J Korean Med Sci.  2009 Apr;24(2):189-196. 10.3346/jkms.2009.24.2.189.

DNA Microarray-Based Gene Expression Profiling in Porcine Keratocytes and Corneal Endothelial Cells and Comparative Analysis Associated with Xeno-related Rejection

• Kim MK
• Oh JY
• Ko JH
• Lee HJ
• Jung JH
• Wee WR
• Lee JH
• Park CG
• Kim SJ
• Ahn C
• Kim SJ
• Hwang SY

• KMID: 1779117
• DOI: http://doi.org/10.3346/jkms.2009.24.2.189

Abstract

Porcine to rat corneal xenotransplantation resulted in severe inflammation and rejection of the corneal stroma, whereas an allograft showed mainly endothelial cell-associated rejection. We, therefore, investigated and compared the gene expression between porcine keratocytes and corneal endothelial cells. RNA was isolated from primary cultured porcine or human keratocytes and porcine corneal endothelial cells. Gene expression was comparatively analyzed after normalization with microarray method using Platinum pig 13 K oligo chip (GenoCheck Co., Ltd., Ansan, Korea). Real-time polymerase chain reaction (PCR) was performed for C1R, CCL2, CXCL6, and HLA-A in porcine keratocytes and corneal endothelial cells. As a result, upregulated expression more than 2 folds was observed in 1,162 genes of porcine keratocytes versus porcine endothelial cells. Among the immune-regulatory genes, SEMA3C, CCL2, CXCL6, F3, HLA-A, CD97, IFI30, C1R, and G1P3 were highly expressed in porcine keratocytes, compared to porcine corneal endothelial cells or human keratocytes. When measured by real-time PCR, the expression of C1R, CCL2, and HLA-A was higher in porcine keratocytes compared to that in porcine corneal endothelial cells. In conclusion, the increased expression of C1R, CCL2, and HLA-A genes in porcine keratocytes might be responsible for the stromal rejection observed in a porcine to rat corneal xenotransplantation.

Keyword

Cornea; Microarray analysis; Porcine; Polymerase Chain Reaction; Transplantation, Heterologous

MeSH Terms

Animals
Cells, Cultured
Chemokine CCL2/metabolism
Complement C1r/metabolism
Corneal Transplantation/*immunology/pathology
Endothelium, Corneal/*metabolism/pathology
*Gene Expression Profiling
Graft Rejection/*immunology/pathology
HLA-A Antigens/metabolism
Humans
Keratinocytes/*metabolism
Oligonucleotide Array Sequence Analysis
Rats
Reverse Transcriptase Polymerase Chain Reaction
Swine
Transplantation, Heterologous
Up-Regulation

Figure

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  Fig. 1 The hematoxylin-eosin staining of cornea at the postoperative 3 (A), 7 (B), 10 (C), and 13 (D) days after porcine to rat corneal transplantation. Early infiltration of neutrophils and monocytes into the stroma were observed. Red arrows indicate neutrophils and white arrows monocytes. Original magnification ×200.

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  Fig. 2 RT-PCR for vimentin as a marker for keratocytes and endothelin as a marker for endothelial cells, indicating that porcine keratocytes and corneal endothelial cells did not cross-contaminate.

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  Fig. 3 Hybridization image of the genes of keratocytes (A) versus those of endothelial cells (B) using platinum pig 13 K biochip.

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  Fig. 4 Log signal intensity ratio of gene expression in porcine keratocytes versus those in porcine corneal endothelial cells or human keratocytes. (A) 1,162 genes were upregulated more than 2 folds in expression when porcine keratocytes were examined versus porcine corneal endothelial cells. (B) 6,060 genes were upregulated more than 2 folds when porcine keratocytes were evaluated versus human keratoctyes.

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  Fig. 5 Comparison of gene expression levels between porcine keratocytes and corneal endothelial cells using real-time PCR. Significant up-regulation of C1R, CCL2, and HLA-A was observed in porcine keratocytes compared to porcine corneal endothelial cells, while there was no difference in the the level of CXCL6.

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