Expression Of Muscarinic Receptors On Human Limbus, Cornea And Conjunctiva

Purpose: To characterize in human limbus, cornea and conjunctiva the expression of muscarinic receptors (m1-5).Materials and Methods:Epithelial cells from human limbus were isolated and cultured to passage 1 or 2. Confluent cells were airlifted to form multiple layers. The expression of PCNA, p63 and corneal epithelial differentiation markers cytokeratin 3 (K3), cytokeratin 12 (K12), involucrin, connexin 43(Cx43), were studied on cultured limbal epithelial cells by immunocytochemistry. Expression levels of K3 and K12 mRNA were examined by RT-PCR. Fibroblasts were grown from corneal stroma explants. RT-PCR, western blot and immunofluorescent staining for m1-5 were carried out on cultured limbal epithelial cells and corneal fibroblasts. The distribution of m1-5 were studied on human limbus and cornea tissue by immunohistochemistry.Epithelial cells and fibroblasts from human conjunctiva were isolated and cultured to passsage 1 or 2. RT-PCR, western blot and immunofluorescent staining for m1-5 were carried out on cultured conjunctival epithelial cells and corneal fibroblasts. The distribution of m1-5 were analysed on human conjunctiva tissue by immunohistochemistry'. The effect of muscarinic receptors agonist-carbachol on conjunctival fibroblasts' proliferation was studied by Cell Proliferation ELISA.Results:Limbal epithelial cells showed clonal growth when cultured on mitomycin C treated 3T3 fibroblasts feeder layer. The transcription levels of genes for K3 and K12 in cultured cells were lower than those in tissue. A few cells in some colonies were stained positively for K3, K12, involucrin. However, most of the cells in the colonies did not show positive labelling for the differentiation markers K3, K12, involucrin, but were positive for PCNA, and p63. Connexin 43 was positive for some cells in the colonies. In cells that were positive for K3, weakly positive p63 staining was observed. After airlifting, involucrin showed positive staining in cells on the upper part of the cell sheet while staining for connexin 43 was observed in the basal layer. The staining of K3 remained sparse.The message RNA for m1-5 was found in the cultured human limbal epithelial cells and corneal fibroblasts. Western blot detected in cultured human limbal epithelialcells and corneal fibroblasts immunoreactive bands for m1-5 at the following molecular weight: ml 55 kDa ; m2 55 kDa; m3 65 kDa ; m4 55 kDa; m5 60 kDa. Immunohistochemistry produced positive results for ml-5 proteins in cultured limbal epithelial cells and corneal fibroblasts. Epithelial layer and endothelial layer in human limbus and cornea tissue were stained positive by ml-5.RT-PCR analysis showed that the message RNA for ml-5 was found in the cultured human conjunctival epithelial cells and fibroblasts. Western blot detected in cultured human limbal epithelial cells and corneal fibroblasts immunoreactive bands for ml-5. Immunohistochemistry produced positive results for ml-5 proteins in cultured conjunctival epithelial cells and fibroblasts. Epithelial layer including goblet cells in human conjunctiva tissue was stained positive by ml-5. Cell proliferation ELISA showed that carbachol promoted proliferation of cultured conjunctival fibroblasts.Conclusions: Cultured human limbal epithelial cells exhibited a phenotype similar to that of the basal layer of the limbus. Muscarinic receptors were broadly expressed on human limbal, cornea and conjunctiva, carbachol promoted proliferation of cultured conjunctival fibroblasts. Muscarinic receptors' agonist carbachol promoted proliferation of cultured conjunctival fibroblasts, which suggests that these G-protein receptors may contribute to the control of cell function.Ph.D Student: Liu Shao Hui (Ophthalmology) Directed by Prof. Wang Chuan Fu;Assoc. Prof. Donald Tan; Prof. Roger Beuerman;...