Yang Wei, Gu Guo-zhen, Song E, Cui Zhi-hua, Dong Yu, Sui Dong-ming, Ma Yan-ling
Department of Ophthalmology, First Affiliated Hospital, Jilin University, Changchun 130021, China.
Zhonghua Yan Ke Za Zhi. 2007 Feb;43(2):134-41.
To investigate the biological characters of limbal cells and evaluate the effect of cultivated human limbal epithelial cells transplantation on ocular surface reconstruction.
Human limbal cells were isolated and cultivated in vitro. Immunofluorescence staining and RT-PCR were used to study the phenotype of the cells, BrdU labeling test was used to identify the slow-cycling cells in the cultures. Limbal stem cell deficiency (LSCD) was established in rat cornea by alkali burn. Two weeks after the injury, the rats received transplantation of cultivated human limbal epithelial cells with amniotic membrane carrier, and then the therapeutic effects were evaluated by slit lamp observation, HE staining and immunofluorescent staining.
On day 7, p63 and K19 were strongly expressed by most cells, only a few cells expressed K3. On day 14 and day 21, p63 and K19 were still expressed by a majority of cells, while the proportion of K3 positive cells increased, some cells co-expressed p63 and K3. RT-PCR showed that gene expression of both p63 and K12 were positive in cultivated limbal cells, but in mature superficial epithelial cells only K12 was detected. Slow-cycling cells were observed after cultured for 21 days with BrdU free medium. Four weeks after limbal stem cells combined amniotic membrane transplantation (LSAT), both slit lamp observation and HE staining showed that LSAT relieved the pathological changes of rat cornea notably as compared with the amniotic membrane transplantation (AMT) group and control group. The rats that received LSAT exhibited reconstructed corneas with the intact epithelium and improved transparency. Immunofluorescence staining showed that a majority of the rat corneal epithelial cells stained positively to anti-human nuclear antibody and K3 antibody.
P63 is not exclusively expressed by limbal stem cells (LSCs), a certain amount of p63 may also expressed by transient amplifying cells, LSCs are identified as p63 and K19 positive, K3/K12 negative cells. The detection of slow-cycling cells in the culture confirms that LSCs can be cultivated in vitro. Cultivated LSCs combine with amniotic membrane transplantation can functionally reconstruct the cornea suffered with LSCD.
研究角膜缘细胞的生物学特性，评估培养的人角膜缘上皮细胞移植对眼表重建的效果。
体外分离培养人角膜缘细胞。采用免疫荧光染色和逆转录-聚合酶链反应（RT-PCR）研究细胞表型，用5-溴脱氧尿嘧啶核苷（BrdU）标记试验鉴定培养物中的慢循环细胞。通过碱烧伤建立大鼠角膜缘干细胞缺乏（LSCD）模型。损伤后两周，大鼠接受带羊膜载体的培养人角膜缘上皮细胞移植，然后通过裂隙灯观察、苏木精-伊红（HE）染色和免疫荧光染色评估治疗效果。
第7天时，大多数细胞强烈表达p63和角蛋白19（K19），只有少数细胞表达角蛋白3（K3）。第14天和第21天时，大多数细胞仍表达p63和K19，而K3阳性细胞比例增加，部分细胞同时表达p63和K3。RT-PCR显示，培养的角膜缘细胞中p63和角蛋白12（K12）基因表达均为阳性，但在成熟的表层上皮细胞中仅检测到K12。在无BrdU培养基中培养21天后观察到慢循环细胞。角膜缘干细胞联合羊膜移植（LSAT）四周后，裂隙灯观察和HE染色均显示，与羊膜移植（AMT）组和对照组相比，LSAT显著减轻了大鼠角膜的病理变化。接受LSAT的大鼠角膜重建，上皮完整，透明度提高。免疫荧光染色显示，大多数大鼠角膜上皮细胞抗人核抗体和K3抗体染色呈阳性。
P63并非仅由角膜缘干细胞（LSCs）表达，一定量的P63也可能由短暂扩增细胞表达，LSCs被鉴定为p63和K19阳性、K3/K12阴性细胞。培养物中慢循环细胞的检测证实LSCs可在体外培养。培养的LSCs联合羊膜移植可在功能上重建患有LSCD的角膜。
