Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province 430030, China.
Tissue Tech Inc, Miami, Florida, 33126 USA.
Int J Med Sci. 2020 Feb 10;17(4):549-557. doi: 10.7150/ijms.40881. eCollection 2020.
: To compare the difference in gene expression between human limbal niche cells (LNC) and bone marrow derived mesenchymal stem cells (BMMSC). : LNC were isolated by collagenase and expanded in modified embryonic stem cell medium (MESCM) on a Matrigel coated plastic plate. Cell diameters were measured with Image J software. Relative gene expression levels between LNC and BMMSC were compared using Affymetrix Human Primer View Gene Expression Array. A subset of differentially expressed genes was verified by RT-qPCR. The protein level of LAMA1 and COL4A1 was confirmed by Western blot and immunostaining. : The average diameter of LNC was 10.2±2.4 μm, which was significantly smaller than that of BMMSC (14 ±3.4 μm) (p<0.0001). Expression of 20,432 genes was examined by Gene Expression Array, among which expression of 349 genes in LNC was 10-fold or higher than that of BMMSC and expression of 8 genes in LNC was 100-fold or higher than that of BMMSC, while expression of 3 genes in BMMSC was 100-fold higher than that of LNC. GO analysis and pathway analysis showed that the differentially expressed genes were mainly enriched in the extracellular matrix receptor interaction pathway and Wnt signaling pathway. In addition, RT-qPCR results demonstrated that the expression of CD73, CD90, CD105, PDGFRβ, Vimentin, SCF, KIT (CD117), COL14A1, LAMA2, THBS2, FZD1, BMP2 and CXCL12 genes in LNC were at least 2 folds higher than BMMSC. The protein level of LAMA1 was higher but the protein level of COL4A1 was lower in LNC than that in BMMSC. : LNC exhibit differential gene expression from BMMSC in the extracellular matrix (ECM) receptor interaction pathway and Wnt signaling pathway, suggesting that LNC have their unique signaling pathways to support limbal stem cell niches.
: 比较人角膜缘龛细胞（LNC）和骨髓间充质干细胞（BMMSC）之间的基因表达差异。 : LNC 通过胶原酶分离，并在涂有 Matrigel 的塑料板上的改良胚胎干细胞培养基（MESCM）中扩增。使用 Image J 软件测量细胞直径。使用 Affymetrix Human Primer View Gene Expression Array 比较 LNC 和 BMMSC 之间的相对基因表达水平。通过 RT-qPCR 验证了一部分差异表达基因。通过 Western blot 和免疫染色证实了 LAMA1 和 COL4A1 的蛋白水平。 : LNC 的平均直径为 10.2±2.4 μm，明显小于 BMMSC（14±3.4 μm）（p<0.0001）。通过基因表达谱分析检查了 20432 个基因的表达，其中 LNC 中 349 个基因的表达是 BMMSC 的 10 倍或更高，LNC 中 8 个基因的表达是 BMMSC 的 100 倍或更高，而 BMMSC 中 3 个基因的表达是 BMMSC 的 100 倍。GO 分析和途径分析表明，差异表达基因主要富集在外细胞基质受体相互作用途径和 Wnt 信号通路。此外，RT-qPCR 结果表明，LNC 中 CD73、CD90、CD105、PDGFRβ、Vimentin、SCF、KIT（CD117）、COL14A1、LAMA2、THBS2、FZD1、BMP2 和 CXCL12 基因的表达至少是 BMMSC 的 2 倍。LNC 中 LAMA1 的蛋白水平较高，但 COL4A1 的蛋白水平较低。 : LNC 在细胞外基质（ECM）受体相互作用途径和 Wnt 信号通路中表现出与 BMMSC 不同的基因表达，表明 LNC 具有独特的信号通路来支持角膜缘干细胞龛。
