|
RESULTS
ong>β1integrinong>GFP Fusion Gene Expression in Epithelial Cells Figure 1A shows the RTPCR ong>ofong> mock transfected cells and representative ong>β1integrinong> transfected cells. mRNA expression ong>ofong> GFP in the two groups were detected, and 785bp strip was found in β1 group, which indicated that β1 GFP gene was successfully transferred into corneal epithelial cells and expressed in the mRNA level. Moreover, mRNA expression level ong>ofong> ong>β1integrinong> and GFPfused detected in β1 group was obviously upregulated compared to mock group. Increased ong>β1integrinong> mRNA was also found in ong>β1integrinong> transfected cells compared to mock group. Figure 1B showed in Western blot, 140000Da band (which represents ong>β1integrinong> and GFPfused protein) and 113000Da band (which represents ong>β1integrinong> protein) were both found in β1 group, while only 113000Da band was found in mock group, which indicates fused expression ong>ofong> ong>β1integrinong> and GFPfused protein in corneal epithelial cells.
Impact ong>ofong> ong>β1integrinong>GFP Gene Transfection to the Adhesive Force ong>ofong> Corneal Epithelial Cell In Vitro Figure 2 shows increased adhesive ability ong>ofong> transfected cells to ECM proteins but not 10g/L BSA in β1 group than in mock group (P<0.05).
Impact ong>ofong> ong>β1integrinong>GFP Gene Transfection to Corneal Epithelial Cell Apoptosis Within 5 days, apoptosis percentage was the same in mock and β1 group. At 610 days after transfection, apoptosis was inhibited in β1 group (Figure 3).
Impact ong>ofong> ong>β1integrinong>GFP Gene Transfection to MAP kinase in Corneal Epithelial Cell Figure 4 shows that ong>β1integrinong> and GFPfused gene transfection induced the phosphorylation ong>ofong> three MAP kinases, indicating MAP kinase plays an important role in resistence ong>ofong> ong>β1integrinong> to apoptosis.
DISCUSSION
The precondition ong>ofong> corneal cell transplantation is obtaining corneal epithelial cell capable ong>ofong> longterm survival and proliferation in vitro. However, apoptosis has always been the pressing issue during the process ong>ofong> cell culture in vitro. ong>β1integrinong> family is the major cellular surface receptor mediating extracellular matrix signal, and is related to the cell proliferation, differentiation, apoptosis and migration [2]. Overexpression ong>ofong> ong>β1integrinong> by the way ong>ofong> transfecting ong>β1integrinong>GFP gene into corneal epithelial cell significantly increases the adhesive force ong>ofong> corneal epithelial cell to ECM, which indicates certain biological function ong>ofong> overexpressed ong>β1integrinong> in corneal epithelial cell. And GFP is currently an ideal molecular probe analyzing protein function and dynamics at cellular level. RTPCR and Western blot confirm that insertion and expression ong>ofong> exogenous GFP gene. It provides an ideal cell model for observing and localizing the distribution and transportation ong>ofong> ong>β1integrinong> real time in viable cells. It has advantages including simplicity, specificity and lower cost over the traditional way ong>ofong> studying integrin by ECM protein coated culture plate.
Figure 1 The expression ong>ofong> transgene in transfected cells detected by RTPCR and Western blot（略）
A:RTPCR. ong>β1integrinong> and GFPfused mRNA expression was detected in ong>β1integrinong> transfected cells. Increased ong>β1integrinong> mRNA was also found in ong>β1integrinong> transfected cells compared to mock transfected cells;B:Western blot. ong>β1integrinong> and GFPfused protein expression was detected in ong>β1integrinong> transfected cells
Figure 2 The adhesive ability ong>ofong> 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 ong>β1integrinong> transfected cells. aP<0.05, vs mock cells（略）
Although there has been reports that ong>β1integrinong> is closely related to resistance to apoptosis in certain celltype cells [35]. However, the study on relationship ong>ofong> ong>β1integrinong> 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 ong>β1integrinong> as laminin receptor is possible related to the apoptosis resistence. Our results proved the possibility ong>ofong> overexpression ong>ofong> ong>β1integrinong> inhibits corneal epithelial cell apoptosis. It is reported that the mechanism ong>ofong> ong>β1integrinong> resisting apoptosis is related to the upregulation ong>ofong> Bcl2, activation ong>ofong> MAP and PI3 kinase [79]. Our data for the first time proves that ong>β1integrinong> overexpression induced MAP kinase phosphorylation in corneal epithelial cells, and ong>β1integrinong> mediated MAP kinase phosphorylation is possibly the vital mechanism ong>ofong> overexpression ong>ofong> ong>β1integrinong> inhibiting corneal epithelial cell apoptosis. MAP kinase presents with wide catalytic acitivity. It regulates gene transcription, cell growth and apoptosis through phospharylating residue ong>ofong> transacting actor in the nucleus. It is key enzyme in the apoptosis pathway [10]. We will further focusing on the role ong>ofong> three members ong>ofong> MAP kinase, i.e. ERK, p38 and JNK, played in ong>β1integrinong> antiapoptosis effect to investigate the molecular mechanism ong>ofong> ong>β1integrinong> inhibiting corneal epithelial cell apoptosis.
