EDTA: a promoter of proliferation in human corneal endothelium.
EDTA: a promoter of proliferation in human corneal endothelium.
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发表时间：
2000-09
影响因子：
4.4
通讯作者：
Tadashi Senoo;Tadashi Senoo;Yoshitaka Obara;N. Joyce
中科院分区：
文献类型：
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作者：
Tadashi Senoo;Tadashi Senoo;Yoshitaka Obara;N. Joyce
目的 确定是否有可能通过用 EDTA 处理完整的单层角膜来诱导老年供体角膜的内皮细胞增殖。方法 从眼库获取 52 至 75 岁捐赠者的角膜，通常将其切成四份以增加样本量。通过细胞连接标记 ZO-1 染色评估 EDTA 剂量（0.02-2.0 mg/ml）和孵育时间（6、30 和 60 分钟）对内皮细胞-细胞接触的影响。通过商业活死测定来测试细胞死亡。在 EDTA 处理之前，将角膜片在培养基（M-199、10%胎牛血清、10 ng/ml 表皮生长因子、20 ng/ml 成纤维细胞生长因子）中孵育 0、24、48 或 60 小时。处理后，将碎片在相同的培养基中孵育24、48、72或96小时以允许细胞周期进入。组织被固定、Ki67（G1 期晚期到 M 期的标记物）染色，并固定在含有碘化丙啶的培养基中以可视化所有细胞核。通过计算机（图像软件；NIH，贝塞斯达，马里兰州）评估共焦图像以对 Ki67 阳性和碘化丙啶染色的细胞进行计数。结果 EDTA 以剂量和时间依赖性方式释放角膜内皮细胞-细胞接触。在测试的剂量和孵育时间下，EDTA 不会诱导显着的细胞死亡。内皮细胞需要在培养基中预孵育 24 小时，才能有效地启动响应 EDTA 的增殖。在不含 EDTA 处理的情况下，在含有丝裂原的培养基中孵育长达 108 小时的角膜内皮没有被 Ki67 染色。 2.0 mg/ml 的 EDTA 作用 60 分钟似乎效果最佳，可刺激 16% 至 18% 的细胞增殖。暴露于 EDTA 后 48 小时可见 Ki67 阳性有丝分裂图。 Ki67 和 ZO-1 双重染色后可以看到子细胞的形成。结论 EDTA 使细胞免受接触抑制，并促进老年供体角膜内皮细胞的增殖。作者假设，在细胞暂时从接触抑制中释放的条件下，当暴露于正生长因子时，老年人的角膜内皮会原位分裂。
PURPOSE To determine whether it is possible to induce proliferation in the endothelium of older donor corneas by treatment of the intact monolayer with EDTA. METHODS Corneas from donors 52 to 75 years of age were obtained from an eye bank and were usually cut in quarters to increase sample size. The effect of EDTA dose (0.02-2.0 mg/ml) and incubation time (6, 30, and 60 minutes) on endothelial cell-cell contacts was evaluated by staining for ZO-1, a cell junction marker. Cell death was tested by a commercial live-dead assay. Corneal pieces were incubated for 0, 24, 48, or 60 hours in culture medium (M-199, 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 20 ng/ml fibroblast growth factor) before EDTA treatment. After treatment, pieces were incubated in the same medium for 24, 48, 72, or 96 hours to permit cell cycle entry. Tissue was fixed, stained for Ki67 (a marker for late G1-phase through the M-phase), and mounted in medium containing propidium iodide to visualize all nuclei. Confocal images were evaluated by computer (Image software; NIH, Bethesda, MD) to count Ki67-positive and propidium iodide-stained cells. RESULTS EDTA released corneal endothelial cell-cell contacts in a dose- and time-dependent manner. At doses and incubation times tested, EDTA did not induce significant cell death. Preincubation in culture medium for 24 hours was needed for endothelial cells to efficiently initiate proliferation in response to EDTA. The endothelium of corneas incubated in mitogen-containing medium for up to 108 hours without EDTA treatment did not stain for Ki67. EDTA at 2.0 mg/ml for 60 minutes appeared optimal and stimulated 16% to 18% of the cells to proliferate. Ki67-positive mitotic figures were visible 48 hours after exposure to EDTA. Formation of daughter cells was visible after double-staining for Ki67 and ZO-1. CONCLUSIONS EDTA released cells from contact inhibition and promoted proliferation in corneal endothelium from older donors. The authors hypothesize that corneal endothelium from older individuals divide in situ when exposed to positive growth factors under conditions in which cells have been transiently released from contact inhibition.