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Empirical Study of CIK Cells Combined with Oxaliplatin Against Oxaliplatin-induced Human Gastric Cancer Drug Fast Cell Line

Author: HuXiaoJie
Tutor: ZhaoQun；LiYong
School: Hebei Medical University
Course: Surgery
Keywords: Gastric cancer CIK cells Oxaliplatin Multidrug resistance Tetrazolium salt ( MTT ) colorimetric
CLC: R735.2
Type: Master's thesis
Year: 2008
Downloads: 186
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Abstract

Objective: Gastric cancer is the most common gastrointestinal cancer, the mortality rate in our country ranks first in the malignant tumor. Chemotherapy tumor surgery is the most effective means of adjuvant therapy, has a pivotal role in the prevention and treatment of tumor recurrence and metastasis. Oxaliplatin (oxaliplatin, L-OHP) is a third-generation platinum-based anticancer drugs, following cisplatin and carboplatin has been gradually as the primary drug for the treatment of advanced gastric cancer, and shows a good prospect. Although the chemotherapy of gastric cancer continues to improve, but some of the patients with gastric cancer chemotherapy is not ideal, multidrug resistance (multidrug resistance, MDR) is the failure of chemotherapy lies. With the rapid development of tumor immunology, molecular biology and genetic engineering techniques, cytokine therapy as a means of anti-tumor Molecular Therapeutics by the wide attention of scholars at home and abroad, showing good prospects. Cytokine-induced killer cells (cytokine-induced killer, CIK) is a new, efficient, broad-spectrum immune cells, research shows that CIK cells with strong in vitro and in vivo anti-gastric cancer cell activity, may be used in clinical advanced gastric cancer adoptive immunotherapy. MDR tumor cell killing activity of immune effector cells compared with the parental cells similar or even higher. At present, less killing effect of CIK cells combined with chemotherapy for MDR tumor cells in vitro and in vivo studies. In this study, on the basis of the establishment of a human gastric cancer invasion and metastasis of the oxaliplatin resistant cells OCUM-2MD3/L-OHP, the application Pathomorphology MTT method to observe the CIK cells combined with L-OHP gastric cancer invasion and metastasis Osage platinum resistant cell OCUM-2MD3/L-OHP in vitro anti-tumor effect, to provide experimental basis for clinical application of CIK cells combined with chemotherapy in the treatment of drug-resistant gastric cancer. Materials and Methods: 1 Materials 1.1 experimental cell: high human gastric cancer invasion transfer cell lines the cell lines OCUM-2MD3, families and eight generations by the Medical University in Osaka, Japan, and kindly provided by Prof.. Human gastric cancer invasion and metastasis of oxaliplatin resistant cell line OCUM-2MD3/L-OHP established by the laboratory for culture, resistance index (resistant index, RI) 4.3, and passaged or frozen three months after the recovery remains Similar resistance. Resistance protein P-gp expression in the resistant cell line than the parental cell line. Two cells were placed in DMEM containing 10% FBS, 37 ℃, 5% CO ₂ were incubated, respectively, to take the two cells of the logarithmic growth phase, the preparation of a concentration 1 × 106 / mL of the cell suspension set aside. 1.2 Experiment drugs: oxaliplatin (oxaliplatin, L-OHP), the Jiangsu Hengrui Pharmaceutical Factory production, diluted with 0.9% saline 1200μg/mL, 600μg/mL, 300μg/mL, 150μg/mL, 75μg/mL spare. 1.3 effector cells (CIK cells) Preparation and in vitro amplification: take normal heparinized peripheral lymphocytes stratified fluid density gradient centrifugation separated PBMC, PBS washed cells were resuspended and saved by RPMI-1640 , the culture medium consisting of 10% human AB serum, 25mmol/LHEPES, 2mmol/L- Glutamine, 100μg/mL penicillin, 100μg/mL streptomycin, 50 micromol / L 2 - mercaptoethanol, PBMC was adjusted to 1 × 10 After ^{6 / mL, the first day of the concentration of 100μg/mL rhIFN-gamma, after 24 hours of incubation, and the concentration of 50μg/mL CD3McAb 100U/mL of rhIL-1 and 300U/mL rhIL-2, placed 37 ° C, 5% CO ₂ hatched box culture, cell changed every 2 days fresh medium subculture, while additional rhIL-2 1000U / mL, cells harvested 14 days 2.1 L-OHP intervention group before adjustment effector to target ratio 40:1,20:1 and 10:1. 