The Anti-tumor Effects Of5-fluorouracil Combined With DC-CIK Cell Against Gastric Carcinoma Cell And The SP Cell In Vitro

Gastric cancer (GC) is one of the cancers with higher morbidity in theworld. There is surgical operation, radiotherapy, chemotherapy of the GC’treatment, but relapse and drug resistance of gastric cancer has limited efficacy.However, recurrence and drug resistance have important relationship withgastric cancer stem cell. Cancer stem cells (CSC) is not sensitive to thechemotherapy, which may be the root of malignant tumor recurrence andmetastasis. The side population (SP) cells are rich in undifferentiated cells, tosome extent, are similar to the characteristics of tumor stem cells, so they arethe ideal object of cancer stem cells.In recent years, the biological immune therapy in the treatment ofmalignant tumor has gradually attracted people’s attention, and cytokine-induced killer cells (CIK) and dendritic cells (DC) of cellular immunotherapyin basic and clinical trials achieved the exact curative effect. The DC and CIKcells co-culture, both can play a synergistic antitumor effect. This experimentwe will combined chemotherapy drugs with DC-CIK cells, then we watch thekilling effect of them on gastric carcinoma cell and the SP cells.PartⅠ The cultivation of the gastric cancer BGC-823side population celland the study of its biological characteristicsObjective:Chemotherapy is one of the important means in the treatment of gastriccancer, but the existence of drug resistance makes the treatment effect is poor.However, the existence of the gastric cancer stem cells may be the root of thegastric cancer drug resistance. Because of SP cell with the certaincharacteristics of CSC, so it can be studied in the aspects of gastric cancerstem cells. Method：(1) Gastric carcinoma cell line BGC-823from ultra-low temperaturefreezer was thawed out rapidly, gave passage after resuscitation.(2) Took BGC-823cells to be vaccinated in serum free medium (SFM) insuspension culture.(3) Directed counting method drew the growth curve of BGC-823cellsand SP cells.(4) MTT colorimetric assay was used to evaluate the killing effect of5-fluorouracil against BGC-823cells and SP cells.(5) Flow cytometry was used to detect the cell cycle of SP cells.Result：1BGC-823-SP cells was isolated from BGC-823cells.In serum free conditions, BGC-823cells formed tumor cell spheres about7d, and they were good refraction, suspended growth. The cells were roundbright.2The growth curve of BGC-823cells and BGC-823-SP cells.The proliferation of BGC-823-SP cells was faster than BGC-823cells,and the difference was statistically significant (P <0.01).3Killing effect of5-FU against BGC-823cells and BGC-823-SP cells.After four different concentrations of5-FU’s48h effect, the killing rate ofBGC-823cells was higher than that of BGC-823-SP cells, and the differencewas statistically significant (P <0.05).4Flow cytometry was used to detect the cell cycle of SP cells.Compared with BGC-823cells, BGC-823-SP cells in the percentage ofG0/G1phase were obviously increased.Conclusion：The SP cell, which is isolated from BGC-823cell, is much stronger onthe aspect of proliferation and drug resistance, and most of SP cells are in thecell cycle quiescence period. part Ⅱ To study the killing effect of DC-CIK cell combined with5-FUon gastric cancer cell and the SP cell in vitroObjective:In recent years, the biological immunotherapy in antitumor effect drawthe attention of people, especially the DC, CIK cells and their co-culturedDC-CIK cells become a hot topic of research. This experiment is to train thepreparation of DC-CIK cells, to observe its killing effect on gastric cancercells and the SP cells, to study the effect of chemotherapy combined withDC-CIK cells to gastric cancer cells and the SP cells, so as to provide a newstrategy and a theoretical basis for cell immune chemotherapy combinedclinical gastric cancer therapy.Method:(1) Took healthy human peripheral blood mononuclear cells (PBMCs),and add to different cytokines, then prepare DC-CIK cells.(2) Observed morphology of DC-CIK cell under microscope.(3) Flow cytometry was used to detect DC cell, DC-CIK cell phenotype.(4) MTT colorimetric assay was used to evaluate the killing rates ofdifferent effect target ratio DC-CIK cells against the BGC-823cells andBGC-823-SP cells.(5) MTT colorimetric assay was used to evaluate the killing rates of thecombination of5-FU and DC-CIK cells against the BGC-823cells andBGC-823-SP cells.Result:1Observed morphology of DC-CIK cell under inverted microscope.DC-CIK cells were spherical, uniform and transparent. And cell spacingis small, so the cells were leant close.2Flow cytometry was used to detect DC cell, DC-CIK cell phenotypeTo detect the expression of cell surface markers CD83, CD86, HLA-DRof the3d DC cells and the7d DC cells were (5.79±2.47)%,(18.95±3.21)%,(22.48±3.45)%and (63.67±1.29)%,(70.23±4.06)%,(86.43±2.78)%. The7dDC cells expression of CD83, CD86, HLA-DR were much more than that of the3d DC cells, and the difference was statistically significant (P <0.01). Thatwas mean the7d DC cells have been mature.To detect the expression of cell surface markers CD3+, CD8+,CD3+CD56+of the0d DC-CIK cells and the14d DC-CIK cells were(60.72±3.24)%,(53.87±3.25)%,(8.01±4.67)%and (79.54±2.56)%,(73.91±2.43)%,(43.45±2.30)%. The7d DC-CIK cells expression of CD3+、CD8+、CD3+CD56+were much more than that of the0d DC-CIK cells, and thedifference was statistically significant (P <0.01). That was mean the7dDC-CIK cells have been mature.3The killing effect of DC-CIK cells on BGC-823and BGC-823-SP cellsDifferent effect target ratio of DC-CIK cells had both killing effect onBGC-823and BGC-823-SP cells. At the same effect target radio, thekilling rate of DC-CIK cells against BGC-823and BGC-823-SP cells wassimilar (P>0.05).4Killing effect of5-FU combined with DC-CIK against BGC-823cells andBGC-823-SP cellsKilling effects of5-FU combined with DC-CIK cells to BGC-823andBGC-823-SP cells were stronger than the individual group, and the killingeffect was dependent increase with the DC-CIK cells effect target ratioincreased and concentration of5-FU increased.Conclusion:(1) DC-CIK cell has both killing effect on gastric cancer cells and the SPcells, and the killing rate is similar at the same effect target ratio. That is meanDC-CIK cell can kill the SP cell of gastric cancer.(2) Killing effect of5-FU combined with DC-CIK cell to gastric cancercell and SP is stronger than the single group. So chemotherapy drugscombined with cell immune therapy can enhance anti-cancer effect.