The Influence Of CD147 On Retinal Pigment Epithelium Cells Under High Glucose Culture And The Inhibition Effect Of Tunicamycin On CD147

The influence of CD 147 on retinal pigment epithelium cells under high glucose culture and the inhibition effect of tunicamycin on CD 147Background Diabetic retinopathy(DR) is a severe complication of diabetes mellitus, and is the main cause of blindness of diabetes mellitus patients.The research on mechanism of DR and searching effective treatment is hot point.It is not entirely clear about the mechanism of DR. Many studies indicate that VEGF,MMPs and other cytokines play a important role during the DR.CD147 is a member of immunoglobulin superfamily,and it works by protein glycosylation.The expression of CD147 is more higher in malignant tumor and corneal ulcer etc..CD147 can stimulate fibroblast and endothelial cells to secrete matrix metalloproteinases(MMPs), then destroy basement membrane and cause series of pathological reaction. RNA interference(RNAi) means degradation of homologous mRNA induced by endogenous or exogenous short double-stranded RNA(dsRNA) molecule,its effector molecule is 21-23 nucleotide(nt) dsRNA called small interfering RNA(siRNA).The gene knockout effect caused by RNAi technology can inhibit the expression of oncogene,tumor suppression gene and gene mutatin et al.The short hairpin RNA(shRNA) expressed by vector transfected into target cells can be recognized and splitted into siRNA.Use the plasmid vector system which can steadily express shRNA can inhibit gene expression permanently. Tunicamycin is a natural nucleoside antibiotic, can inhibit the production of dolichol of the protein glycosylation pathway, hamper the generation of sugar chain, produce desugar protein, is a effective tool in sugar protein research. In this study, the expression and distribution of CD147 on each tissue of normal eyeball was detected by immunohistochemistry; detect the changes of CD 147 expression of RPE cells cultured by different concentration glucose in vitro; then construct the CD147-shRNA vector and transfect RPE cells and detect the inhibition effect of CD147-shRNA on the expression of CD147,and observe the migration ability of RPE cell and the effect on the secretion of MMP-2,MMP-9 and VEGF.Add tunicamycin on RPE cells,detect the deglycosylation effect on effect, and the changeof proliferation,migration and secretion ability of RPE cells.Methods1 Detect the expression and distribution of CD147 on each tissue of normal human eyeball by immunohistochemistry.2 Detect the changes of CD147 expression of RPE cells cultured by different concentration glucose in vitro and the influence of CD 147 on biological behavior of RPE cells.Detect the changes of expression of CD147 under different concentration glucose culture by immunofluorescent staining, study the migration ability by wound-healing assays,detect the expression of CD147,MMP-2,MMP-9 and VEGF by Western blot.3 Construction and identification of CD147-shRNA and the inhibition effect of CD147-shRNA on the expression of CD 147 of RPE cells.According to the CD147 mRNA sequence in Genebank,use the siRNA design software of Promega to find the right target sequence,BLAST to confirm no homologous with known human gene.Three pairs of oligonucleotide strand were synthesized.Cloned the sequence to pBS/U6 vector.Rename the recombinant plasmid pBS/U6/CD147-shRNA1,pBS/U6/CD147-shRN2,pBS/U6/CD147-shRNA3,and restriction enzyme digestion and sequencing to confirm the correctness.Transfect the plasmid into RPE cells to detect the expression of CD147 by Western blot, and the expression of MMP-2,MMP-9 and VEGF.Also the migration ability was detected by wound-healing.4 The inhibition effect of tunicamycin on CD 147 of RPE cellsAdd lOug/ml tunicamycin to RPE cells cultured by high and low glucose,detect the expression of CD147 by immunofluorescent staining;detect the degree of CD147 protein glycosylation and the secretion of MMP-2,MMP-9 and VEGF.Study the migration ability by wound-healing.Detect the influence of tunicamycin on cell cycle of RPE cell by flow cytometry.Results 1 CD147 was expressed on human cornea epithelial cell, iris epithelial cell, lens epithelial cell and fibre suface, sclera vascular endothelial cell, choroid membranes pigment epithelial cell and small vascular endothelial cell, optic nerve fibre and all layers of retina.2 The expression of CD 147 of RPE cell was increased under the high concentration glucose culture,and the migration ability was strengthened, more MMP-2,MMP-9 and VEGF was secreted.3 CD147-shRNAs recombinant plasmid is completely correct comfirmed by restriction enzyme digestion and sequencing.All the plasmids have gene silencing effect and the pBS/U6/CD147-shRNA3 is most significant.The result of Westernblot indicates that CD 147 expression was significantly decreased after RPE cells was transfected by pBS/U6/CD147-shRNA3, and the migration ability was declined, and the secretion of MMP-2,MMP-9 and VEGF was significantly decreased.4 CD147 was deglycosylated by tunicamycin, CD147 on the cell membrane was decreased showed by immunofluorescence.And the results of Western blot showed glycosylated CD147 significantly declined, and also the MMP-2,MMP-9 and VEGF. RPE cell migrated slowly.Conclusions1 CD 147 was expressed on human cornea epithelial cell and endothelial cells, lens epithelial cell and suface fibre cell, iris surface pigment cell, choroid membranes pigment epithelial cell, optic nerve fibre and all layers of retina and vascular endothelial cell of all tissues, and located mainly in cell membrane and cytoplasm.So in eyeball CD147 located mainly in pigment epithelium,epithelial cell and vascular endothelial cell.2 Under high glucose culture,migration ability of RPE cell was strengthened, express more CD147, and the expression of MMP-2,MMP-9 and VEGF was increased, there is a positive correlation between them. pBS/U6/CD147-shRNA3 can inhibit the migration of RPE cell and the secretion of MMP-2,MMP-9 and VEGF.It indicates that high glucose circumstance can upgrade the expression of MMP-2,MMP-9 and VEGF, and promote the migration of RPE cell. 3 Tunicamycin works on the N terminal of CD147 protein, makes CD147 deglycosylation, and them inhibit its biological activity, result in a weakness of proliferation,migration and secretion of RPE cells.It indicates that as a membrane protein CD147 works by the degree of glycosylation.