Russo Alessandra, Bonci Paola, Bonci Paolo
Eye Bank, Department of Ophthalmology, S. Maria della Scaletta Hospital, Via Montericco 4, Imola, Bologna, Italy.
Cell Tissue Bank. 2012 Jun;13(2):353-61. doi: 10.1007/s10561-011-9261-5. Epub 2011 Jun 18.
The aim of this work is to quantify the total protein and growth factors content in a tissue-suspension obtained from processed human amniotic membrane (hAM). hAM was collected, frozen, freeze dried, powdered and sterilized by γ-irradiation. At each step of the process, samples were characterized for the total protein amounts by a Bradford protein assay and for the growth factor concentrations by ELISA test of the tissue suspensions. Frozen-hAM samples show higher release of total proteins and specific growth factors in the tissue suspension in comparison with freeze-dried hAM. We observed that even if the protein extraction is hindered once the tissue is dried, the powdering process allows a greater release in the tissue suspension of total proteins and growth factors after tissue re-solubilization in comparison with only the freeze-drying process (+91 ± 13% for EGF, +16 ± 4% for HGF, +11 ± 5% for FGF, +16 ± 9% for TGF-β1), and a greater release of EGF (85 ± 10%) in comparison with only the freezing process, because proteins become much readily solubilized in the solution. According with these results, we describe a protocol to obtain a new sterile biological product from hAM tissue, with well-known effects of thermal, mechanical and physical processes on the total protein and grow factors contents.
这项工作的目的是对从经过处理的人羊膜（hAM）获得的组织悬液中的总蛋白和生长因子含量进行定量。收集hAM，冷冻、冻干、粉碎并通过γ射线辐照灭菌。在该过程的每个步骤中，通过考马斯亮蓝蛋白测定法对样品的总蛋白量进行表征，并通过组织悬液的ELISA试验对生长因子浓度进行表征。与冻干的hAM相比，冷冻的hAM样品在组织悬液中显示出更高的总蛋白和特定生长因子释放量。我们观察到，即使组织干燥后蛋白质提取受到阻碍，但与仅冻干过程相比，粉碎过程在组织重新溶解后能使组织悬液中总蛋白和生长因子有更大的释放（表皮生长因子（EGF）增加91±13%，肝细胞生长因子（HGF）增加16±4%，成纤维细胞生长因子（FGF）增加11±5%，转化生长因子-β1（TGF-β1）增加16±9%），并且与仅冷冻过程相比，EGF的释放量更大（85±10%），因为蛋白质在溶液中更容易溶解。根据这些结果，我们描述了一种从hAM组织获得新的无菌生物制品的方案，以及热、机械和物理过程对总蛋白和生长因子含量的已知影响。
