Shahidullah Mohammad, Tamiya Shigeo, Delamere Nicholas A
Department of Physiology, University of Arizona, Tucson, Arizona 85724, USA.
Curr Eye Res. 2007 Jun;32(6):511-22. doi: 10.1080/02713680701434899.
Primary culture of nonpigmented ciliary epithelium (NPE) has proved difficult in the past. Here we report development of a method of growing and maintaining primary cultures of NPE from porcine eye. Studies were conducted to confirm that the cultured NPE expressed proteins characteristic of native NPE.
Intact rings of NPE were isolated from adult pig eyes. A mixture of hyaluronidase and collagenase was used to detach the cells from the basement membrane and vitreous. Dispersed cells were seeded at high density and grown in DMEM with 20% fetal bovine serum under 5% CO2 and 95% air. Protein expression was examined by immunohistochemistry and immunoblot analysis.
NPE cells were grown in primary culture and maintained up to 10th passage. Analysis of the ciliary body showed three Na, K-ATPase isoforms (alpha 1, alpha 2, alpha 3) and three nitric oxide synthase isoforms (eNOS, nNOS, iNOS) enriched in the NPE layer but weaker or absent in the PE layer. Each of these proteins as well as the tight junction-specific protein ZO-1 was detected in the cultured NPE.
We developed a simple and reliable way to isolate, culture, and maintain NPE cells from porcine eyes. Success of the method hinged on our ability to isolate pure NPE in large number, detach the cells from the vitreous, and seed the cells at high density. The cultured cells express several proteins that are characteristic of native NPE. NPE cells cultured in this way may prove to be valuable for the study of ciliary body function.
过去已证明非色素睫状上皮（NPE）的原代培养很困难。在此我们报告一种从猪眼生长和维持NPE原代培养物的方法的开发。进行研究以确认培养的NPE表达天然NPE的特征性蛋白质。
从成年猪眼中分离出完整的NPE环。使用透明质酸酶和胶原酶的混合物将细胞从基底膜和玻璃体上分离下来。将分散的细胞高密度接种，并在含有20%胎牛血清的DMEM中于5%二氧化碳和95%空气条件下培养。通过免疫组织化学和免疫印迹分析检测蛋白质表达。
NPE细胞在原代培养中生长，并维持到第10代。睫状体分析显示三种钠钾ATP酶同工型（α1、α2、α3）和三种一氧化氮合酶同工型（内皮型一氧化氮合酶、神经元型一氧化氮合酶、诱导型一氧化氮合酶）在NPE层中富集，但在色素上皮（PE）层中较弱或不存在。在培养的NPE中检测到了这些蛋白质中的每一种以及紧密连接特异性蛋白质ZO-1。
我们开发了一种简单可靠的方法来从猪眼中分离、培养和维持NPE细胞。该方法的成功取决于我们大量分离纯NPE、将细胞从玻璃体上分离下来以及高密度接种细胞的能力。培养的细胞表达几种天然NPE的特征性蛋白质。以这种方式培养的NPE细胞可能被证明对睫状体功能的研究有价值。
