PURPOSE:Amniotic membrane (AM) transplantation is an accepted procedure in ocular surgery. However, little is known of the interdonor and intradonor variability within the membrane. In addition, the effects of the methods of processing, storage, and preoperative preparation on the membrane are not fully elucidated. The purpose of this study was to use TGF-beta as an example to investigate interdonor and intradonor variability and to determine the effect of "handling " on TGF-beta1 within fresh, processed and stored, and transplantation-ready AM (TRAM). METHODS:Seventeen human AMs, both fresh and handled, were analyzed for TGF-beta1 by real-time polymerase chain reaction, immunohistochemistry, SDS-PAGE, and Western blotting. RESULTS:TGF-beta1 was the highest normalized expressed isoform of TGF-beta in all samples, but it varied between membranes of different donors and at different sites within the same membrane. The highest concentration was noted in the spongy layer. Removal of the spongy layer successfully removed the bulk of TGF-beta1 from TRAM. Latency-associated protein (LAP) and a latent TGF-beta-binding protein (LTBP) were also detected. CONCLUSION:TGF-beta1 is present in various regulatory forms in the AM. A degree of intermembrane and intramembrane variation is modified by handling. Unless a standardized protocol is adopted that delivers a membrane with consistent constituents, clinical outcomes may vary and comparisons may be invalid.

译文

目的:羊膜移植术是眼科手术中公认的手术方法。然而,对于膜内的供体间和供体内变异性知之甚少。另外,尚未完全阐明处理,储存和术前准备方法对膜的影响。这项研究的目的是以TGF-β为例,研究供体间和供体内的变异性,并确定“处理”对新鲜,加工和储存以及准备好移植的AM(TRAM)中TGF-beta1的影响。
方法:通过实时聚合酶链反应,免疫组织化学,SDS-PAGE和Western印迹分析了17例新鲜和处理过的人类AMs的TGF-beta1。
结果:TGF-β1是所有样品中TGF-β的最高标准化表达同工型,但在不同供体的膜之间以及同一膜内的不同位置上,TGF-β1均存在差异。在海绵层中观察到最高浓度。去除海绵层成功地从TRAM中去除了大部分TGF-beta1。还检测到潜伏期相关蛋白(LAP)和潜在的TGF-β结合蛋白(LTBP)。
结论:TGF-beta1以各种调控形式存在于增材制造中。膜间和膜内的变化程度通过处理而改变。除非采用标准化的方案来递送具有一致成分的膜,否则临床结果可能会有所不同,并且比较可能无效。

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