作者：YouDong Wang
作者单位：Foundation item: Supported by S&T Research Project ong>ofong> Liaoning Provincial Department ong>ofong> Education (No. 20061000)
【摘要】 AIM: To investigate the effect ong>ofong> ong>β1integrinong> overexpression on the apoptosis ong>ofong> rabbit corneal epithelial cells and the related mechanism.
METHODS: The plasmid expressing ong>β1integrinong>GFP fusion protein was constructed by polymerase chain reaction (PCR), and this plasmid (β1 group) or the empty vector (mock group) was transfected into rabbit corneal epithelial cells, respectively. The expression ong>ofong> ong>β1integrinong>GFP fusion gene was confirmed by reverse transcriptionpolymerase chain reaction (RTPCR) and Western blot. The adhesion ong>ofong> transfected cells to extracellular matrix (ECM) proteins was determined by adhesion assay. The apoptosis ong>ofong> rabbit corneal epithelial cells was assayed by Hoechst 33342 staining and DNA ladder. The phosphorylation ong>ofong> mitogenactivated protein (MAP) kinase was examined by Western blot.
RESULTS: Rabbit corneal epithelial cells overexpressing ong>β1integrinong>GFP fusion gene were successfully established. Compared with mock group, ong>β1integrinong> transfection significantly promoted the adhesive ong>ofong> rabbit corneal epithelial cells to ECM proteins such as laminin, fibronectin, collagen I and collagen IV. ong>β1integrinong> overexpression inhibited apoptosis and induced MAP kinase phosphorylation in rabbit corneal epithelial cells (P<0.05).
CONCLUSION: These data suggest that overexpression ong>ofong> ong>β1integrinong> confers resistance to apoptosis in rabbit corneal epithelial cells, and MAP kinase pathway may play an important role in this process.
KEYWORDS: corneal epithelial cells; ong>β1integrinong>; apoptosis; MAP kinase
【关键词】 corneal epithelial cells ong>β1integrinong> apoptosis MAP kinase
INTRODUCTION
Corneal transplantation utilizing cultured corneal epithelial cell in vitro overcomes the unavailability ong>ofong> donor corneas and postoperative transplantation rejection ong>ofong> penetrating keratoplasty, which shows its broad prospects. However, how to overcome cellular apoptosis in vitro, and obtain corneal epithelial cell with longterm survival and proliferation has been the bottleneck ong>ofong> corneal cell transplantation and the topic ong>ofong> great interest to researchers. ong>β1integrinong> family is one ong>ofong> the factors that most correlated to the survival ong>ofong> epithelial cells [1]. In the present article we investigated the possibility and mechanism ong>ofong> inhibition ong>ofong> corneal epithelial cell apoptosis by the overexpression ong>ofong> ong>β1integrinong>, and provided theoretical basis for the prevention ong>ofong> corneal epithelial cell apoptosis and clinical application ong>ofong> corneal cell transplantation.
MATERIALS AND METHODS
Reagents and Materials HamsF12 and DMEM (Gibco, Langley, OK, USA) mixed at 1∶1,with 100mL/L fetal bovine serum (FBS) (Gibco), 100U/mL penicillin, 100mg/L streptomycin and 0.02g/L glutamine then added and pH value adjusted to be 7.27.4, served as cell culture solution. Phosphorylated specific antiERK, p38 MAPK, JNK antibody, and total antiERK, p38 MAPK, JNK antibody (Cell Signaling, Danvers, MA, USA).
Methods
Rabbit corneal epithelial cell culture Eyeballs ong>ofong> adult Holland rabbits weighing 2.53.0kg were extracted aseptically and washed with normal saline after they were executed with air embolism. Corneal tissue which contains only epithelium and stroma was separated on the superclean blenches, and then was digested in 3mL 2.5g/L trypsin at 4 ℃ overnight. On the next day corneal epithelium was scraped gently with scraper and washed in Hanks solutions for three times. 2mL 100mL/L FBS contained culture solution were then added and when cells dispersed evenly, they were transferred to 24 and 6well culture plate and incubated in 50mL/L CO2 incubator at 37℃. Cell concentration is adjusted to 4×105/mL with culture solution.
