being elucidated, the development of in vivo imaging platform is indispensable for murine ocular surface. Here, we have developed an intravital imaging system to image different cell types and various cell components in ocular surface using multiphoton microscopy in transgenic mice of which specific cell populations are labeled with fluorescent proteins (FPs). With our imaging platform and the expression of FPs, we obtained the three-dimensional images across the whole cornea from epithelium to endothelium and in conjunctiva with subcellular resolution in vivo. Specified EGFP expression in corneal epithelium of K5-H2B-EGFP mice helped to identify both corneal and limbal epithelial cells while ubiquitous nuclear FP expression in R26R-GR mice allowed us to visualized nuclei of all cell types. Universal membrane-localized FP in mT-mG mice outlined all cell boundaries, nerve fibers, and capillaries. The simultaneously collected second harmonic generation signals from collagenous stroma provided architectural contrast. Time-lapsed recording enabled monitoring the mitotic activity of corneal epithelial cells and limbal epithelial cells. The 3D nucleus volume of basal cells significantly increases from the central cornea to the peripheral cornea through manually segmenting the boundary of the nucleus from 2D slices and further refinement. Interesting, the basal cell density in peripheral cornea is closed to that in central cornea. Through FUCCI mice, the number of corneal epithelial cells entry into S/G2/M phases in peripheral is more than that in the central cornea and the limbus. Therefore, we propose a balance between cell proliferation and cell loss achieves basal cell density in corneal epithelium. In addition, basal cells detached from the basal layer within 24 hours using K5creER; mT-mG mice. According to the high proliferation rate in peripheral cornea, we further investigate the role of the mammalian target of rapamycin complex 1 (mTORC1) in corneal epithelium. The mTORC1 only expressed in the cornea, including wing cells and superficial cells during homeostasis while the mTORC1 in limbus is rapidly activated after mechanical debridement. Although raptor deletion in corneal epithelium maintains barrier function, the p63 expression decreases results in the thickness of corneal epithelium reduction. Hence, the mTORC1 signals are supposed to decrease thickness of corneal epithelium via impaired cell proliferation. In this study, we have developed an in vivo imaging platform to trace a single cell, which is potential for ophthalmologic drug screening system. We also have characterized the mTORC1 signaling in corneal epithelium, which is vital for ophthalmologic research.