Our study found ong>β1integrinong> overexpression in corneal epithelial cell by transfecting ong>β1integrinong>GFP gene can inhibit corneal epithelial cell apoptosis in vitro during which phosphorylation ong>ofong> MAP kinase may play an important role. ong>β1integrinong> overexpression is an indispensable factor ong>ofong> improving longterm survival ong>ofong> corneal epithelial cells and provides important theoretical basis for the prevention ong>ofong> corneal epithelial cell apoptosis and clinical application ong>ofong> corneal cell transplantation.
Figure 3 The result ong>ofong> apoptosis in transfected cells（略）
A:Hoechst 33342 staining;B:The apoptosis cells percentage ong>ofong> mock transfected cells and ong>β1integrinong> transfected cells;C:DNA ladder. Compared with mock transfected cells, ong>β1integrinong> transfected cells showed resistance to apoptosis after 610 days transfection
Figure 4 The result ong>ofong> mitogenactivated protein kinase (MAP) kinase phosphorylation in transfected cells ong>β1integrinong> and GFPfused gene transfection induced the phosphorylation ong>ofong> MAP kinase in RCE cells（略）
【参考文献】
1 PajooheshGanji A, PalGhosh S, Simmens SJ, Stepp MA. Integrins in slowcycling corneal epithelial cells at the limbus in the mouse. Stem Cells 2006;24:10751086
2 Hasenson S, Maatta M, Rousselle P, Kekkawa Y, Miner JH, Tervo T, Virtamen I. The immortalized human corneal epithelial cells adhere to laminin10 by using Lutheran glycoproteins and integrin alpha3beta1. Exp Eye Res 2005;81:415421
3 Park CC, Zhang H, Pallavicini M, Gray JW, Baehner G, Park CJ, Bissell MJ. Beta1 integrin inhibitory antibody induces apoptosis ong>ofong> breast cancer cells, inhibits growth, and distinguishes malignant from normal phenotype in three dimensional cultures and in vivo. Cancer Res 2006;66:15261535
4 Wang R, Li J, Lyte K, Yashpal NK, Fellows F, Goodyer CG. Role for beta1 integrin and its associated alpha3, alpha5, and alpha6 subunits in development ong>ofong> the human fetal pancreas. Diabetes 2005;54:20802089
5 Pinkse GG, Voorhoeve MP, Noteborn M, Terpstra OT, Bruijn JA, De Heer E. Hepatocyte survival depends on beta1integrinmediated attachment ong>ofong> hepatocytes to hepatic extracellular matrix. Liver Int 2004;24:218226
6 Esco MA, Wang Z, McDermott ML, KurpakusWheater M. Potential role for laminin 5 in hypoxiamediated apoptosis ong>ofong> human corneal epithelial cells. J Cell Sci 2001;114:40334040
7 Tiberio R, Marconi A, Fila C, Fumelli C, Pignatti M, Krajewski S, Giannetti A, Reed JC, Pincelli C. Keratinocytes enriched for stem cells are protected from anoikis via an integrin signaling pathway in a Bcl2 dependent manner. FEBS Lett 2002;524:139144
8 Aoudjit F, Vuori K. Integrin signaling inhibits paclitaxelinduced apoptosis in breast cancer cells. Oncogene 2001;20:49955004
9 Jin A, Kurosu T, Tsuji K, Mizuchi D, Arai A, Fujita H, Hattori M, Minato N, Miura O. BCR/ABL and IL3 activate Rap1 to stimulate the BRaf/MEK/Erk and Akt signaling pathways and to regulate proliferation, apoptosis, and adhesion. Oncogene 2006;25:43324340
10 Lu L, Wang L, Shell B. UVinduced signaling pathways associated with corneal epithelial cell apoptosis. Invest Ophthalmol Vis Sci 2003;44:51025109 上一页 [1] [2] |
