CTL Induced From Human Umbilicus Blood Dendritic Cell Inhibit BGC823 Tumor Cells

Background and objectIn recent years, with the population aging and environmental deterioration, the incidence and mortality of malignant tumor continue to show an upward tendency. In clinical practice, surgery, radiotherapy and chemotherapy is the traditional method of treatment of cancer. However, these methods are difficult to eliminate small focuses inside the body, and moreover, cause great damage to human body. The malignant tumor was wiped out, at the expense of the normal tissues and cells of our body were damaged. With further understanding of the mechanism of tumor and developing of oncomolecularbiology and biotechnology, cancer biotherapy is developing rapidly. It has become the forth method for cancer treatment. Dendritic cells have the unparalleled ability which can present antigen, active and proliferate the naive T cell (Tn). With the unique features, DCs have become the hot research. However, DCs in the peripheral blood and tissues are lower. It is difficult to achieve therapeutic dose in vitro, even though amplification mrthod. In this paper, we try to find a new way that separating and deducing DCs from umbilicus blood which come from healthy maternity. Study anti-tumor effect and mechanism of DCs in vivo.Lay the foundation for clinical application of DCs and CTL.MethodsCollect the umbilicus blood 50-80ml from healthy term delivery maternity then separate human umbilicus blood mononuclear cell by centrifugation. Place the culture flask at rest for 2 hours then collect adherent cells from the bottom. The adherent cells was cultured in fetal calf serum culture fluid with the presence of multiple cytokines (GM-CSF,IL-4,SCF) to generate HUBDCs. Tumor antigen BCG823 was prepared from the exponential phase of growth tumor cells thorough freeze-thawing method. After HUBDCs were pulsed with tumor antigen, morphology dyed by HE was observed under optical microscope and the phenotypes was analyzed by flow cytometry. T cells were separated by nylon fiber from non-adherent cells and identify the purity. The activation and proliferation ability of active T lymphocyte was analyzed through Mixed lymphocyte reaction (MLR). Detect the killing power of active T lymphocyte by methyl thiazolyl tetrazolium (MTT) colorimetry. For in vivo experiment, animal models were built:transplant BGC823 cells to nude mice. Recode the status of tumor-bearing nude mice and the growth of tumor.Divide tumor-bearing mice into 4 groups in accordance with the random principle. Treatment of each group are as follow:A group:antigen-pulsed HUBDCs and inactive T lymphocyte, B group: antigen specific CTLs; C group:HUBDCs pulsed with tumor antigen; D group: inactive T lymphocyte which separated freshly from umbilicus blood. Tumor growth of each group was recorded respectively.ResultHUBDCs can be induced from HUBMC with the presence of cytokines (GM-CSF, IL-4, SCF). Tumor antigens were added to mature HUBDCs. The Morphology of HUBDCs dyed by HE conformed with typical morphology features of DCs:dendritic and extensively branched. The phenotypes were analyzed by flow cytometry. Compared with before induction, tumor antigen pulsed HUBDCs expressed high-level phenotypes, including:CD86(co-stimulatory molecules),CD80, CD54(adhesion molecule),MHC-ⅠandⅡ.The positive percentage of CD86 was 40.59%±3.27%, CD54 was 59.21%±6.32% CD11c was 67.01%±5.17% MHC-Ⅰand MHC-Ⅱwere:42.37%±10.11% and 56.31%±6.76%. The difference was statistically significant compared with before induction. The result of Mixed lymphocytes reaction (MLR) shows that:HUBDCs can stimulate and proliferate antigen-specific T lymphocyte into cytotoxic T lymphocyte (CTL).In vitro; CTL can inhibit and kill the BGC823 target cells. In zoopery part, Both A group which injected with tumor antigen pulsed HUBDCs and allogeneic inactive T lymphocyte and B group which injected with cytotoxic T lymphocyte have obvious antitumor effect. At 30 days after, A group of tumor size was 211mm3, B group was:153 mm3, B Group is more obvious effect. C group which inject with tumor antigen pulsed HUBDCs and D group inject with inactive T lymphocyte have no effect. To 30 days, C and D group of tumor size was 1093 mm3 and 1022 mm3.ConclusionHUBDCs that have morphology and phenotypes feature can be induced successfully from HUBMCs, and they have the capacities of stimulating native T cell activation, proliferation and differentiation into antigen specific CTLs.Transplantation of antigen pulsed HUBDCs and allogeneic T lymphocyte can enhance the anti-tumor ability in tumors bearing nude mice.