Lindstrom R L
University of Minnesota Department of Ophthalmology, Minneapolis.
Trans Am Ophthalmol Soc. 1990;88:555-648.
The functional status of the endothelium and sustained corneal deturgescence after corneal preservation are of great clinical importance and have been primary goals in the development of corneal storage media. In our investigational studies we have specifically addressed the improvement of the quality of donor tissue after 4 degrees C storage, the extension of corneal preservation time, the enhancement of corneal wound healing, and the reduction of the normal progressive loss of endothelial cells postkeratoplasty. Specifically we have developed in vitro HCE cell and epithelial cell culture models that can accurately reflect the response of human corneal tissue in vivo. These models have been utilized to study the effects of growth factors and medium components in relation to their biocompatibility and efficacy in the development of improved corneal preservation solutions. Our laboratory investigated in vitro conditions that allowed human corneal endothelium to shift from a nonproliferative state, in which they remain viable and metabolically active, to a proliferative, mitotically active state. Isolation techniques developed in our laboratory have enabled the establishment of primary and subsequent subcultures of human corneal endothelium that retain the attributes of native endothelium. These in vitro conditions maintain HCE cells in a proliferative state, actively undergoing mitosis. A quantitative bioassay has been developed to determine the effects of various test medium in the stimulation or inhibition of DNA synthesis. In attempting to learn more about the events that occur during in vitro endothelial cell isolation, cell reattachment, extracellular matrix interaction and migrating during subculture, SEM was done on isolated HCE cells incubated in CSM. These studies suggest that the components of the extracellular matrix modulate the growth response of HCE cells, and play a role in regulating proliferation and migration. These observations are important in view of the fact that anterior chamber environment limits cell regeneration of the endothelium, and supports wound healing via cell migration. In vivo, it is the complex interaction of the HCE cell and the extracellular matrix that signal the cell to respond to cell loss in this manner. As our knowledge of human corneal endothelium has increased so has our anticipation of developing the "optimum" medium. Thus additional components have been added to this basic medium to address specific complications encountered with 4 degrees C corneal preservation. Antioxidants, additional energy sources, and other nutritive substrates have been used to supplement and further define a chondroitin sulfate-based medium. These changes have been a part of our new awareness that, even at 4 degrees C, the cornea is metabolically active.(ABSTRACT TRUNCATED AT 400 WORDS)
内皮细胞的功能状态以及角膜保存后的持续角膜消肿在临床上具有重要意义，并且一直是角膜保存介质研发的主要目标。在我们的研究中，我们特别关注了4℃保存后供体组织质量的改善、角膜保存时间的延长、角膜伤口愈合的增强以及角膜移植术后内皮细胞正常渐进性丢失的减少。具体而言，我们建立了体外人角膜内皮细胞（HCE）和上皮细胞培养模型，该模型能够准确反映人角膜组织在体内的反应。这些模型已被用于研究生长因子和培养基成分在开发改良角膜保存液方面的生物相容性和功效。我们实验室研究了体外条件，该条件可使人角膜内皮细胞从非增殖状态（在此状态下它们保持存活且代谢活跃）转变为增殖、有丝分裂活跃状态。我们实验室开发的分离技术已能够建立人角膜内皮细胞的原代培养及后续传代培养，这些细胞保留了天然内皮细胞的特性。这些体外条件使HCE细胞保持在增殖状态，积极进行有丝分裂。已开发出一种定量生物测定法来确定各种测试培养基对DNA合成的刺激或抑制作用。为了更多地了解体外内皮细胞分离、细胞重新附着、细胞外基质相互作用以及传代培养期间迁移过程中发生的事件，我们对在角膜保存液（CSM）中孵育的分离HCE细胞进行了扫描电子显微镜（SEM）检查。这些研究表明，细胞外基质的成分调节HCE细胞的生长反应，并在调节增殖和迁移中发挥作用。鉴于前房环境限制内皮细胞的再生，并通过细胞迁移支持伤口愈合，这些观察结果具有重要意义。在体内，正是HCE细胞与细胞外基质的复杂相互作用以这种方式向细胞发出应对细胞丢失的信号。随着我们对人角膜内皮细胞了解的增加，我们对开发“最佳”培养基的期望也在增加。因此，已在这种基础培养基中添加了其他成分，以解决4℃角膜保存中遇到的特定并发症。抗氧化剂、额外的能量来源和其他营养底物已被用于补充并进一步确定一种基于硫酸软骨素的培养基。这些变化是我们新认识的一部分，即即使在4℃时，角膜仍具有代谢活性。（摘要截选至400字）