2 packets (the groups were OCUM-2MD3 cells as a control): Calculation of different concentrations of L-OHP alone of OCUM-2MD3/L-OHP cells 24h, 48h, 72h in vitro killing activity. 2.2 CIK cells intervention group: OCUM-2MD3/L-OHP cells as effector cells, CIK cells as target cells, calculate the separate application of different effector to target ratio CIK cells of OCUM-2MD3/L-OHP cells 12h, 24h, 48h in vitro anti- activity. 2.3 CIK cells combined L-OHP intervention group: effector to target ratio 40:1 CIK 12h and then different concentrations of L-OHP combination of both calculated after 24 hours on in vitro of OCUM-2MD3/L-OHP cells destruction activity. 3 3.1 Application inverted phase contrast microscope to observe the above three groups OCUM-2MD3 / L-OHP cell morphological changes. 3.2 application MTT assay above three groups at a wavelength of 570nm optical density (OD) values ??were calculated at different times L-OHP, CIK cells alone and the combination of the two the OCUM-2MD3/L-OHP cells in vitro activity. 4 statistical methods of experimental data processing with SPSS 12.0 statistical software, P lt; 0.05 when the difference was significant. Results: 1 in vitro cytotoxic activity of pathological morphology observed under inverted phase contrast microscope OCUM-2MD3/L-OHP cell lines with the than cell lines OCUM-2MD3 cells, no significant morphological difference. 1.1 L-OHP on OCUM-2MD3/L-OHP cells in vitro cytotoxic activity of pathological morphology observed under an inverted microscope observation, the dosing of the OCUM-2MD3/L-OHP cells cultured cell number after 24h decreased slightly, loosely arranged cells form rounded, smaller size, refraction nuclear cytoplasm ratio larger, floating cells, and the cytoplasm of larger particles; after 48h the cells continue to reduce the number of the intracytoplasmic particles increased, cell debris and visible; continue to reduce the number of cells cultured for 72h, seen more cell debris. L-OHP the cytotoxicity of OCUM-2MD3 strong in OCUM-2MD3/L-OHP cells, of each period OCUM-2MD3 cells to reduce the amount of the number and cell debris much larger than OCUM-2MD3/L-OHP cells. Control group gastric cancer cells still adherent growth, in good condition. 1.2 CIK cells of OCUM-2MD3/L-OHP cells in vitro cytotoxic activity of pathological morphology observed The CIK cells grown in suspension cells were round, active cell growth increase in BaoTuan rate. CIK cells observed under an inverted microscope a certain direction athletic ability, gradually gathered to around the OCUM-2MD3/L-OHP cells OCUM-2MD3/L-OHP cytoplasmic granular material, the cells gradually blurred and disappeared , affixed to the wall of the growth of cancer cells floating. Effector to target cells co-cultured the 12h OCUM-2MD3/L-OHP cells significantly reduced, significantly floating 24h cells and debris in the culture medium. 48h appears a large amount of debris in the culture medium. The CIK cells the cytotoxicity of OCUM-2MD3 and similar to OCUM-2MD3/L-OHP cells. Control group gastric cancer cells still adherent growth, in good condition. CIK cells alone no-driven phenomenon, uniform with the field of vision. 1.3 CIK cells combined L-OHP on OCUM-2MD3/L-OHP cells in vitro cytotoxic activity of pathological morphology observed under an inverted microscope observation, CIK cells gradually gathered to around the OCUM-2MD3/L-OHP cells, OCUM-2MD3 / L -OHP cytoplasmic granular material, the cells gradually blurred, disappeared, the adherent growth of cancer cells floating effector to target cells co-cultured cells were significantly reduced in 12h OCUM-2MD3/L-OHP, L-OHP after intracytoplasmic particles increased, showing a large number of cell debris, CIK cells combined with L-OHP the OCUM-2MD3 cell killing effect similar to OCUM-2MD3/L-OHP cells. 