Baharvand Hossein, Heidari Manzar, Ebrahimi Marzieh, Valadbeigi Tahereh, Salekdeh Ghasem Hosseini
Department of Stem Cells, Royan Institute, Tehran, Iran.
Mol Vis. 2007 Sep 18;13:1711-21.
A new strategy of treating ocular surface reconstruction is to transplant a bioengineered graft by expanding limbal stem cells (SCs) ex vivo on the amniotic membrane (AM). The reasons for the exceptional success on the AM are not fully understood but are believed to be related to its unique composition. We investigated the proteome of the epithelium-denuded AM to increase our understanding of the mechanisms by which AM may confer its beneficial effects.
We compared the epithelially denuded-human AM with matrigel and collagen on the expansion of limbal SCs by evaluating the expression of specific markers. The protein pattern of the epithelium-denuded AM was analyzed using two dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS) identification of proteins.
Epithelial outgrowth of limbal explants on AM expressed more p63 and K19 (SC markers) and less K3 and connexin 43 (corneal differentiation markers) in comparison with other extracellular matrices (ECMs). Moreover, in all groups, the cells expressed ABCG2, K19, K12, p63, and Pax6 as shown by reverse transcription polymerase chain reaction (RT-PCR). Out of about 600 protein spots analyzed on six 2-DE gels, 515 spots could be detected in all replicates. A high average correlation coefficiency (CC) of 0.926 implied good intra-sample reproducibility. Forty major proteins of AM were identified using MALDI TOF/TOF MS of which different isoforms of lumican and osteoglycin were responsible for around 23% of the total proteome on gels.
Our results showed that epithelium-denuded AM provides a superior niche for limbal SC proliferation and phenotype maintenance in vitro and the denuded human AM is a protein enriched ECM. This will prove critical to the future understanding of the biological and therapeutic mechanisms involved in AM transplantation and regeneration. The identification of highly abundant proteins in denuded-AM, such as lumican, osteoglycin/memican, collagen alpha type IV, and fibrinogen, further explains its unique properties and will assist in the efforts to generate bioengineered and artificial AM constructs.
治疗眼表重建的一种新策略是通过在羊膜（AM）上体外扩增角膜缘干细胞（SCs）来移植生物工程移植物。AM取得非凡成功的原因尚未完全明确，但据信与其独特的组成有关。我们研究了去上皮AM的蛋白质组，以增进对AM可能产生有益作用机制的理解。
我们通过评估特定标志物的表达，比较了去上皮人AM与基质胶和胶原蛋白对角膜缘SCs扩增的影响。使用二维电泳（2-DE）结合蛋白质质谱（MS）鉴定分析去上皮AM的蛋白质图谱。
与其他细胞外基质（ECM）相比，角膜缘外植体在AM上的上皮生长表达更多的p63和K19（SCs标志物），而K3和连接蛋白43（角膜分化标志物）表达较少。此外，通过逆转录聚合酶链反应（RT-PCR）显示，所有组中的细胞均表达ABCG2、K19、K12、p63和Pax6。在六块2-DE凝胶上分析的约600个蛋白点中，所有重复实验均能检测到515个点。平均相关系数（CC）高达0.926，表明样本内重复性良好。使用基质辅助激光解吸电离飞行时间串联质谱（MALDI TOF/TOF MS）鉴定了AM的40种主要蛋白质，其中核心蛋白聚糖和骨形成蛋白聚糖的不同异构体占凝胶上总蛋白质组的约23%。
我们的结果表明，去上皮AM为角膜缘SCs在体外增殖和表型维持提供了优越的微环境，去上皮人AM是一种富含蛋白质的ECM。这对于未来理解AM移植和再生所涉及的生物学和治疗机制至关重要。去上皮AM中高丰度蛋白质如核心蛋白聚糖、骨形成蛋白聚糖/膜形成蛋白聚糖、IV型胶原α链和纤维蛋白原的鉴定，进一步解释了其独特性质，并将有助于生成生物工程和人工AM构建体的努力。
