Hypoxia enhances the expansion of human limbal epithelial progenitor cells in vitro
Hypoxia enhances the expansion of human limbal epithelial progenitor cells in vitro
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DOI：
10.1167/iovs.07-0077
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发表时间：
2007-08-01
影响因子：
4.4
通讯作者：
Shimmura, Shigeto
中科院分区：
文献类型：
--
作者：
Miyashita, Hideyuki;Higa, Kazunari;Shimmura, Shigeto
目的。目的：探讨缺氧对人角膜缘上皮细胞体外增殖和分化的影响。原代人角膜缘上皮细胞来自供体角膜边缘。在标准（20% O-2）和低氧（2% O-2）培养条件下观察集落形成效率（CFE）和细胞增殖。流式细胞术分析细胞周期、前向散射（FSC）和侧向散射（SCC）。用脉冲标记（2小时）BrdU和Ki67染色观察增殖细胞。TUNEL法检测细胞凋亡。分离的菌落通过免疫组化检测对K15、p63、involucrin和K3的抗性。Western blot法检测天合蛋白的表达。缺氧时，CFE和角膜缘上皮细胞的增殖均明显增强。流式细胞术显示，与常氧相比，G(0)/G(1)期缺氧细胞比例更高，s期细胞比例更低。然而，在2小时的脉冲中，BrdU的摄取没有差异，这表明缺氧菌落含有快速循环的细胞。两组细胞凋亡稀疏，缺氧细胞FSC低于常氧细胞。虽然K15、p63和Ki67的染色模式没有差异，但在常氧环境下培养的细胞表达了更高水平的分化标记物involucrin和K3。Western blot也观察到大鼠血清中天花素的表达明显升高。低氧培养（2%）可促进体外角膜缘上皮细胞的增殖，同时抑制其分化。
PURPOSE. To demonstrate the effects of hypoxia on proliferation and differentiation of human limbal epithelial cells in vitro.METHODS. Primary human limbal epithelial cells were harvested from the rim of donor corneas. Colony-forming efficiency (CFE) and cell proliferation were observed in standard (20% O-2) or hypoxic (2% O-2) culture conditions. Cell cycle, forward scatter (FSC) and side scatter (SCC) of cells were analyzed by flow cytometry. Proliferating cells were also observed by pulse labeling (2 hours) with BrdU and Ki67 staining. Apoptosis was detected by TUNEL assay. Isolated colonies were examined by immunohistochemistry against K15, p63, involucrin, and K3. Involucrin expression was also analyzed by Western blot analysis.RESULTS. Both CFE and proliferation of limbal epithelial cells was significantly enhanced in hypoxia. Flow cytometry revealed a higher fraction of hypoxic cells in the G(0)/G(1)-phase and fewer cells in the S-phase, compared with normoxia. However, there was no difference in the uptake of BrdU during a 2-hour pulse, suggesting that hypoxic colonies contained rapidly cycling cells. Apoptotic cells were sparse in both groups, and hypoxic cells showed lower FSC compared with normoxic cells. Although there was no difference in the staining pattern of K15, p63, and Ki67, cells cultivated in normoxia expressed higher levels of the differentiation markers involucrin and K3. Significantly higher involucrin expression was also observed by Western blot.CONCLUSIONS. Hypoxic culture (2%) enhances proliferation while inhibiting differentiation of limbal epithelial cells in vitro.