Cook E B, Stahl J L, Miller S T, Gern J E, Sukow K A, Graziano F M, Barney N P
Department of Medicine, School of Medicine, University of Wisconsin-Madison 53792, USA.
Invest Ophthalmol Vis Sci. 1998 Feb;39(2):336-43.
To isolate and purify mast cells and epithelial cells from human cadaveric donor conjunctival tissue and to characterize interactions between these cell types in vitro.
Monodispersed cell suspensions obtained by enzymatic digestion of conjunctival tissue were applied to a single-density Percoll gradient. Epithelial cells obtained from the top layer of the gradient were cultured to confluence. Mast cells obtained from the pellet were equilibrated in culture medium and further purified using a two-step Percoll gradient. Using reverse transcription-polymerase chain reaction (RT-PCR), RNA from the purified mast cell preparation was probed for tumor necrosis factor-alpha (TNF alpha) message. Fluorescence activated cell sorting (FACS) analysis of intracellular immunostained mast cells was used to detect the TNF alpha protein. An examination for intercellular adhesion molecule 1 (ICAM-1) on epithelial cells was performed after 24-hour incubations with either recombinant TNF alpha supernatants from calcium ionophore A23187 (CaI)-stimulated mast cells or appropriate controls using FACS analysis.
Highly purified human conjunctival mast cells and epithelial cells (each > 95%) were obtained from human cadaveric donor tissue. RT-PCR analysis of purified mast cell RNA revealed the expression of TNF alpha mRNA. An evaluation of mast cells for intracellular protein demonstrated positive staining for tryptase and TNF alpha. ICAM-1 was found on purified epithelial cells, and incubation of epithelial cell monolayers with supernatants from Cal-stimulated mast cells resulted in upregulation of this receptor. This upregulation was blocked by incubation with TNF alpha-neutralizing antibody.
This work provides the methods for isolating and purifying mast cells and epithelial cells from human donor tissue and the opportunity for studying mechanisms of conjunctival inflammation by evaluating the interactions between these cells.
从人尸体供体结膜组织中分离和纯化肥大细胞和上皮细胞，并在体外表征这些细胞类型之间的相互作用。
将通过酶消化结膜组织获得的单分散细胞悬液应用于单密度Percoll梯度。从梯度顶层获得的上皮细胞培养至汇合。从沉淀中获得的肥大细胞在培养基中平衡，并使用两步Percoll梯度进一步纯化。使用逆转录聚合酶链反应（RT-PCR），检测纯化的肥大细胞制剂中的RNA是否存在肿瘤坏死因子-α（TNFα）信息。对细胞内免疫染色的肥大细胞进行荧光激活细胞分选（FACS）分析，以检测TNFα蛋白。在用钙离子载体A23187（CaI）刺激的肥大细胞的重组TNFα上清液或使用FACS分析的适当对照孵育24小时后，对上皮细胞上的细胞间粘附分子1（ICAM-1）进行检测。
从人尸体供体组织中获得了高度纯化的人结膜肥大细胞和上皮细胞（每种均>95%）。对纯化的肥大细胞RNA进行RT-PCR分析，显示有TNFα mRNA表达。对肥大细胞的细胞内蛋白进行评估，结果显示色氨酸酶和TNFα呈阳性染色。在纯化的上皮细胞上发现了ICAM-1，用CaI刺激的肥大细胞的上清液孵育上皮细胞单层会导致该受体上调。用TNFα中和抗体孵育可阻断这种上调。
这项工作提供了从人供体组织中分离和纯化肥大细胞和上皮细胞的方法，以及通过评估这些细胞之间的相互作用来研究结膜炎症机制的机会。
