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角膜缘干细胞是角膜上皮更新与修复的来源,角膜上皮受损严重常会导致角膜盲。尽管近几年通过角膜缘干细胞移植术(LSCT)治愈角膜上皮受损的临床应用已被推广,但是对于角膜缘干细胞移植受损机体后的修复机理并不明确。为了实现角膜缘干细胞移植后的活体追踪,使用G418筛选标记有Venus荧光蛋白的角膜缘干细胞株(GLSC-V),并以其为种子细胞接种于去上皮羊膜上,体外培养21d构建成荧光角膜上皮植片。荧光倒置显微镜下观察GLSC-V的细胞质和细胞核均有绿色荧光表达,在体外培养荧光至少持续3个月。免疫荧光检测GLSC-V细胞P63、Integrinβ1均呈阳性表达,对GLSC-V细胞及未转染的GLSCs进行半定量RT-PCR检测显示,两组细胞皆未表达终末分化角膜上皮细胞基因k3、k12,GLSC-V中p63及pcna较未转染组细胞略上调,venus强表达。经HE染色观察构建的人工角膜组织由5～6层上皮细胞组成,组织中上表皮细胞个数少、体积大且呈扁平状;基底部细胞密集、体积小且成立方状。经免疫荧光检测仅组织基底部最基层细胞表达P63,上表皮细胞不表达。该人工角膜与正常角膜上皮组织结构特性相似,可用于移植,为研究角膜缘干细胞修复严重受损角膜上皮机理奠定基础。
Limbal stem cells are the source of corneal epithelial renewal and repair, corneal epithelial damage often lead to corneal blindness. Although the clinical application of limbal stem cell transplantation (LSCT) to cure corneal epithelial lesions has been promoted in recent years, the mechanism of repair after damaged limbal stem cell transplantation is not clear. In order to achieve the in vivo tracking after limbal stem cell transplantation, the limbal stem cell line (GLSC-V) labeled with Venus fluorescent protein was screened by G418 and seeded onto the de-epithelial amnion using the G418 as a seed cell, and cultured in vitro for 21 days to construct a fluorescent cornea Epithelial plant. Under fluorescence inverted microscope, GLSC-V showed green fluorescence in both cytoplasm and nucleus, and cultured in vitro for at least 3 months. The positive expression of P63 and Integrinβ1 in GLSC-V cells was detected by immunofluorescence. Semiquantitative RT-PCR analysis of GLSC-V cells and untransfected GLSCs showed that both groups did not express terminal differentiated corneal epithelial cells k3, k12, p63 and pcna in GLSC-V were slightly up-regulated compared with untransfected cells, and venus was strongly expressed. The corneal tissue constructed by HE staining consisted of 5 to 6 layers of epithelial cells. The number of epithelial cells in the tissues was small, large and flat, and the basal cells were dense and small in size. Only the most basal cells in the basal part of the tissue expressed P63 by immunofluorescence, while the epithelial cells did not express. The artificial cornea and normal corneal epithelial tissue structure similar to the structure, can be used for transplantation, in order to study the corneal limbal stem cells repair damaged corneal epithelial mechanism.