影响兔完全角膜缘缺陷模型中羊膜上体外扩增角膜缘上皮移植术后结局的因素

Factors affecting outcome following transplantation of ex vivo expanded limbal epithelium on amniotic membrane for total limbal deficiency in rabbits.

作者信息

Ti Seng-Ei, Anderson David, Touhami Amel, Kim Charles, Tseng Scheffer C G

机构信息

Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida, USA.

出版信息

Invest Ophthalmol Vis Sci. 2002 Aug;43(8):2584-92.

DOI:

PMID:12147589

Abstract

PURPOSE

To determine factors affecting the outcome of corneal surface reconstruction in rabbits with total limbal stem cell deficiency (LSCD), by using autologous limbal epithelial stem cells (LSC) ex vivo, expanded on rabbit amniotic membrane (AM).

METHODS

Left eyes of 52 rabbits were rendered totally limbal stem cell deficient by n-heptanol debridement of the entire corneal epithelium followed by surgical removal of 360 degrees of limbal rim. After cytologic verification of LSCD, the fibrovascular pannus of each cornea was removed. Group I (n = 10) received a rabbit AM transplant, whereas groups II, III, and IV (n = 42) underwent transplantation of LSCs cultured on rabbit AM (LSC-AM graft) derived from a small limbal biopsy specimen from the right eye. Clinical outcome was graded as a success if a smooth, avascular corneal surface was restored, a partial success if more than two quadrants of corneal surface were smooth, or a failure if the corneal surface was revascularized and irregular.

RESULTS

A long-term follow-up of more than 1 year was achieved. Compared with the 100% failure rate in group I, inclusion of expanded LSCs resulted in variable success rates in groups II, III, and IV (all P < 0.001). Kaplan-Meier survival analysis showed that different suturing techniques, subconjunctival injection of long-acting steroid, and tarsorrhaphy used in groups II (n = 17) and III (n = 13) did not significantly alter the outcome (P = 0.89). However, the use of a larger graft and human AM as a temporary patch with the explant retained for 1 week in group IV (n = 12) significantly improved the success rate to 83% (P = 0.002). Among eyes showing clinical failure, there was a significant correlation between the logarithm of the first day when an epithelial defect was noted and the time of graft failure (r(2) = 0.60, P < 0.001). Furthermore, the presence of severe lid deformity was borderline significant when correlated with failure cases in all four groups (P = 0.069).

CONCLUSIONS

Ex vivo expansion of LSCs can be achieved by using rabbit AM culture. Such expanded LSCs can successfully reconstruct corneal surfaces affected by total LSCD. This animal model is useful to investigate culturing variables affecting epithelial stemness so that surgical reconstruction of corneas with total LSCD can be successfully performed. Furthermore, this model can be used to test the feasibility of gene therapies targeting LSCD in the future.

摘要

目的

通过使用在兔羊膜（AM）上体外扩增的自体角膜缘上皮干细胞（LSC），确定影响完全角膜缘干细胞缺乏（LSCD）兔角膜表面重建结果的因素。

方法

52只兔的左眼通过用正庚醇清除整个角膜上皮，然后手术切除360度角膜缘，造成完全角膜缘干细胞缺乏。在通过细胞学验证LSCD后，去除每个角膜的纤维血管性血管翳。I组（n = 10）接受兔羊膜移植，而II、III和IV组（n = 42）接受移植来自右眼小角膜缘活检标本在兔羊膜上培养的LSCs（LSC-AM移植物）。如果恢复光滑、无血管的角膜表面，则临床结果评为成功；如果超过两个象限的角膜表面光滑，则评为部分成功；如果角膜表面血管化且不规则，则评为失败。

结果

实现了超过1年的长期随访。与I组100%的失败率相比，II、III和IV组中加入扩增的LSCs导致成功率各不相同（所有P < 0.001）。Kaplan-Meier生存分析表明，II组（n = 17）和III组（n = 13）中使用的不同缝合技术、结膜下注射长效类固醇和睑裂缝合并未显著改变结果（P = 0.89）。然而，IV组（n = 12）中使用更大的移植物和人羊膜作为临时贴片并将外植体保留1周，显著提高成功率至83%（P = 0.002）。在显示临床失败的眼中，首次发现上皮缺损的第一天的对数与移植物失败时间之间存在显著相关性（r(2) = 0.60，P < 0.001）。此外，严重眼睑畸形的存在与所有四组中的失败病例相关时接近显著（P = 0.069）。