|
Apoptosis measured by Hoechst 33342 staining 1×105 of β1integrin transfected cells or mock transfected cells were cultured in 35mm culture dish. After 10 days, newly prepared Hoechst33342 (Wako, Osaka, Japan) was added to incubate for another 10 minutes at 37℃. Cell morphology was observed under fluorescence microscope. Cells with pyknosis or karyorrhexis were identified as apoptotic. Number of apoptotic cells in every 1 000 cell, known as apoptosis index was counted in randomly chosen 10 high power fields.
Apoptosis detected by DNA ladder Transfected corneal epithelial cells were collected and rinsed in PBS. After centrifuging, the supernatant was discarded. Cells were resuspended in proper volume of lysis buffer [10mmol/L ethylene diamineN, Ntetraacetic acid (EDTA), 10mmol/L Tris (pH8.0), 5g/L Triton] for 10 minutes at 4℃. Supernatant was collected after centrifuge at 15 000 r/min for 20 minutes. RNAase (100mg/L) was placed in 37℃ water bath for 1 hour. Then proteinase K (100mg/L) was added at 50℃ for 30 minutes. Solution was sedimented in isopropanol at 20℃ overnight. Centrifuge rotated at 15 000 r/min for 10 minutes to get sediment which was then washed in 700mL/L ethanol twice. Solution was centrifuged followed by air drying of the sediment which was resolved in pH 8.0 TE buffer. 20g/L agarose gel (containing 0.5mg/L EB) electrophoresis analysis was conducted for 3 hours with 1×TBE as electrophoresis buffer under persistent voltage of 50V.
Results were observed under ultraviolet light and photographed.
The phosphorylation of mitogenactivated protein (MAP) kinase examined by Western blot Corneal epithelial cells 48 hours after transfection were collected and rinsed in PBS. Supernatant was discarded after centrifuge. Cells were resuspended in proper volume of lysis buffer [50mmol/L TrisHCl (pH 7.5), 150mmol/L NaCl, 5mmol/L EDTA (pH 8.0), 1mmol/L phenylmethylsulfonal fluoride (PMSF), 1g/L SDS, 1mg/L aproptinin, 10mg/L pepstatin, 10mg/L leupeptin, 10mg/L peptin, 10mg/L soybean trypsin inhibitor], and sonicated for 45s. Supernatant was preserved at 70℃ after centrifuge at 15 000 r/min for 20 minutes at 4℃. Protein concentration was detected by BCA protein assay kit (BioRad, Hercules, CA, USA). Equal protein sample were applied to the lanes, and electrophoresed in 100g/L sodium dodecyl sulfatepolyacrylamide gel and electrophoretic transferred to polyvinylidene fluoride (PVDF) membrane. Blocking buffer was added. 1∶500 phosphorylated specific antiERK, p38 MAPK, JNK antibody, and total antiERK, p38 MAPK, JNK antibody were added and incubated at 4℃ overnight, and then in 1∶2 000 enzymelabeled secondary antibody at room temperature for 1 hour. Solution was rinsed in TBS, and developed color with ECL. Film was pressed in dark room.
Statistical Analysis Data were analyzed by SPSS 12.0 statistical package (SPSS, Chicago, IL, USA). ANOVA and Dunnett t was used for intergroup comparison. P<0.05 was considered to be significant.
RESULTS
β1integrinGFP Fusion Gene Expression in Epithelial Cells Figure 1A shows the RTPCR of mock transfected cells and representative β1integrin transfected cells. mRNA expression of 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 of β1integrin and GFPfused detected in β1 group was obviously upregulated compared to mock group. Increased β1integrin mRNA was also found in β1integrin transfected cells compared to mock group. Figure 1B showed in Western blot, 140000Da band (which represents β1integrin and GFPfused protein) and 113000Da band (which represents β1integrin protein) were both found in β1 group, while only 113000Da band was found in mock group, which indicates fused expression of β1integrin and GFPfused protein in corneal epithelial cells.
Impact of β1integrinGFP Gene Transfection to the Adhesive Force of Corneal Epithelial Cell In Vitro Figure 2 shows increased adhesive ability of transfected cells to ECM proteins but not 10g/L BSA in β1 group than in mock group (P<0.05).
Impact of β1integrinGFP 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 of β1integrinGFP Gene Transfection to MAP kinase in Corneal Epithelial Cell Figure 4 shows that β1integrin and GFPfused gene transfection induced the phosphorylation of three MAP kinases, indicating MAP kinase plays an important role in resistence of β1integrin to apoptosis.
DISCUSSION
The precondition of corneal cell transplantation is obtaining corneal epithelial cell capable of longterm survival and proliferation in vitro. However, apoptosis has always been the pressing issue during the process of cell culture in vitro. β1integrin family is the major cellular surface receptor mediating extracellular matrix signal, and is related to the cell proliferation, differentiation, apoptosis and migration [2]. Overexpression of β1integrin by the way of transfecting β1integrinGFP gene into corneal epithelial cell significantly increases the adhesive force of corneal epithelial cell to ECM, which indicates certain biological function of overexpressed β1integrin 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 of exogenous GFP gene. It provides an ideal cell model for observing and localizing the distribution and transportation of β1integrin real time in viable cells. It has advantages including simplicity, specificity and lower cost over the traditional way of studying integrin by ECM protein coated culture plate.
上一页 [1] [2] [3] 下一页 |
