Yin Jiqing, Liu Wenqiang, Liu Chao, Zhao Guimin, Zhang Yihua, Liu Weishuai, Hua Jinlian, Dou Zhongying, Lei Anmin
Shaanxi Stem Cell Engineering and Technology Research Center, College of Veterinary Medicine, Northwest A & F University, Yangling 712100, China.
Sheng Wu Gong Cheng Xue Bao. 2010 Dec;26(12):1636-44.
The integrity and transparency of cornea plays a key role in vision. Limbal Stem Cells (LSCs) are precursors of cornea, which are responsible for self-renewal and replenishing corneal epithelium. Though it is successful to cell replacement therapy for impairing ocular surface by Limbal Stem Cell Transplantation (LSCT), the mechanism of renew is unclear after LSCT. To real time follow-up the migration and differentiation of corneal transplanted epithelial cells after transplanting, we transfected venus (a fluorescent protein gene) into goat LSCs, selected with G418 and established a stable transfected cell line, named GLSC-V. These cells showed green fluorescence, and which could maintain for at least 3 months. GLSC-V also were positive for anti-P63 and anti-Integrinbeta1 antibody by immunofluorescent staining. We founded neither GLSC-V nor GLSCs expressed keratin3 (k3) and keratinl2 (k12). However, GLSC-V had higher levels in expression of p63, pcna and venus compared with GLSCs. Further, we cultivated the cells on denude amniotic membrane to construct tissue engineered fluorescent corneal epithelial sheets. Histology and HE staining showed that the constructed fluorescent corneal epithelial sheets consisted of 5-6 layers of epithelium. Only the lowest basal cells of fluorescent corneal epithelial sheets expressed P63 analyzed by immunofluorescence, but not superficial epithelial cells. These results showed that our constructed fluorescent corneal epithelial sheets were similar to the normal corneal epithelium in structure and morphology. This demonstrated that they could be transplanted for patents with corneal impair, also may provide a foundation for the study on the mechanisms of corneal epithelial cell regeneration after LSCT.
角膜的完整性和透明度在视觉中起着关键作用。角膜缘干细胞（LSCs）是角膜的前体细胞，负责角膜上皮的自我更新和补充。尽管角膜缘干细胞移植（LSCT）用于治疗眼表损伤的细胞替代疗法取得了成功，但LSCT后角膜上皮更新的机制尚不清楚。为了实时跟踪角膜移植上皮细胞移植后的迁移和分化，我们将venus（一种荧光蛋白基因）转染到山羊LSCs中，用G418筛选并建立了一个稳定的转染细胞系，命名为GLSC-V。这些细胞呈现绿色荧光，并且可以维持至少3个月。通过免疫荧光染色，GLSC-V对抗P63和抗整合素β1抗体也呈阳性。我们发现GLSC-V和未转染的LSCs均不表达角蛋白3（k3）和角蛋白12（k12）。然而，与未转染的LSCs相比，GLSC-V中p63、增殖细胞核抗原（pcna）和venus的表达水平更高。此外，我们将这些细胞接种在去上皮羊膜上构建组织工程荧光角膜上皮片。组织学和苏木精-伊红（HE）染色显示，构建的荧光角膜上皮片由5-6层上皮细胞组成。通过免疫荧光分析，仅荧光角膜上皮片最底层的基底细胞表达P63，表层上皮细胞不表达。这些结果表明，我们构建的荧光角膜上皮片在结构和形态上与正常角膜上皮相似。这表明它们可用于角膜损伤患者的移植，也可能为研究LSCT后角膜上皮细胞再生的机制提供基础。
