Mass Spectrometry Reveals a-2-HS-Glycoprotein as a Key Early Extracellular Matrix Protein for Conjunctival Cells.
Mass Spectrometry Reveals a-2-HS-Glycoprotein as a Key Early Extracellular Matrix Protein for Conjunctival Cells.
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质谱分析揭示 a-2-HS-糖蛋白是结膜细胞早期关键的细胞外基质蛋白。
DOI：
10.1167/iovs.61.3.44
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发表时间：
2020
影响因子：
4.4
通讯作者：
Makuloluwa AK
中科院分区：
文献类型：
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作者：
Makuloluwa AK
目的：确定结膜上皮细胞系分泌的细胞外基质（ECM）蛋白的组成，并鉴定有助于结膜上皮细胞培养的成分。方法：在无血清培养基中培养人结膜上皮细胞（hcye - gi），并用氢氧化铵分离其ECM。将新鲜的hcye - gi细胞镀在3- 28天细胞培养的ecm上，评估其生长特性。质谱法测定了42天的ECM成分。研究了预吸附的纤维连接蛋白和α-2- hs -糖蛋白（α-2-HS-GP）对细胞的粘附和生长情况。结果：与细胞培养5- 28天的ECM相比，第3天的ECM为细胞生长提供了最好的基质。质谱分析发现，在整个时间过程中，主要是层粘连蛋白332基质，随着时间的推移，基质成分逐渐发生变化：基质结合生长因子成比例减少，蛋白酶增加。纤维连接蛋白和α-2-HS-GP在早期ECM中的含量分别是晚期ECM的5倍和200倍。对这些蛋白的分离实验表明，纤维连接蛋白支持细胞快速粘附，而与组织培养聚苯乙烯相比，纤维连接蛋白和α-2-HS-GP都支持细胞生长。结论：这些数据表明α-2-HS-GP是促进结膜上皮细胞生长的候选蛋白，并提高了利用这些发现靶向改进合成组织工程结膜底物的可能性。
Purpose: To determine the composition of extracellular matrix (ECM) proteins secreted by a conjunctival epithelial cell line and to identify components that aid conjunctival epithelial cell culture.Methods: Human conjunctival epithelial cell line (HCjE-Gi) cells were cultured in serum-free media and their ECM isolated using ammonium hydroxide. Growth characteristics were evaluated for fresh HCjE-Gi cells plated onto ECMs obtained from 3-to 28-day cell cultures. Mass spectrometry was used to characterize the ECM composition over 42 culture days. Cell adhesion and growth on pre-adsorbed fibronectin and α-2-HS-glycoprotein (α-2-HS-GP) were investigated.Results: Day 3 ECM provided the best substrate for cell growth compared to ECM obtained from 5-to 28-day cell cultures. Mass spectrometry identified a predominantly laminin 332 matrix throughout the time course, with progressive changes to matrix composition over time: proportional decreases in matrix-bound growth factors and increases in proteases. Fibronectin and α-2-HS-GP were 5-and 200-fold enriched as a proportion of the early ECM relative to the late ECM, respectively. Experiments on these proteins in isolation demonstrated that fibronectin supported rapid cell adhesion, whereas fibronectin and α-2-HS-GP both supported enhanced cell growth compared to tissue culture polystyrene.Conclusions: These data reveal α-2-HS-GP as a candidate protein to enhance the growth of conjunctival epithelial cells and raise the possibility of exploiting these findings for targeted improvement to synthetic tissue engineered conjunctival substrates.