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


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 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.


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.
Yang
Wei
W
Department of Ophthalmology, First Affiliated Hospital, Jilin University, Changchun 130021, China.
Gu
Guo-zhen
GZ
Song
E
E
Cui
Zhi-hua
ZH
Dong
Yu
Y
Sui
Dong-ming
DM
Ma
Yan-ling
YL
D000650:Amnion / Q000637:transplantation*; D000818:Animals; D002057:Burns, Chemical / Q000601:surgery*; D017690:Cell Transplantation / Q000379:methods*; D002478:Cells, Cultured; D065306:Corneal Injuries; D019573:Epithelium, Corneal / Q000637:transplantation*; D005126:Eye Burns / Q000601:surgery*; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley