眼科FAN, I-MOREI-MOREFANYANG, CHANG-HAOCHANG-HAOYANGYANG, CHUNG-MAYCHUNG-MAYYANGCHEN, MUH-SHYMUH-SHYCHEN2010-07-082018-07-122010-07-082018-07-122009http://ntur.lib.ntu.edu.tw//handle/246246/188716Transplantation of retinal pigment epithelium (RPE) following removal of choroidal neovascular membranes has been attempted in patients with age- related macular degeneration (AMD). However, inability of transplanted RPE to initially attach and subsequently proliferate on Bruch's membrane may lead to failure of RPE transplants and poor visual outcomes. Integrin alpha(6)beta(4) functions as a receptor for laminin, the major component of Bruch's membrane, and mediates the stable attachment of most epithelial cells to the underlying basement membrane. To improve adhesion and proliferation of transplanted RPE on Bruch's membrane, we elucidated the roles of integrin alpha( 6)beta(4) in RPE adhesion to extracellular matrix and investigated whether ex vivo gene transfer of integrin alpha (6) and beta(4) in RPE could promote adhesion and proliferation of transplanted RPE on Bruch's membrane. The expression of integrin alpha(6) and beta(4) mRNA and surface protein in ARPE-19 cells was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric analysis. We generated point mutation in the ligand binding domain of integrin alpha(6) and beta(4) by using site-directed mutagenesis and transfected these mutated constructs into ARPE-19 cells. Adhesion assay was used to determine the roles of integrin alpha(6) and beta(4) in RPE adhesion to extracellular matrix. In addition, we transfected full-length alpha(6) cDNA or beta(4) cDNA into ARPE-19 cells. The reattachment and proliferation ratios of alpha(6)-cDNA- or beta(4)-cDNA-transfected ARPE-19 cells on different layers of Bruch's membrane were determined by cell adhesion and proliferation assays. Cell morphology and surface coverage were evaluated by scanning electron microscopy 7 days after plating on various layers of Bruch's membrane. We found that integrin alpha(6) and beta(4) mRNA and proteins were constitutively expressed in ARPE-19 cells. Decreased endogenous integrin alpha(6) and beta(4) expression by selective mutation of amino acid residues caused a significant reduction in adhesion of ARPE-19 cells to laminin 5. Modification of integrin expression by transfection of alpha(6) cDNA into ARPE-19 cells induced a significant increase in cell adhesion to laminin 5, fibronectin, whereas transfection with beta(4) cDNA caused increased adhesion only to laminin 5 . alpha(6)-cDNA- transfectants increased cell attachment and proliferation on all layers of Bruch's membrane, whereas beta(4)-CDNA- transfectants enhanced adhesion and proliferation on basal lamina and inner collagenous layers. These data indicate that integrin alpha(6) and beta(4) play a role in adhesion of ARPE-19 cells to extracellular matrix. Modification of integrin expression by ex vivo genetic manipulation in RPE might be an alternative strategy to increase the success of RPE transplantation.en-USage-related macular degenerationintegrin alpha(6)beta(4)retinal pigment epithelium transplantationBruch's membrane