| Objective: Gastric cancer is the most common enteron malignant tumor. The mortality of gastric cancer is in the first range of all kinds of malignant tumors in our country. Chemotherapy, as one of the major methods to anticancer, has got influential effect in the prevention and cure recurrence and metastasis of malignant tumor. L-OHP, the third generation drug followed by C-DDP and carboplatin, has been used in advanced gastric cancer widely and shows great prospect. Chemotherapy is constant consummated, however, the curative effect of chemotherapy to parts of gastric cancer patients is not ideal because of the multi-drug resistance (MDR). With the development of tumor immunology, molecular biology, and gene engineering, cytokine therapy, as a method of anticancer, which has been paid attention to, presents a good prospect. Cytokine-induced killer (CIK), which is a new-type of immunological competent cell with high-efficiency and board-spectrum in the aspect of killing gastric cancer cells in vivo and in vitro, may be used in the adoptive immunotherapy of advanced gastric cancer. It has been conformed by vitro experiment that the killing activity of immune effector cell to the MDR tumor cell is similar or even stronger than the sensitive cell. Until now, few experiment that utilizing CIK combined chemotherapy to MDR tumor cells in vivo and vitro has been launched. In our study, pathological morphology and MTT will be utilized to observe the anticancer effect of CIK combined with L-OHP on OCUM-2MD3/L-OHP in vitro in order to provide relative experiment foundation for clinical treatment.Materials and Methods:1 Materials1.1 Experiment cells lines: OCUM-2MD3 cell line, given by Professor Badaizhenghe, Osaka medical university.OCUM-2MD3/L-OHP cell line, established by our division, the resistant index(RI)is 4.3, and it maintaines the similar drug resistance when passaging or revitalization after being freezed for3 months. The expression of P-gp will be higher than the sensitive cells.cells were incubated in DMEM culture fluid, at 37℃in a humidified atmosphere of 5% carbon dioxide. The cells in exponential phase of growth are needed, prepare them to cell suspension in the density of 1×10~6/mL before experiment.1.2 Experiment medicine: L-OHP, made by Jiasuhengrui drug manufactory. Dilute to different density of 1200μg/mL,600μg/mL,300μg/mL,150μg/mL,75μg/mL before experiment.1.3 Effector cells'(CIK cells)preparation and amplification extraorgan: We collected health adults'periphery blood anticoagulated by heprin. And then we separated PBMC through lymphocyte demixing fluid density gradient centrifugation, and washed cells through PBS fluid. Then cells were resuspensed and saved in culture fluid made of RPMI-1640, 10% adults'AB serum, 25mmol/LHEPES, 2mmol/L-GLN-glutamine, 100μg/mL penicilin, 100μg/mL streptomycin, 50μmol/L 2- mercaptoethanol. The density of PBMC was 1×106/mL, on the first day, we added 100μg/mL rhIFN-γand incubated 24 hours. Then we added 50ng/mL CD3McAb,100U/mL rhIL-1 and 300U/mL rhIL-2 and cells were incubated at 37℃in a humidified atmosphere of 5% carbon dioxide. Cells were incubated in fresh culture fluid changed each 2 days, and added rhIL-2 1000 U/mL. The cells were reaped and spared on the 14th day. Adjusted the E:T ratio to 40:1, 20:1 and 10:1 before experiment.2 Group2.1 L-OHP intervention group: Calculated the inhibition ratio of L-OHP under different density against OCUM-2MD3/L-OHP cell at 24 hours, 48 hours and 72 hours in vitro.2.2 CIK intervention group: Calculated the inhibition ratio of CIK against OCUM-2MD3/L-OHP under diffentent E:T ratios at 12 hours, 24 hours and 