The Clone Of Human P27kiplcDNA And The Study Of Its Cellular Biology Effect In Human Tongue Cancer Cell Line Tca8113 Cells

Objective: Tongue squamous cell carcinoma (TSCC) is one of the most common malignancies in oral and maxillofacial region. It grows fast and invades tissues beside soon. It often has lymph nodes transfer in earlier period and has a bad therapeutic efficacy. Recently, with the rapid development of molecular biology, gene therapy exhibits an immeasurable role in tumor therapy.Our investigative object is the human TSCC cell Tca8113 that cultured in vitro. We linked cloned human p27Kip1 gene to pcDNA3 vector and transfected it to Tca8113 cells mediated by liposomes. We discussed the biological action of p27Kip1 gene in TSCC cell line Tca8113.Methods: Extracts the RNA of the normal liver tissue. Reverse transcription the RNA to cDNA by RT-PCR method. Design the two primers in accordance with the cDNA sequence of p27Kip1 gene in Gene Bank and make PCR to amplify p27Kip1 cDNA. Undertake TA clone with the production of PCR and sequencing. Constructed the recombinated expression plasmid with eukaryon expression vector pcDNA3 after sequencing. Coating the pcDNA3-p27Kip1 and pcDNA3 plasmid DNA with liposomes separately and transfected them to Tca8113 cells. Selected the transfected cell in G418 culture medium and evaluated the Tca8113 cells strain transfected by p27Kip1 gene with PCR. We used flow cytometer to detect the expression of p27Kip1 protein, apoptosis related protein Bcl-2, cell cycle and cell apoptosis. Detected the effects on the growth of Tca8113 cells that transfected by p27Kip1 gene with MTT method. Detected the effects of chemical therapy medicine on the growth of Tca8113 cells transfected by p27Kip1 gene.Results:1. The sequence of p27Kip1 cDNA that we obtained was confirmed coincide with the sequence of Gene Bank. This result proved that it was the p27Kip1 gene with integrated read framework. We constructed the recombination expression plasmid pcDNA3-p27Kip1 successfully and it was correct after of enzyme cut. We screened positive cell clone that anti-G418 and established two new cell strains that we called them Tca-P27 and Tca-P3. Tca-P27 is the cell strain that transfected by pcDNA3-p27Kip1and Tca-P3 is the cell strain that transfected by empty vector pcDNA3. Evaluated by PCR, the genome DNA of Tca8113 cells transfected by pcDNA3-p27Kip1 plasmid appeared p27Kip1 cDNA amplification strip. The PCR production of genome DNA of Tca8113 cells and Tca-P3 cells transfected empty vector pcDNA3 were negative. This proved the pcDNA3- p27Kip1 plasmid transfected to Tca8113 cells successfully.2. We detected the expression of p27Kip1 protein with flow cytometry and registered the results by the percentage of the positive cells that expressed the p27Kip1 protein. The percentage of the positive cells that expressed the p27Kip1 protein in Tca-P27 cells is higher than Tca-P3 cells and Tca8113 cells distinctly and the difference was prominence(P<0.05). There was no prominent difference (P>.05) in the percentage of the positive cells that expressed the p27Kip1 protein in Tca-P3 cells and Tca8113 cells. These results indicated that the Tca8113 cells transfected by p27Kip1 gene obtained the high expression of p27Kip1 protein.3. Measure the OD value of Tca8113 cells, Tca-P3 cells and Tca-P27 cells with MTT method at 24 hours, 48 hours, 72 hours and 96 hours. The OD value represents the cell relative number. The results indicated that the proliferative speed of Tca-P27 cells was slower than Tca8113 cells and Tca-P3 cells distinctly. The proliferative speed of Tca-P27 cells was slower than Tca8113 cells and Tca-P3 cells at 24 hours and 48 hours. Tca-P27 cells appeared a state of descending of proliferation at 72 hours and 96 hours. Tca8113 cells and Tca-P3 cells proliferated continually at 24 hours, 48 hours, 72 hours and 96 hours and the difference was prominence(P<0.05)in the speed of proliferation among them. This investigation shown that the growth of Tca8113 cells was inhibited after transfected by p27Kip1 gene and the proliferation of Tca8113 cells presented stasis. 4. The results of cell cycle detected...