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Xiaoqing Q. Guo, Audrey E. Hutcheon, James D. Zieske; Comparison of Fibrosis-Related mRNA in Human Corneal Fibroblasts Treated with TGF-β1 or TGF-β3. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1089.
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© ARVO (1962-2015); The Authors (2016-present)
In our in vitro stroma-like model, we have observed that TGF-β1 (T1) stimulates human corneal fibroblasts (HCF) to self-assemble a fibrotic matrix; whereas, TGF-β3 (T3) appears to stimulate the formation of a non-fibrotic matrix. The goal of this study was to screen HCF stimulated with either T1 or T3 to determine which genes were differentially regulated by T1 and T3.
Human corneal fibroblasts (HCF) were cultured on 100mm culture dishes in basic media (EMEM with 10% FBS). Two time points were examined: (1) 4 hours with 0.5mM Vitamin C (VitC control) ± 10ng/ml T1 or T3, and (2) 3 days with VitC ± 2ng/ml T1 or T3. HCF were also cultured without VitC (No VitC control) for 4 hours. At the appropriate time, cells were harvested and processed for qRT-PCR fibrosis array (Qiagen). The array was run on an Eppendorf Multiplex 2 Real Time PCR machine and the data was analyzed (SABiosciences RT2 Profiler PCR Array Data Analysis software, v. 3.4).
With the addition of VitC to the HCF, 10 genes were downregulated and 1 was upregulated. In the 4-hour samples as compared to VitC controls, 7 genes were downregulated and 12 upregulated in the T1 samples, and 5 genes were downregulated and 10 upregulated in the T3 samples. However, when comparing T3 to T1, only 1 gene was downregulated and 2 genes were upregulated in the T3 samples. In the 3-day samples as compared to VitC controls, 19 genes were downregulated and 19 upregulated in the T1 samples, and 13 genes were downregulated and 29 upregulated in the T3 samples. However, when comparing T3 to T1, 2 genes were downregulated and 19 were upregulated in the T3 samples.
In our initial analysis, there were multiple genes that appeared to be regulated by T1 and/or T3 as compared with the VitC Control. The subset of genes that were different when T3 was compared with T1 was much smaller. Differentially regulated genes included Fas Ligand, TGF-βRI and II, Smad 4, and Integrin beta 6. These genes, as well as others, are being pursued further.
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