Human Canstatin Gene Transfection On Hepg₂ Cell In Vitro Apoptosis And Migration

Objective:To study the expression of recombinant plasmid canstatin gene product impact liver cells in vitro of migration and apoptosis.Methods:Animals were divided into three parts. The first part contains canstatin recombinant plasmid, including:1.Canstatin obtain genes from the human placenta, RT-PCR amplification Canstatin cDNA and gel recovery and purification, restriction enzyme digestion pcDNA3.1 (-)-3flag vector plasmid, Construction of directional connection technology using DNA recombinant plasmid containing canstatin pcDNA3.1 (-)-3flag/canstatin; 2. pcDNA3.1 (-)-3flag/ canstatin recombinant plasmid was transformed into competent Escherichia coli DH5α; 3. plasmid amplification and extraction of plasmid DNA; 4. plasmid DNA and screened by restriction analysis is made of glycerol bacterial plasmid sequence sent to the company; second part pcDNA3.1 (-)-3flag/canstatin plasmid DNA was transfected into HepG2 hepatoma cells and the expression of mRNA and protein identification, including:1. liposome hepatoma HepG2 cells were transfected, G418 selection stably transfected hepatoma HepG2 cells; 2. real-time quantitative PCR to detect and more stable turn in after canstatin hepatocellular carcinoma HepG2 cells and empty vector transfected group and untransfected group expression; 3.Western blot detection of empty vector transfected group, plasmid transfection group, simple group of hepatocellular carcinoma HepG2 cells canstatin protein; 4. collection canstatin containing the recombinant plasmid stably transfected HepG2 hepatoma cells after expression of canstatin protein; third part cantatin protein on cultured hepatoma HepG2 cell apoptosis and migration of detection, including:1. experimental groups:supernatant of empty vector transfected group, the expression plasmid transfected supernatant group, hepatocellular carcinoma HepG2 cells after stable transfection group, without hepatocellular carcinoma HepG2 cells transfected group (control group); 2. the role of each group in cultured hepatoma HepG2 cells; 3. apoptotic cells detected by flow cytometry, the apoptosis rate; 4.Trance well scratch experimental determination of small rooms and cells transfected with recombinant basement membrane migration ability.Results:1. In the human placenta was 684bp canstatin gene sequencing canstatin cDNA coding sequence with that gene pool in the registry of Chinese canstatin sequence comparison, the rate of 100 percent. Construction of eukaryotic expression vector secreted pcDNA3.1 (-)-3flag/canstatin obtained by digestion two bands 5000bp and 700bp, and pcDNA3.1 (-)-3flag plasmid 5400bp and canstatin684bp consistent.2. The constructed recombinant plasmid pcDNA3.1 (-)-3flag/canstatin transfected hepatoma HepG2 cells and screened with 400μg/mlG418 concentrations can be stabilized on the passage of transfected hepatoma HepG2 cells, RT-PCR confirmed that plasmid pcDNA3.1 (-)-3flag/canstatin has been successfully transfected into HepG2 hepatoma cells, real-time quantitative PCR results showed that the transfected gene copy number is 842128, Western blot detection of transfected cell lysate and supernatant, the SDS-PAGE the gel are to get a new treaty 26KD protein, and protein size was close to canstatin.3. Plasmid transfected HepG2 liver cancer cells in the role of cell supernatant group and hepatoma HepG2 cells after transfection, apoptosis group (respectively 15.50±0.77 and 13.33±0.92) was significantly higher than empty vector transfected the role of cell supernatant group and untransfected group cultured apoptosis rate (respectively 0.48±0.05 and 0.36±0.09) p<0.01, and the plasmid transfected cells in the supernatant of apoptotic action group rate is higher than the stable transfected HepG2 liver cancer cell apoptosis group (p<0.01);the above 24h and 48h of cell migration rate was not statistically different between (p> 0.05).Conclusion:1. A successful acquisition or construction of recombinant plasmid pcDNA3.1 (-)-3flag/canstatin;2. pcDNA3.1-Canstatin-3Flag recombinant plasmid liposome transfected into HepG2 hepatoma cells obtained canstatin gene expression in m RNA levels, and cell culture supernatant obtained canstatin protein.3. Transfected with pcDNA3.1-Canstatin-3Flag hepatoma HepG2 cells of recombinant plasmid for in vitro culture supernatant of HepG2 liver cancer cell apoptosis, and migration of hepatocellular carcinoma HepG2 cells has no effect.