山羊角膜缘干细胞荧光蛋白(Venus)细胞株的建立及角膜上皮植片构建

Establishment of goat limbal stem cell strain expressing Venus fluorescent protein and construction of limbal epithelial sheets

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。

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-Integrinβ1 antibody by immunofluorescent staining.We founded neither GLSC-V nor GLSCs expressed keratin3（k3） and keratin12（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.