Construction ong>ofong> the plasmid expressing ong>β1integrinong>GFP fusion protein Plasmid pBJ1, provided by Dr. Y. Takada (Scripps Research Institute, USA), contains fulllength encoding sequence ong>ofong> ong>β1integrinong> cDNA. Fulllength cDNA sequence ong>ofong> ong>β1integrinong> lacking termination codon was obtained by polymerase chain reaction (PCR), then inserted with restriction BamH1 and XhoI restriction enzyme cutting sites at ends, and then was assembled into plasmid pT7Blue vector (Novagen, Madison, WI, USA) to form pT7GFβ1. Sequence ong>ofong> cDNA ong>ofong> ong>β1integrinong> were determined by DNA sequencer. pT7GFβ1 was digested by BamH1 and XhoI
restriction enzyme, and 2.4kb fragment was recycled, and connected to the same restriction enzyme cutting site in pEGFPN1 vector (Clonetech, Palo Alto, CA, USA) to form ong>β1integrinong>green fluorescein protein (GFP) fusion plasmid pEGFPN1/GFβ1, and was confirmed by restriction enzyme.
Gene transfection Cell concentration ong>ofong> the passage 24 cells were adjusted to 4×105/mL and these cell were transferred to the culture plate until 80% ong>ofong> cells fused. pEGFPN1/GFβ expressing plasmid (β1 group) and pEGFPN1 empty vector (mock groupwere transfected into cells using Lipong>ofong>ectamine gene transfection technology (GIBCOBRL, Grand Island, NY, USA) (refering to company specifications). Transfection solution was aspirated after 12 hours ong>ofong> incubation in 50mL/L CO2 incubator at 37℃ and was added into serum contained culture solution for 2 days incubation.
Reverse transcriptionpolymerase chain reaction (RTPCR) detection ong>ofong> mRNA expression ong>ofong> transfected gene in transfected cells Total RNA is extracted by total RNA extraction kit (Nippon Gene, Toyama, Japan) following its instructions. RNA quality is detected by electrophoresis and optic density is detected at 260nm wavelength. All the primers are known as the gene specific by consulting Genbank. GFP primer sequence: 5GCAAGCTGACCCTGA AGTTCATC3, reverse sequence: 5’GGATCTTGAAAT TCACCTTGATGC3’, length ong>ofong> PCR product is 384bp. Primer sequence ong>ofong> ong>β1integrinong>: 5’AGAATCCAGAGTGT CCCACTGG3’, reverse sequence: 5’TTCCCTCATAC TTCGGAT TGA3’, length ong>ofong> PCR product is 238bp. Glyceraldehyde 3phosphate dehydrogenase (GAPDH) primer sequence: 5’ACGCATTTGGTCGTATTGGG3’, reverse sequence: 5’TGATTTTGGAGGGATCTCGC3’, length ong>ofong> PCR product is 231bp. ong>β1integrinong>GFP fusion gene expression is detected using sense ong>β1integrinong> and antisense GFP as primers. Product length is 785bp. RTPCR is conducted by the instructions ong>ofong> firststrand synthesis kit (Takara, Shiga, Japan). The amplifying conditions: degeneration at 95 ℃ for 1 minute, annealing at 55℃ for 1 minute, extension at 72 ℃ for 1 minute, 30 cycles. PCR products are detected by 20g/L agarose gel electrophoresis, and observed at longwavelength light after Ethidium bromide staining.
Cell adhesion test Laminin, fibronectin, type I and IV collagen coated 24well culture plate (BD Company, Franklin Lakes, NJ, US). In the control group, culture plate was coated with 10g/L BSA. At 1 hour after 1g/L BSA blocking all the wells at 37℃. Cells transfected by ong>β1integrinong>GFP fusion gene expression plasmid or empty vector were added to 24well plate coated with different extracellular matrix proteins with concentration ong>ofong> 5×104 cells in each well. These cells were incubated in 50mL/L CO2 incubator at 37℃ for 1 hour and rinsed in PBS three times and suspension cells were removed. Number ong>ofong> adhesion cells was evaluated by the absorbance measured by WST1 proliferation kit.
Apoptosis measured by Hoechst 33342 staining 1×105 ong>ofong> ong>β1integrinong> 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 ong>ofong> 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 ong>ofong> 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 ong>ofong> 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 ong>ofong> 50V.
Results were observed under ultraviolet light and photographed.
The phosphorylation ong>ofong> 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 ong>ofong> 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.
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