新鲜人羊膜负载构建兔角膜上皮

Cultivation of rabbit limbal stem cell on human fresh amnion

目的新鲜人羊膜负载构建兔角膜上皮，为全角膜组织工程作基础。方法采用组织块法培养兔角膜缘干细胞，以新鲜人羊膜为载体，形成单层细胞膜片后用于传代；采用相差显微镜进行形态观察及AE5、p63免疫荧光鉴定。结果培养24h，兔角膜缘组织块边缘可见有细胞长出，之后形成“沙滩样”移形带，培养至7～8d细胞在羊膜上形成单层膜状，细胞透明，边缘整齐，形态呈多样化，以卵圆形和多边形为主；AE5抗体、p63抗体染色阳性细胞比例分别为（41．72±11．35）％、（21．57±6．36）％；2代AE5和p63阳性细胞比例与原代比较，无显著性差异（P〉0．05）；3代AE5和p63阳性细胞比例与原代比较，差异有统计学意义（P〈0．05）；但4代AE5阳性细胞比例又明显升高。结论以新鲜羊膜为载体、采用组织块法培养角膜缘干细胞可获得较好的角膜上皮片，在传代细胞中，3代细胞分化程度最低、增殖能力最强，最适合移植。

Rabbit limbal epithelium was cultured on human fresh amnion by tissue block method. It was passage time when the primary cells formed monolayer cell patch. Keratin 3 （ AE5 ） and p63 were detected on the primary and four generation cells with immunofluorescence. Contrast phase microscope was used to observe the appearance of cells. Results Cells outgrew from the limbal tissue block within 24 hours, then ＇ sandy beach＇ band formed, and confluent monolayer cell patch appeared when cultivated 7 - 8 days. The positive percentage of AE5 and p63 was （41.72 ± 11.35）% and （21.57 ±6. 36）% respectively in primary cells. Positive percentages of AE5 and 1363 were no of statistical difference between the second generation cells and primary cells （ P 〉 0. 05 ）, while there was significant difference between the third generation and primary cells （P 〈 0. 05 ）, and AE5 positive cells increased in the fourth generation. Conclusion Corneal epithelium is produced with cultivation of rabbit limbal stem cell by method of tissue block on human fresh amnion. Among all generations, the third generation cells are most suitable to be translated, because of the lowest differential degree and the strongest proliferative ability.