48 hours in vitro.2.3 CIK cells combined with L-OHP intervention group: First kept CIK cells agaist the target cells for 12 hours (E:T ratio is 40:1), then rejoined different density of L-OHP. Calculated the inhibition ratio of CIK cells combined with L-OHP against OCUM-2MD3/L-OHP 24 hours later.3 Methods3.1 To observe pathological morphology of OCUM-2MD3/ L-OHP in the three groups montioned above utilizing inverted phase contrast microscope.3.2 To detect the OD value of the three groups mentioned above utilizing MTT in 570nm and calculate the inhibition ratio of OCUM-2MD3/L-OHP in different groups respectively.4 Statistical methodAll statistical analyses were carried out with SPSS 12.0 and P<0.05 were considered significant.Results:1 The observation of killer-activity's pathological morphology in vitro cultureThere was no morphological different between OCUM- 2MD3/L-OHP and OCUM-2MD3 cell lines observed through the inverted phase contrast microscope.1.1 The observation of killer-activity's pathological morphology about L-OHP agaist OCUM-2MD3/L-OHP in vitro cultureUnder the inverted microscope we can see that ,after givin L-OHP for 24 hours, OCUM-2MD3/L-OHP cell population slightly grew downwards, ranked rarefaction, cell shape changed round and volume changed small, short of refraction and karyoplasmic ratio greaten, showed up supernatant intracytoplasm had more bead; 48 hours later the cell population to continue grew downwards, intracytoplasm bead increased and cell debris showed up; 72 hours later cell population still continue grew downwards and we kan see more cell debris. The killer-activity that L-OHP agaist OCUM-2MD3/L-OHP cells is higher than that agaist OCUM-2MD3 cells. The decrease of cell population and the capacity of cell debris of OCUM-2MD3/L-OHP cells was more smaller than OCUM-2MD3 cells. The control group of gastric carcinoma cells still to remain grew adherence well.1.2 The observation of killer-activity's pathological morphology about CIK cells agaist OCUM-2MD3/L-OHP in vitro cultureCIK cell was round, float growing cell. Some of the cells got together when they grew actively. Under the inverted phase contrast microscope, CIK cells had determinate directionality locomotivity. They removed to the target cells and revolred round cancer cells. In the intracytoplasm of OCUM-2MD3/L-OHP cell granulo-piece appeared, the cell gradually vague, disappear, adherence cells floated. The cell population obviously grew downwards after effect cells and target cells cultivated 12 hours. Cells floated obviously and fragment appeared in the culture fluid at 24 hours. There was a lot of fragment in the culture fluid at 48 hours. CIK cells'killer activity to OCUM-2MD3 cell was similar to OCUM-2MD3/L-OHP cells. The control group of gastric carcinoma cells was still to remain. Only the CIK cells had no tendency move, bespread distributed in the field of vision.1.3 The observation of killer-activity's pathological morphology about CIK cells combined with L-OHP against OCUM-2MD3/L-OHP in vitro cultureUnder the inverted microscope we can see that, CIK cells gradually removed to OCUM-2MD3/L-OHP cells and revolred round them. In the intracytoplasm of OCUM-2MD3/L-OHP cells granulo-piece appeared, the cell gradually vague, disappear, adherence cells floated. The cell population of OCUM-2MD3/L-OHP obviously grew downwards after effect cells and target cells cultivated 12 hours. When added in L-OHP, granulo-piece In the intracytoplasm of OCUM-2MD3/L-OHP increased and there were a lot of cell debris. killer-activity of CIK cells combined with L-OHP against OCUM-2MD3 cells is similar to OCUM-2MD3/L-OHP cells.2 