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Figure 1 The expression of transgene in transfected cells detected by RTPCR and Western blot(略)
A:RTPCR. β1integrin and GFPfused mRNA expression was detected in β1integrin transfected cells. Increased β1integrin mRNA was also found in β1integrin transfected cells compared to mock transfected cells;B:Western blot. β1integrin and GFPfused protein expression was detected in β1integrin transfected cells
Figure 2 The adhesive ability of transfected cells to xtracellular matrix (ECM) proteins (F=29.198,P<0.001). Compared with mock transfected cells, the adhesive ability to ECM proteins but not 10g/L BSA was increased in β1integrin transfected cells. aP<0.05, vs mock cells(略)
Although there has been reports that β1integrin is closely related to resistance to apoptosis in certain celltype cells [35]. However, the study on relationship of β1integrin and corneal epithelial cell apoptosis has been in the initial stage. Esco et al[6] found that when antilaminin antibody acting on corneal epithelial cell, and blocking intragenous and exogenous laminin, mass apoptosis occurred in primary cell. Adding laminin could obviously resist apoptosis, which indicates β1integrin as laminin receptor is possible related to the apoptosis resistence. Our results proved the possibility of overexpression of β1integrin inhibits corneal epithelial cell apoptosis. It is reported that the mechanism of β1integrin resisting apoptosis is related to the upregulation of Bcl2, activation of MAP and PI3 kinase [79]. Our data for the first time proves that β1integrin overexpression induced MAP kinase phosphorylation in corneal epithelial cells, and β1integrin mediated MAP kinase phosphorylation is possibly the vital mechanism of overexpression of β1integrin inhibiting corneal epithelial cell apoptosis. MAP kinase presents with wide catalytic acitivity. It regulates gene transcription, cell growth and apoptosis through phospharylating residue of transacting actor in the nucleus. It is key enzyme in the apoptosis pathway [10]. We will further focusing on the role of three members of MAP kinase, i.e. ERK, p38 and JNK, played in β1integrin antiapoptosis effect to investigate the molecular mechanism of β1integrin inhibiting corneal epithelial cell apoptosis.
Our study found β1integrin overexpression in corneal epithelial cell by transfecting β1integrinGFP gene can inhibit corneal epithelial cell apoptosis in vitro during which phosphorylation of MAP kinase may play an important role. β1integrin overexpression is an indispensable factor of improving longterm survival of corneal epithelial cells and provides important theoretical basis for the prevention of corneal epithelial cell apoptosis and clinical application of corneal cell transplantation.
Figure 3 The result of apoptosis in transfected cells(略)
A:Hoechst 33342 staining;B:The apoptosis cells percentage of mock transfected cells and β1integrin transfected cells;C:DNA ladder. Compared with mock transfected cells, β1integrin transfected cells showed resistance to apoptosis after 610 days transfection
Figure 4 The result of mitogenactivated protein kinase (MAP) kinase phosphorylation in transfected cells β1integrin and GFPfused gene transfection induced the phosphorylation of MAP kinase in RCE cells(略)
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