| Part Ⅰ:Isolation and culturing dendritic cells of F344 rat Objective: To establish a convenient and high-performance method of isolating and culturing dendritic cells (DCs) from the bone marrow of F344 rats. Methods: 16 inbred F344 rats were anaesthed by butaylone, sacrificed and sterilized by 75% alcoholic solution. Dendritic cells obtained from the bone marrow of F344 rats'limbs were cultured in the RPMI 1640 in vitro under the condition of recombinant rat granulocyte-macrophage colony-stimulating factor (rrGM-CSF) and recombinant rat interleukin-4 (rrIL-4). Dendritic cells were purified by density gradient separation and were identified by invesion microscope, electron microscope and flow cytometry. Results: On the 4th cultured day, DCs displayed typical morphology with elongated dendritic processes viewed by inversion microscope as well as electron microscope. DCs expressed surface antigens, including OX62 (8.92%), MHC-II (20.9%), CD86 (16.98%) on the 4th cultured day; OX62 (58.07%), MHC-II (60.49%), CD86 (62.94%) on the 8th day; OX62 (84.68%), MHC-II (88.03%), CD86 (62.80%) on the 15th day. The quantity of getatable DCs was 1.0×107~2.0×107 from a rat. Conclusion: 1.The purity of DCs is above 80% by density gradient separation and culturing. 2.The quantity of getatable DCs was 1.0×10~7~2.0×10~7 from a rat by this method, as well as monoclonal antibody exclusion and monoclonal antibody magnetic beads. 3.This method is a convenient and high-performance method of isolating and culturing dendritic cells. Part Ⅱ: Empirical study sensitized dendritic cells in the treatment of bladder tumor Objective: To study the effects of dendritic cells sensitized by freeze thawing supernatant of bladder tumor cells in the treatment of bladder tumor on F344 rats and further discuss the mechanism of this immunotherapy. Methods: 44 female F344 rats, which irrigated 0.1 mL 20 mg/mL N-methyl-N-nitrosourea (MNU) into bladders every other week (0, 2nd, 4th, 6th and 8th week) for a total of five doses, were induced to bladder tumor. They were treated subcutaneously with either 0.1 mL PBS, (3.0×105) unsensitized DCs, 0.1 mL freeze thawing supernatant of tumor cells, or (3.0×105) sensitized DCs with freeze thawing supernatant of tumor cells respectively every week (11th, 12th, 13th and 14th week) for a total of four times. In the fifteenth week, their bladders were weight and harvested for observation by naked eye and microscope, their blood was harvested for examination CTL by flow cytometry (FCM) and their blood-serum was harvested for examination IFN-γand IL-2 by ELISA. Results: The weight of bladders in sensitized DC group was (0.15±0.06 g) lower than those in PBS group (0.32±0.07 g), unsensitized DC group(0.25±0.08 g) and freeze thawing supernatant of bladder tumor cells group(0.25±0.05g) (P<0.05=. The stages and grades of bladder tumor in sensitized DCs group were statistically descended compared with those in PBS group (P<0.05=. The interferon-γ(IFN-γ) in blood serum of sensitized DCs group (6.23±3.23 pg/mL) was higher than those in PBS group (0.70±0.12 pg/mL), unsensitized DC group(1.62±1.64 pg/mL) and freeze thawing supernatant of bladder tumor cells group(1.10±0.88 pg/mL) (P<0.001=, while the interleukin-2 (IL-2) in blood serum of 4 groups has not obviously changes. The CD3+ T cells in sensitized DC group (56.28±3.55%) was lower than those in PBS group (63.44±6.00%), unsensitized DC group (62.45±7.05%) and freeze thawing supernatant ofbladder tumor cells group (67.00±4.36%) (P<0.05). The CD3+CD8+CD28+ T cells in sensitized DC group (8.97±2.17%) was higher than those in PBS group (1.02±0.71%), unsensitized DC group (2.00±1.05%) and freeze thawing supernatant of bladder tumor cells group(5.92±1.90%) (P<0.001). Conclusion : 1.Sensitized DCs injecting subcutaneously can reduce the stages and grades of F344 rats'bladder tumor. 2.Unsensitized DCs injecting subcutaneously have not effect in the treatment of bladder tumor; while the effect of freeze thawing supernatant of tumor cells injecting sub... |
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