The killer activity detected via MTT2.1 The killer activity of L-OHP against OCUM-2MD3/ L-OHP cell in vitroAt 24 hours , The IC50 that L-OHP against OCUM-2MD3 cells was 111.3μg/mL. The IC50 that L-OHP against OCUM-2MD3/L-OHP was 354.4μg/mL. OCUM-2MD3/L-OHP cells'tolerance to L-OHP cells were 3.2 times more than sensitive strain OCUM-2MD3 cells.At 48 hours , The IC50 that L-OHP against OCUM-2MD3 cells was 71.2μg/mL. The IC50 that L-OHP against OCUM-2MD3/L-OHP was 235.2μg/mL. OCUM-2MD3/L-OHP cells'tolerance to L-OHP cells were 3.3 times more than sensitive strain OCUM-2MD3 cells.At 72 hours , The IC50 that L-OHP against OCUM-2MD3 cells was 522.3μg/mL. The IC50 that L-OHP against OCUM-2MD3/L-OHP was 1057.0μg/mL. OCUM-2MD3/ L-OHP cells'tolerance to L-OHP cells were 2.0 times more than sensitive strain OCUM-2MD3 cells.The inhibition in vitro of L-OHP against OCUM-2MD3/ L-OHP or OCUM-2MD3 cells was the most powerful at 48 hours. The inhibition grew followed the density of L-OHP.2.2 The killer activity of CIK cells against OCUM-2MD3/L-OHP cell in vitroAt 12 hours , CIK cells'killer activity to OCUM-2MD3/ L-OHP cells was obviously higher than OCUM-2MD3 cells (P<0.05), and killer activity reinforced with the E: T ratio's growing.At 24 hours , CIK cells'killer activity to OCUM-2MD3/ L-OHP cells was obviously higher than OCUM-2MD3 cells (P<0.05), and killer activity reinforced with the E: T ratio's growing.At 48 hours , CIK cells'killer activity to OCUM-2MD3/ L-OHP cells was obviously higher than OCUM-2MD3 cells (P<0.05), and killer activity reinforced with the E: T ratio's growing.CIK cells'killer activity to the two target cells was the highest at 24 hours, and killer activity reinforced with the E: T ratio's growing. CIK cells'killer activity to OCUM-2MD3/ L-OHP was obviously higher than OCUM-2MD3 at all the given times(P<0.05), It showed that, CIK cells'killer activity to OCUM-2MD3/L-OHP was higher than OCUM-2MD3 in vitro.2.3 The killer activity of CIK cells combined with L-OHP against OCUM-2MD3/L-OHP in vitro The killer activity of CIK cells combined with L-OHP against OCUM-2MD3/L-OHP or OCUM-2MD3 were obviously higher than they worked alone(P<0.05), and the killer activity reinforced with the E: T ratio's or density of L-OHP growing. Combined with CIK cells, the IC50 of L-OHP agaist OCUM-2MD3/L-OHP cells can obviously reduced. Compared with OCUM-2MD3 cells, CIK cells combined with L-OHP had better killer effect to OCUM-2MD3/L-OHP cells.Conclusion:1 Compared to OCUM-2MD3 cells, OCUM-2MD3/L-OHP has not so much change in morphologically. In the intracytoplasm of OCUM-2MD3/L-OHP cells granulo-piece increased and cell debris appeared after L-OHP effected. In the intracytoplasm of OCUM-2MD3/L-OHP cells granulo-piece appeared and the cells gradually vagued, disappeared after CIK cells effected. The effect of CIK cells combined with L-OHP is more obviously2 Compared to OCUM-2MD3 cells, the anti-tumor activity of L-OHP against OCUM-2MD3/L-OHP was lower. The inhibition grew follow the concentration of L-OHP. And it was the highest at 48 hours.3 Compared to OCUM- 2MD3 cells, the anti-tumor activity of CIK cells against OCUM-2MD3/L-OHP was higher. CIK cells'killer activity to the two target cells was the highest at 24 hours, and killer activity reinforced with the E: T ratio's growing.4 Compared to CIK cells or L-OHP using alone, the anti-tumor activity of CIK cells combined with L-OHP against the two target cells were obviously higher. Compared to OCUM- 2MD3 cells, CIK cells combined with L-OHP against OCUM-2MD3/ L-OHP has better therapeutical effect. |
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