The Effects Of Fibronectin On Cell Proliferation, Attachment And Migration In A Human Lens Epithelial Cell Line

IntroductionFibronectin(FN) is a kind of macromolecule glycoprotein as a important ingredient of extracellular matrix(ECM).A series of domain can bind to fibrin, collagen protein, heparin, actin,DNA and cells,playing a role as a bridge in cell-cell,cell-basal membrane interaction to maintain normal morphous and function of cells.Normal adult capsule of lens is mainly composed of type IV collagen and laminin,also FN in embryonic period. Fibronectin plays a important role in the embryologic development of the lens,which hints that FN may be a significant ingredient in regulating the proliferation , attachment and migration of lens epithelial cells(LECs).After cataract is the most common disorder to affect visual acuity after the operation. After extracapsular cataract extraction, the proliferation , attachment and migration of the remaining LECs on the posterior capsule,succeeded with the transdifferentiation and ECM deposition, is the important mechanism of posterior capsular opacification(PCO). Compared with the intact lens, blood-aqueous barrier is destroyed after surgery,the extracellular matrix (ECM) around the remaining LECs is altered, the introduction and the deposition onto the posterior capsule of fibronectin may play a important role in the development of PCO. Some research shows that FN can significantly promote the proliferation of porcine lens epithelial cells and the attachment and migration of rabbit lens epithelial cells in vitro. In the study on a human lens epithelial cell line SRA 01/04(transfected with large T-antigen of SV40), FN promoting the attachment and migration is significant,but promoting the proliferation is not obvious.There are integrin receptors mediating cell proliferation,attachment and migration on cell surface,which is the important ingredient of cell adherion molecules(CAM).It is a transmembrane glycoprotein heterodimers composed of a subunit andβsubunit,αsubunit is relevant with ligand recognization,βsubunit transmits signals into cytoskeleton,mediating the cell-cell or cell-basal membrane interaction, participating the cell locating and coherence,as well as the information transmitting into and out of cells. The induction and expression of genes, phosphorylation of protein kinase, variation of pH and Ca²⁺ in cells and cyclohexanhexol lipid metabolism are all invoived.Nishi first detected integrinβ1 in lens epithelial cells of cataract patients,accordingly revealed correlation between some ophthalmopathy and integrin. Integrinα5β1 is the major receptor of FN,can bind to RGD(Arg-Gly-Asp)sequence in FN,regulating the attachment and proliferation of LECs, Expression of integrin may play a role in LECs-posterior capsule attachment after surgery.Human lens epithelial cell line HLE-B3 is a immortalized cell line,obtained by infecting cells with adenovirus 12 containing an immortalizing gene derived from simian virus 40(SV40). It shows high proliferative potency with the characteristics of HLECs. Compared with cells in primary culture,the major advantage of this cell line is that a large number of cells are readily available to undertake a wide range of studies concerning the molecular events that occur in these cells.Our study observed the influence of FN on the proliferation,attachment,migration and the expression of integrinα5 of HLE-B3,trying to get a better understanding of the biologic characteristics of HLE-B3 and the role of FN in the development of PCO and to provide evidence for exploring the mechanism of after cataract.ObjectiveTo investigate the influence of fibronectin on the proliferation,attachment and migration of human lens epithelial cell line HLE-B3 and the expression of its receptor integrinα5.MethodsHuman lens epithelial cell line HLE-B3 was cultivated and inoculated in plates coated by FN in different concentration (0μg/ml,10μg/ml,20μg/ml,40μg/ml) .The biologic characteristics of HLE-B3 was observed with inversed microscopy.WST-8 method was used to detect the proliferation and attachment of the cells. Scratch method was used to observe the migration of the cells,the hours of the closure of cell defect domain was recorded. Immunohistochemical method was used to detect the expression of integrinα5.ResultsAfter coating with FN in different concentration,the increasing of absorbance detected by WST-8 method was not obvious, in proliferation essay.As the coating concentration of FN rised,the absorbance increased gradually in attachment essay, the hours of the closure of cell defect domain were shorten gradually in scratch essay. The expression of integrinα5 was enhanced as the coating concentration of FN rised.ConclusionsFN can significantly promote the attachment and miagration of HLE-B3 as well as up-regulating the expression of its receptor integrinα5,but the effect on the proliferation of HLE-B3 was not obvious. Our study further revealed the biologic characteristics of HLE-B3, showed that FN may play a important role in the development of secondary cataract.