Construction of eukaryotic plasmid expressing human TGFBI and its influence on human corneal epithelial cells.

Abstract

To detect the expression of transforming growth factor beta-induced gene (TGFBI) protein in human corneal tissue and overexpress it in the human corneal epithelial cells in order to discuss the function of TGFBI in the pathogenesis of corneal dystrophy. Immunohistochemistry (IHC) was used to detect the expression of TGFBI in the human cornea tissue. TGFBI cDNA was obtained by reverse transcription-PCR from human corneal total RNA extracted from cornea transplant donor and cloned into pCMV-N-HA vector. The recombinant pCMV-N-HA-TGFBI plasmid transfected human corneal epithelial cells. Forty-eight hours later, mRNA and proteins were harvested from cells for real-time PCR analysis and western blot assay respectively. IHC indicated TGFBI mainly exist below the human corneal epithelium layer. Transfection of recombinant pCMV-N-HA-TGFBI into human corneal epithelial cells resulted in effective expression of TGFBI, as shown by increased mRNA level detected by real-time PCR as well as increased protein level detected by Western blot. Meanwhile the result of real-time PCR and Western blot shown the expression of MMP1, MMP3 (matrix metalloproteinases MMP) increased while the expressin of TIMP1 (tissue inhibitors of matrix metalloproteinases TIMP) decreased. TGFBI mainly exists below the corneal epithelial layer, recombinant eukaryotic expression vector harboring human TGFBI cDNA was obtained and efficiently overexpressed in human corneal epithelial cells. Meanwhile the TGFBI overexpression in human corneal epithelial cells result in MMP1, MMP3 increasing and TIMP1 decreasing. The result might be helpful for studying the function and role of TGFBI in pathogenesis of corneal dystrophy.