以兔去上皮浅层角膜为载体体外培养角膜缘上皮细胞的实验研究

目的 建立以兔去上皮浅层角膜为载体体外培养兔角膜缘上皮细胞的培养方法 ,观察细胞生物学特性。方法 用含5%胎牛血清的DMEM/F12（1∶1）培养液对兔角膜缘上皮细胞进行体外组织块法原代培养,待细胞形成单层后,传代种植于去上皮浅层角膜载体上。每天用倒置显微镜观察培养细胞的形态及生长情况,电镜观察培育细胞的超微结构并采用HE染色和抗增殖细胞核抗原（anti-proliferating cell nuclear antigen,PCNA）单克隆抗体免疫细胞化学染色对生长于去上皮浅层角膜载体上的细胞进行检测。结果 原代培养时,48小时后可见组织块边缘有细胞生长游出,10天左右,细胞汇合成单层。传代种植于载体上,细胞24小时贴壁伸展,第5天可见部分区域细胞逐渐融合成片,11天左右细胞形成良好单层。细胞呈卵圆形或多边形,类似角膜上皮细胞;电镜下,可见细胞表面有许多微绒毛,细胞间有桥粒连接;免疫细胞化学染色显示部分细胞PCNA单克隆抗体呈阳性反应。结论 含5%胎牛血清的DMEM/F12培养液适合角膜缘干细胞的体外培养,并能成功地在去上皮角膜浅层载体上培养出类似生理状态下的角膜上皮细胞。

Culture of limbal stem cells on rabbit superficial layer cornea removed epithelium in vitro

Objective To establish a method about culturing limbal stem cells on rabbit superficial layer cornea removed epithelium in vitro and observe biological characteristic of the cells.Methods To culture rabbit corneal limbal stem cells in medium containing 5%fetal bovine serum(FBS)and plant the cells on carrier of rabbit superficial layer cornea removed epithelium after forming intact monolayer cells.To observe shape and growing of cells with inverted microscope every day during culture process and to observe ultrastructure of the cells with scanning electron microscope while adopting HE ataining and anti-proliferating cell nuclear antigen(PCNA)monoclonal antibodies immunochemical staining to examine the cells plantd on superficial layer cornea removed epithelium.Result The limbal stem cells of primary culture began to adhere and grow from edge of tissues after 48 hours and cells formed a confluent layer in 10 days.The cells began to adhere and spread in 24 hours after planting the carrier and formed confluent layer in section in 5 days and converged complete confluent layer in about 11 days.Similarity to corneal epithelium,the cells show orbicular-ovate of polygon.Plenty of microvilli can be observed on cell surface and there are desmosomes to link between cells by scanning electron microscope observation.In some cells,PCNA monoclonal antibody show positive reactions by immunocytochemical stain detection.Conclusion DMEM/F12 culture medium containing five percent fetal bovine serum can be used as the basical medium for culturing corneal limbus stem cell in vitro and with this medium we can culture corneal epithelium similar to raw state on superficial layer cornea removed epithelium.