羊膜为载体的人角膜缘上皮细胞移植治疗大鼠角膜碱烧伤的研究

Transplantation of human limbal cells cultivated on amniotic membrane for reconstruction of rat corneal epithelium after alkali burn

目的研究人角膜缘上皮细胞的生物学特性，探讨体外培养的人角膜缘上皮细胞移植进行眼表重建的临床应用价值及前景。方法体外培养人角膜缘上皮细胞，通过免疫荧光染色法和逆转录聚合酶链反应（RT．PCR）法分析表型，Brdu掺入法检测慢周期（slow—cycling）细胞。建立大鼠角膜碱烧伤模型，随机分为单纯对照组、羊膜移植（AMT）组及羊膜为载体的角膜缘上皮细胞移植（LSAT）组，术后通过裂隙灯显微镜观察、角膜切片HE染色及免疫荧光染色等方法对各组大鼠眼表恢复情况进行评价。结果培养早期大多数角膜缘上皮细胞p63和K19染色阳性，仅少数细胞K3染色阳性；随培养时间的延长，K3阳性细胞增多，部分细胞同时表达p63和K3。RT—PCR示培养的角膜缘上皮细胞中p63和K12均有阳性表达，而角膜上皮细胞仅表达K12。Brdu掺入法检测到慢周期细胞。动物实验表明，LSAT可以显著减轻角膜病变，使角膜恢复完整性和透明性，眼表评分各项指标与AMT组和对照组比较差异均有统计学意义。LSAT组大鼠角膜切片染色示大部分上皮细胞抗人核和K3染色阳性。结论体外培养的人角膜缘上皮细胞中p63不仅表达于角膜缘干细胞，在短暂扩充细胞中也有一定量的表达；慢周期细胞的存在证实了培养的角膜缘上皮细胞中角膜缘干细胞的存在，p63和K19阳性、K3阴性的细胞为角膜缘干细胞；羊膜为载体的体外培养的人角膜缘上皮细胞移植可有效重建眼表，具有较高的临床应用价值与发展前景。

Objective To investigate the biological characters of limbal cells and evaluate the effect of cultivated human limbal epithelial cells transplantation on ocular surface reconstruction. Methods Human limbal cells were isolated and cultivated in vitro. Immunofluorescene 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 anmiotic membrane carrier, and then the therapeutic effects were evaluated by slit lamp observation, HE staining and immunofluorescent staining. Results 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 anmiotic 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. Immunofluorescene staining showed that a majority of the rat corneal epithelial cells stained positively to antihuman nuclear antibody and K3 antibody. Conclusions 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.