CAJ | 학술논문

背景:角膜缘干细胞体外培养的关键在于建立稳定的体外培养体系,包括角膜缘干细胞的定位、培养条件、载体选择和鉴别方法等.目的:探索兔角膜缘上皮干细胞体外扩增方法,并对其生物学特性进行鉴定.方法:采用兔角膜缘组织块培养法,以人羊膜为载体,在体外进行兔角膜缘上皮干细胞原代和传代培养.倒置显微镜观察其体外生长特征;苏木精-伊红染色以及扫描电镜观察其形态;AE5和P63单克隆抗体免疫组织化学染色鉴定其蛋白表达.结果与结论:采用组织块培养法可在体外获得角膜缘上皮干细胞,能成功传代培养且保持较高增殖潜能.培养于去上皮羊膜上的干细胞可融合成片,呈"拉网"现象.原代角膜缘上皮干细胞AE5单克隆抗体染色阳性率低于5%,P63染色阳性达90%;随传代次数增加AE5染色阳性率增高,P63染色阳性率降低.结果显示兔角膜缘组织块培养法可以在体外成功获得角膜缘上皮干细胞,原代和传代细胞均具有干细胞特性,以羊膜为载体培养可形成角膜移植片.
배경:각막연간세포체외배양적관건재우건립은정적체외배양체계,포괄각막연간세포적정위、배양조건、재체선택화감별방법등.목적:탐색토각막연상피간세포체외확증방법,병대기생물학특성진행감정.방법:채용토각막연조직괴배양법,이인양막위재체,재체외진행토각막연상피간세포원대화전대배양.도치현미경관찰기체외생장특정;소목정-이홍염색이급소묘전경관찰기형태;AE5화P63단극륭항체면역조직화학염색감정기단백표체.결과여결론:채용조직괴배양법가재체외획득각막연상피간세포,능성공전대배양차보지교고증식잠능.배양우거상피양막상적간세포가융합성편,정"랍망"현상.원대각막연상피간세포AE5단극륭항체염색양성솔저우5%,P63염색양성체90%;수전대차수증가AE5염색양성솔증고,P63염색양성솔강저.결과현시토각막연조직괴배양법가이재체외성공획득각막연상피간세포,원대화전대세포균구유간세포특성,이양막위재체배양가형성각막이식편.
BACKGROUND: How to establish a stable in vitro culture system, including location of corneal limbal epithelial stem cells, in vitro sample harvest, in vitro culture, vector selection, as well as identification methods, play a key role in corneal limbal epithelial stem cells culture. OBJECTIVE: To culture the isolated rabbit corneal limbal epithelial stem cells and to identify the biological properties of cultured cells. METHODS: The primary rabbit cornel limbal epithelial stem cells were isolated and cultured with tissue inoculation using human amniotic membrane as vector. The growth features of cells were observed under an inverted microscope. The morphology of cells was observed by hematoxylin-eosin staining and a scanning electron microscope. Furthermore, the monoclonal antibody AE5 and P63 two-step immunohistochemical staining were used to identify limbal epithelial stem cell protein expression. RESULTS AND CONCLUSION: The rabbit corneal limbal epithelial stem cells could be successfully cultured and maintained a relatively high value-added potential in vitro. Rabbit corneal limbal epithelial stem cells cultured on the amniotic membrane pull netted cellular layer. The AE5 monoclonal antibody positive rate of primary cultured cells was about 5% and P63 monoclonal antibody positive up to 90%. AE5-positive rate increased and P63-positive rate decreased with the increase in the number of subculture. The rabbit limbal epithelial stem cells can be successful culture and amplified on human amniotic membrane in vitro by limbal tissue culture method. The cultured cells maintain the characteristics of corneal epithelial cells. The rabbit corneal limbal epithelial stem cells can form grafts on the amniotic membrane.