2 in vitro cytotoxic activity of the MTT assay results of 2.1 L-the OHP on OCUM-2MD3/L-OHP cells in vitro cytotoxic activity of 24h, L-OHP on the role of OCUM-2MD3 cells the IC50 111.3μg/mL on OCUM-2MD3 / L-OHP cells and the IC50 tolerance of 354.4μg/mL OCUM-2MD3/L-OHP L-OHP more parental the sensitive strains OCUM-2MD3 increase 3.2 times. 48h, L-OHP on OCUM-2MD3 cells and the IC50 71.2μg/mL, the the IC50 role of OCUM-2MD3/L-OHP cells 235.2μg/mL, the OCUM-2MD3/L-OHP L-OHP tolerated than the parental the sensitive strains OCUM-2MD3 increased by 3.3 times. 72h, L-OHP on OCUM-2MD3 cells and the IC50 522.3μg/mL, L-OHP the IC50 role of OCUM-2MD3/L-OHP cells of 1057.0μg/mL, OCUM-2MD3/L-OHP tolerated than the parent the sensitive strains OCUM-2MD3 increased 2.0 times. L-OHP on OCUM-2MD3/L-OHP the cells and OCUM-2MD3 cells in vitro cytotoxic activity of the strongest in 48h. And enhanced with the increase of the concentration of L-OHP. 2.2 CIK cells on in vitro cytotoxic activity of cells OCUM-2MD3/L-OHP 12h, of CIK cells resistant cell line OCUM-2MD3/L-OHP the killing activity was significantly higher than that the parental sensitive strains the cell lines OCUM-2MD3 (P lt; 0.05). And cytotoxic activity with effector to target ratio was enhanced. 24h CIK cell killing activity was significantly higher than that of OCUM-2MD3/L-OHP OCUM-2MD3 (P lt; 0.05). And cytotoxic activity with effector to target ratio was enhanced. 48h CIK cell killing activity was significantly higher than that of OCUM-2MD3/L-OHP OCUM-2MD3 (P lt; 0.05). And cytotoxic activity with effector to target ratio was enhanced. CIK cells against two target cell killing activity in the 24 hours maximum, and killing activity with effector to target ratio was enhanced the CIK cell in each time period the cytotoxic activity of the resistant cell line OCUM-2MD3/L-OHP are significantly higher than the parental sensitive strain the cell lines OCUM-2MD3 (P lt; 0.05). Prompt and OCUM-2MD3 cells compared to the CIK cells of OCUM-2MD3/L-OHP cells have strong in vitro activity. 2.3 CIK cells combined with L-OHP the OCUM-2MD3/L-OHP the in vitro cytotoxic activity of CIK cells combined with L-OHP on OCUM-2MD3/L-OHP cells and OCUM-2MD3 of in vitro activity than those of pure L-OHP killing activity of CIK cells increased significantly (P lt; 0.05). Joint destruction increase the size of the activity with L-OHP concentration increased. United CIK cell therapy, L-OHP on the OCUM-2MD3 OCUM-2MD3/L-OHP cells and the IC50 reduced. CIK cells combined L-OHP with OCUM-2MD3 cells resistant cell line of human gastric cancer invasion and metastasis OCUM-2MD3/L-OHP significant synergistic effect (P lt; 0.05) Conclusion: 1 parental cells OCUM-2MD3 compared, resistant cells OCUM-2MD3 / L-OHP in morphology was no significant difference. On the role of L-OHP, intracytoplasmic particles increased and visible cell debris; CIK cells effect, cytoplasmic granular material, the cells gradually blurred and disappeared. The combined effects of both more effective. 2 and OCUM-2MD3 cells, L-OHP on in vitro of OCUM-2MD3/L-OHP cells cytotoxic activity of low. L-OHP the OCUM-2MD3/L-OHP cells and cells OCUM-2MD3 vitro activity is enhanced with the increase of the concentration of L-OHP, and 48 hours after administration of the highest. 3 than OCUM-2MD3 cells, CIK cell killing activity of the cells in vitro OCUM-2MD3 / L-OHP. CIK cell killing activity of effector to target ratio increased and enhanced. Killing activity of CIK cells against the two types of target cells in 24 hours maximum. Compared with L-OHP or CIK cells and CIK cells combined with different concentrations of L-OHP two tumor cell killing activity were significantly increased. And with OCUM-2MD3 cells, CIK cells combined L-OHP the OCUM-2MD3/L-OHP has significant synergistic killing effect.}

Empirical Study of CIK Cells Combined with Oxaliplatin Against Oxaliplatin-induced Human Gastric Cancer Drug Fast Cell Line

Abstract

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