| The proliferation of dendritic cells (DCs) in vitro has been common in recent years. There are many expanding approaches, and the principles of them are also not all the same. Different research objectives have different requirements for DCs quantity and purity, so researchers could choose certain methods accordingly. But on the whole, the proliferation of DCs in vitro is complex and costly. Meanwhile, though primary progresses in anti-tumor immunotherapy have been done either in animal or in clinical experiments over the past years, many problems still exist. For example, most animal models were built by tumor cell lines inoculation, which has dramatic differences compared with autologous tumor in oncogenesis and progression; so far clinically immunotherapeutic cases are not many yet; the dosage of DCs, how to pulse DCs, transfusion routes, long term effects, are controversial; and so on. Now the studies about administration routes of DCs are few, and the changes of tumor's characteristics in immunotherapy and its relations to long term therapeutic effects are still not reported. In order to explore more economical DCs expansion methods ex vivo, the best route of DCs transfusion, the changes of tumor's characteristics in immunotherapy and its possible meaning to long term anti-tumor effects, we carry out three stages of research work. Part One Induction and Expansion of Rat Bone Marrow Derived Dendritic Cells in Vitro Objective: To explore the more convenient induction and proliferation methods of rat DCs in vitro. Methods: Four limbs were taken; bone marrow cells were flushed and washed; mononuclear cells were isolated with density gradient centrifugation from marrow and cultured in medium containing rrGM-CSF and rrIL-4. Flow cytometry (FCM) was adopted to detect the relatively specific surface markers of DCs and microscope to identify DCs morphologically. Results: Plenty of DCs with typical shape were observed by microscope. About 0.5×10~7～1.0×10~7DCs can be generated from one F344 rat and the purity was about 70% according to FCM, which could meet most of experiments. Conclusion: The DCs obtained by this method can meet the ordinary experiments either in quantity or in purity. Part Two Sensitized DCs to Treat Rat Bladder Tumor Induced by MNU Objective: To study the treatment effects of DCs loaded with tumor antigen in treating rat bladder tumor induced by MNU. Methods: Inbred line female F344 rats were totally 52. 44 rats out of them were distributed into 4 groups averagely by completely random design; they were PBS group, tumor antigen group, unpulsed DCs group, and pulsed DCs group; other 4 rats were to provide tumor and the rest 4 rats to provide DCs. Except for 4 rats for DCs, they were all irrigated with MNU into bladder to induce bladder tumor every 2 weeks, 2mg MNU/rat every time, and for a total of 5 times (0, 2nd, 4th, 6th, 8th week). Tumor tissue was resected to produce antigen byfreezing and melting tumor tissue. Bone marrow of rats was extracted and mononuclear cells were isolated with density gradient centrifugation. Then the cells were induced, cultured and then developed into DCs in vitro. The pulsed DCs were made by co-culturing with bladder tumor antigen. PBS, tumor antigen, unpulsed DCs and pulsed DCs were separately injected into corresponding group rats subcutaneously every week from 11th week on. All groups were totally treated 4 times. At last, the weight of bladder tumor, ELISA and FCM were adopted to investigate the treatment effect. Results: The difference of bladder weight between pulsed DCs groups(0.15±0.06g) and the other 3 groups was statistically significant(P<0.05); the differences among PBS group ( 0.32±0.07g ) , tumor antigen group(0.25±0.05g) and unpulsed DCs group(0.25±0.08g) had no statistical significance. The IFN-γlevel in serum of pulsed DCs group(6.23±3.32pg/ml) was statistically higher than the other 3 groups that were separately PBS group(0.70±0.12pg/ml), tumor antigen group(1.10±0.88pg/ml)and unpulsed DCs group(1.62±1.64pg/ml). The IL-2...
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