| Over recent years, the effects of dentritic cells (DCs) in tumor immunotherapy is supported by increasing animal experiments as well as initial human trails with the development of in vitro DCs inducing and amplifying technique, and DC has great potential to become a powerful weapon in tumor immunotherapy. The efficacy of routine chemotherapy and radiotherapy in pediatric malignant tumor is far from very good. For example, the pediatric malignant neoplasms, lymphoma, neuroblastoma and nasopharyngeal carcinoma (NPC), which is difficult to diagnose early in clinical practice and usually has been in middle or final phase when the diagnosis is established, shows a limited curative effect of routine therapyincluding chemotherapy and radiotherapy, which commonly accompanied with notable poisonous and ill effects. They can even make a great impact on children's growth and development. And the problem on metastasis and relapse of the neoplasms has not been solved yet. Therefore, clinic practice needs badly to establish an efficient and reliable approach to against the tumor.OBJECTIVE: To improve security and technique of the methods used for DC immunotherapy before clinical practice, including DC in vitro inducing and amplifying methods, DC maturity inducing methods, and in vitro DC loading tumor antigens methods, etc. To research into the antigen presenting and anti-tumor immunologic function of loading-tumor-antigen-information DC (tumor-DC), so as to discover its clinical reliability and effectiveness.METHODS: 1. A pediatric malignant case was chosen, and the patient guardian's consent in written form should be obtained before the experiment. 2. The tumor tissue was prepared by biopsy, and the primary culture of tumor tissue was performed. Then to passage to establish autologous tumor cell line. The cell line was biologically identified by irnmunohistochemistry morphologic observation. 3. Tumor whole cell antigen was generated from collecting logarithm-growth-phase tumor cells, repeatedly freeze-melt for 3 times and exposing in 25 Gy 60Co r ray irradiation. 4. Monocyte-derived dendritic cells(MoDCs) were generated from patients' peripheral blood mononuclear cell(PBMC). In brief, PBMC were separated by density gradient centrifugation, then were seeded in culture bottle for adhering to the wall for 2hours. Non-adherent cell were removed. DCs then were induced and amplified from the adhering cells by applying interlukin-4 and GM-CSF for 9 days. In order to get mature DCs, the tumor whole cell antigen was added to the cell cultures on day 3 and TNF-a on day 7. Then DCs loaded with tumor whole cell antigen information (tumor-DCs) were harvested on day 9. Human homotypic serum was used as culture medium additive in cell culture procedures of this study. 5. Morphologic data of the DCs loaded with tumor whole cell antigen information were obtained through light and scanning or transmission electron microscope. 6. These DCs surface expression information of CDla, CD80, CD86 and HLA-DR were gained by indirect immumofluorescence stain and flow cytometry. 7. Peripheral T cells were separated and used for mixed culture with tumor-DC for 3 days. And then the T cells stimulating index were obtained by MTT methods, which represent the capacity of DCs stimulating T cells to proliferating. 8. Patient peripheral T cells were co-cultured with tumor-DCs (in experimental group) or merely tumor whole cell antigen (in contrast group) for 3 days to induce DC-CTL or Ag-CTL. These two kinds of CTLs were co-cultured with autologous tumor cells, the ratio between effector and target cells was 10, 24 hours later showed color by MTT methods to measure the optical density, and calculated cytotoxicity rate.RESULTS: 1. We chose a 12-year old boy, who suffered from NPC diagnosed by biopsy pathology and classified as clinical phase IV, with themetastasis in both-sides lymph node of his neck and the invasion of his right eye. His guardian agreed to attend the experiment in written form. 2. The NPC biopsy tissue primary culture and passage was...
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