| Objective: The BALB/c nude mice inoculated with cervical cancer cell line Hela-luc were used as nude mouse xenograft model. To study the anti-tumor activity of cytokine-induced killer cells (CIK cells) against Hela-luc cells in vitro and in vivo. To analyse the characteristics of the distribution of CIK cells in cervical cancer nude mouse xenograft model. This research will provide theoretical for clinical application of CIK cells.Methods:1 The preparation and proliferation of CIK cells. The blood samples were obtained from anonymous healthy human volunteers. Peripheral blood mononuclear cells (PBMC) were isolated using standard Ficoll density gradient centrifugation. PBMC were induced to become CIK cells in vitro at the presence of interferon-gamma (IFN-γ), interleukin-2 (IL-2), CD3 monoclonal antibody (CD3McAb) and interleukin- 1α(IL-1α), in a certain sequence. CIK cells were incubated in a culture system that was renewed every 3 days with fresh medium include IL-2. To determinate proliferation response and draw curve of CIK cells growth every 7 days. After 14 days, CIK cells were collected.2 The phenotype and cytokine production of CIK cells were determined. Flow cytometry analysis was used to analyze the phenotype of CIK cells. The expression level of IFN-γmRNA in CIK cells was detected through semi-quantitative RT-PCR assay in 0, 7, 14, 21, 28, 35 days.3 Killing activity of CIK cells were determined in vitro. After 14 days, CIK cells by cytokines were collected in vitro. Logarithmic phase of Hela-luc cells were collected. CIK cells and PBMC were used as effector cells, respectively. Hela-luc cells were used as target cells. CIK cells and PBMC were added at specified effector: target (E: T) ratios (20:1, 40:1) and incubated after 48 hours, respectively. Killing activity of CIK cells and PBMC against Hela-luc cells was measured by MTT assay.4 After Wright-Giemsa's stainning, morphological images of Hela-luc cells after CIK cells treated were observed by light microscope.5 The nude mouse was inoculated Hela-luc cells to establish tumor model. Logarithmic phase of Hela-luc cells were collected into single-cell suspension. To adjust cell concentration 1×108/ml. Hela-luc cells were injected in the BALB/c nude mice's right department of shoulder (0.1ml, 1×107 once) to establish nude mouse xenograft model.6 Anti-tumor effect of CIK cells in vivo. After nude mice were injected tumor cells 7 days, the CIK cells (experiment group) or PBS (control group) was re-infused through tumor adjacent in tumor barring mice. Every group has 6 mice. Tumor changing and treatment effect were detected using in vivo Xenogen IVIS Imaging System. Peripheral blood of tumor barring mice, tumor tissue, lung, liver and spleen were collected after treatmented in two groups.7 The level of IFN-γin peripheral blood of tumor barring mice was detected by ELISA assay.8 After HE staining, the histopathological changed and tumor cells infiltrated of liver, lung and spleen were observed by microscope.9 CIK cells were labled by 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE), and labled situation observed by laser scanning confocal microscope.10 Distribution of CIK cells in nude mouse xenograft model. CIK cells labled by CFSE were injected to tumor barring mice via peritoneal cavity or tumor adjacent. Control group didn't do any treatment. Collected peripheral blood, tumor tissue, lung, liver and spleen in three groups after injected CIK cells 3 hours, 6 hours, 12 hours, 24 hours. Above-mentioned organizations were made of single-cell suspension. A part of single-cell suspension was detected CIK cells concontration by flow cytometry. Another part of single-cell suspension was observed distribution situation of CIK cells in tumor tissue by laser scanning confocal microscope. Results:1 The human peripheral blood mononuclear cells were cultured by different cytokines in vitro. Induced differentiation of CIK cells grew rapidly at 7 days and grew up to the peak at 14 days. The multiplication of CIK cells was highest reached 100 times.2 The CD3+, CD8+, CD3+CD56+ T cells'percentage were largely increased in the