Construct Of SiRNA-MDR1 Eukaryotic Expression Vector And The Expression Of MDR1 Gene In Multidrug Resistance Cell Line TCA8113/CDDP

Part 1 Construct of siRNA-MDRl eukaryotic expression vectorObjective:Construct siRNA-MDR1 eukaryotic expression vector and provide a way to tongue cancer drug resistance reversal.Methods:According to MDR1 gene sequence, we designed and constructed gene targeting MDR1 shRNA eukaryotic expression vector plasmid. By enzyme electrophoresis and Plasmid sequence detection, used to detect recombinant plasmid synthesis. The plasmid was transformed into DH5a competent Escherichia coli. Re-amplified plasmid will use for the next experimentResult:Targeted by pGenesil1.1 vector of shRNA expression plasmid MDR1, which successfully transformed into DH5a host strain. After sequencing, it showed exactly the same sequence. Targeting MDR1 gene was successfully constructed shRNA expression plasmidConclusion:Construction of pGenesil1.1-siRNA-MDR1 eukaryotic vectors can be used for follow-up to reverse the drug resistance test.Part 2 Establishment of drug resistance cell line of human tongue cancer-TCA8113/CDDPObjective:To establish drug resistance cell line of tongue cancer TCA8113/CDDP and study the biological characteristics, and to provide the experimental basis for clinical treatment.Methods:Tongue cancer cell line TCA8113 was induced by Cisplatin with high-dose impact and gradual increase in dose. Sensitivity of TCA8113/CDDP cells and parental cells to different chemotherapeutic drugs was detected by MTT assay. Cell growth curve was drawn according to the results of cell counting.Results:Resistant cell line TCA8113/CDDP was established successfully, which grew well in the 1μg/ml Cisplatin. The results of drug susceptibility test Measured by MTT showed it had different degrees of tolerance to CDDP, PYM, CTX and 5-FU, the resistance index 4.27.3.03,1.41 and 1.09 respectively. Results of cell counting showed that the doubling time of TCA8113 and TCA8113/CDDP cells was 30.1 and 35.2 hours respectively.Conclusion:The acquired multi-drug resistant of the TCA8113/CDDP cell is stable and significantly different from that of parental cells. It is a reliable cell model which can use for the study of multi-drug resistance mechanisms.Part 3 Expression of MDR1 gene in multidrug resistance cell line TCA8113/CDDP and its clinical significance Objective:To explore the expression of MDR1 gene in multidrug(?) resistance cell line of human tongue cancer TCA8113/CDDP and its clinical significance and to provide theoretical basis for the study of tumor multidrug resistance and reversal agents.Methods:Intracellular expression of MDR1 mRNA and P-gp expression were detected by RT-PCR and immunohistochemistry respectively.Results:MDR1 gene was not expressed in TCA8113 cells, but highly expressed in TCA8113/CDDP cells. P-gp was expressed intensely in TCA8113/CDDP, but not in TCA8113 cells.Conclusion:Multi-drug resistance of TCA8113/CDDP cell line induced by Cisplatin is closely related with the expression of MDR1/P-gp. This study provides a theoretical basis to increase the sensitivity to chemotherapy of tongue cancer by MDR1 reversal agents.