| Objective: 1. To establish cisplatin-resistant gastic cancer SGC7901/DDP and provide the cell model for chemosensitive study;2. To observe the chemosensitive change of cisplatin-resistant gastic cancer SGC7901/DDP with adenovirus-mediated human IL-24 gene and investigate the possible mechanism.Methods: A cisplatin-resistant gastic cancer SGC7901/DDP was established by increasing cisplatin concentration step by step and continuing induction. Then we observed the morphological changes of SGC7901/DDP through an inverted microscope, draw the cell growth curve and calculate the population doubling time of SGC7901/DDP by cell counting. The resistance index and cross-resistance characteristics of SGC7901/DDP cells was detected by CCK-8 assay. The drug resistance associated genes expression of SGC7901/DDP cells and SGC7901 cells were detected by RT-PCR.The recombinant adenovirus completed the amplification and the titration was also detected in QBI-293A cells. We detected adenovirus infection efficiency of the drug-resistant cells to determine the appropriate multiplicity of infection. The IL-24 gene expression of the drug-resistant cells infected with Ad-IL-24 was identified by RT-PCR. The growth inhibition and chemosensitivity of drug-resistant cells and drug-sensitive cells which were infected with Ad-IL-24 were tested by CCK-8 essay. The drug resistance associated genes and apoptosis associated genes expression of the drug-resistant cells infected with adenovirus were detected by RT-PCR. The cell cycle of drug-resistant cells infected with Ad-IL-24 was detected by flow cytometry(FCM). The influences of Ad-IL-24 and DDP on drug-resistant cells apoptosis was detected by double fluorescent labeling with Annexin V-PE.7-AAD.Results: We took 6 months to raise the cisplatin concentration 8 times and successfully established the drug-resistant gastric cancer cell SGC7901/DDP which could tolerate 1ug/ml DDP by gradually increasing cisplatin concentration and continuing exposure. Compared with SGC7901 under inverted microscope, SGC7901/DDP cells showed heterogeneity for the size, irregular shape, unclear boundaries, cell volume slightly increased, irregular nuclear and giant cells. Compared with SGC7901 cells, the proliferation rate of SGC7901/DDP cell slowed down and the population doubling time extended approximately 5 hours (P<0.05) from the cell growth curve. The resistance index of SGC7901/DDP to DDP, 5-FU, ADM were 9.77±0.46, 5.71±0.10 and 2.51±0.33 respectively, indicating SGC7901/DDP cross-resistance characteristics. The multidrug-resistance associated genes MDR1, MRP1, GST of SGC7901/DDP cells expressed higher than those of SGC7901 cells by RT-PCR.After the amplification of Ad-IL-24 in QBI-293A cells for 48 hours, we could see the cells becoming round and gathered like grapes under inverted microscope. We could observe the green light under fluorescence microscope which means high efficiency of infection. The titration was 1×109pfu/ml. 50MOI was the most appropriate the multiplicity of infection that the recombinant adenovirus infected SGC7901/DDP cells. The infection efficiency is above 80%. After infected with Ad-IL-24, the IL-24 was proved to be successfully transcribed in SGC7901/DDP cells and SGC7901 cells by RT-PCR. After infected with 50MOI Ad-IL-24, SGC7901/DDP cells and SGC7901 cells growth were inhibited and cell inhibition was significantly increased on the fourth day; The IC50 of SGC7901/DDP cells to DDP, 5-FU, ADM had decreased and their reversal multiple were 1.57±0.13, 1.45±0.08 and 3.76±0.57 respectively. After SGC7901/DDP cells were infected with Ad-IL-24, the expression of multidrug-resistance associated gene MDR1, MRP1 and apoptosis gene Bcl-2, Survivin decreased and the expression of the apoptosis gene Bax enhanced. After SGC7901/DDP cells were infected with Ad-IL-24, the cells of G0/G1 phase and G2/M phase increased, the cells of S phase decreased, and apoptosis peak appeared before G1 phase. The apoptosis rate of Ad-IL-24 and DDP combination group was significantly higher than that of pure drug group or single gene group or control group by FCM with annexin V-PE/7-AAD double staining.Conclusion: 1. The cisplatin-resistance gastric cancer cell line SGC7901/DDP was established by increasing the concentration step by step and continuing induction.2. SGC7901/DDP cells had multidrug resistant characteristics. The possible multidrug resistant mechanism of SGC7901/DDP correlate with the population doubling time extending and multidrug resistance gene MDR1, MRP1 and GST upregulation.3. IL-24 gene mediated by adenovirus vector can increase chemosensitivity on SGC7901/DDP cells. It correlates with the upregulation of Bax gene and downregulation of MDR1, MRP1 gene. |
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