cells culturing process. And they expression rates may reach above 92%, 42.12%, 63.34% at 14 days, respectively. CD3+CD56+ T cells'percentage increased 57.5 times than beginning (1.10%).3 The expression level of IFN-γmRNA in CIK cells was gradually decreased in vitro cultured times. The expression level of IFN-γmRNA in CIK cells up to maximum was 14 days in cultured times. The expression level of IFN-γmRNA in CIK cells was very low in control group.4 At an effector-target cell ratio of 20: 1, 40: 1 after 48 hours, CIK cells showed higher cytotoxic effect for Hela-luc cells with the results are (51.16±2.64)% and (72.14%±4.21)% as measured by the MTT assay, respectively. The killing cytotoxic were (16.33±3.09)% and (21.26±2.71)% in PBMC, respectively.5 After Wright-Giemsa stainning, microscope observed: The normal Hela-luc cells were diamond-shaped or polygons, full shape, nuclear stainning were equal. A number of effector cells arounded the target cells to forming flower ring, and the target cell membrane changed loose, and cytoplasm had many vacuole in experiment group.6 After subcutaneous injected Hela-luc cells 7 days, the nude mice xenograft rate was 100%. Nude mice xenograft model were successful established.7 The tumor size in experiment group were smaller than that in control group after CIK cells treated (p<0.05). The inhibitory rates of the fifth week, eighth week were 47.18% and64.38%, respectiveily.8 The level of IFN-γin experiment group (61.92±6.49) pg/ml was higher than that in control group (34.30±1.78) pg/ml (p<0.05).9 The histopathological changed of tumor had no obviously difference between experiment group and control group. Alveola walls were integrity and cancer nodules didn't find in liver, but there were many inflammatory cells in two groups. Cancer nodules were found in spleen in two groups. Many cancer nodules didn't find in lung, but there were many inflammatory cells infiltrated in experiment group. Many tumor nodules were found in control group lung.10 Lung, liver, spleen, peripheral blood and tumor tissue were observed green fluorescence by laser scanning confocal microscope after injected CIK cells 3 hours via peritoneal cavity. The highest concentration of CIK cells was the lung (32.22%). Second was the liver. The concentration of CIK cells in tumor tissue was only 9.44%. After injected CIK cells 6 hours, the concentrations of CIK cells in lung, liver and spleen were gradually decreased; but the concentrations of CIK cells in peripheral blood and tumor tissue were gradually increased. After injected CIK cells 24 hours, the highest concentration of CIK cells was the tumor tissue (20.56%), and the lowest concentration of CIK cells was the lung (6.73%).11 Lung, liver, spleen, peripheral blood and tumor tissue were also observed green fluorescence by laser scanning confocal microscope after injected CIK cells 3 hours via tumor adjacent. The highest concentration of CIK cells was the lung (36.83%). Second was the tumor tissue (25.75%). After injected CIK cells 6 hours, the highest concentration of CIK cells was peripheral blood (48.02%), and the concentrations of CIK cells in other organs were gradually decreased. After injected CIK cells 24 hours, the concentration of CIK cells in peripheral blood was 23.41% and the concentration of CIK cells in tumor tissue was 13.18%; the concentration of CIK cells in liver, spleen and lung dropped to below 3%.Conclusions:1 CIK cells were highly efficient cytolytic effector cells which have a stronger significant suppression against growth of cervical cancer's Hela-luc cells in vivo and in vitro. The CIK cells showed potent anti-tumor activity in animal experiment,and its mechanisms was maybe relate with IFN-γ.2 The CIK cells were extensively distributed to organs after injected via peritoneal cavity or tumor adjacent. There is relationship between distribution situation of CIK cells and infusion ways. Infusion of CIK cells via peritoneal cavity maybe suit for malignant tumor and malignant effusions in body cavity; and application by means of tumor adjacent should suit for body surface tumor.
|
PDF Full Text Request