In Vitro Experiment Research Of The Anti-glioma Activity Of The Tumor Vaccine Made From The Dendritic Cell

Objective:Active immunotherapy of the glioma has become the key point in the investigation of the glioma. But the first step of the immunity reaction is the capture of the antigen by antigen-presenting cells(APC). After the antigen was disposed and processed by the APC, the messanger is translated to the lymphocyte and then the especially immunity reaction is induced. Dendritic cell (DC) has the strongest function of the APC in body. We discuss the anti-glioma activity of the tumor vaccine made from the dendritic cell. Methods:Kill female SD rats by pulling off the necks and whose weight are about 100-150g.Bilateral femurs and tibias were gotten and both ends of the bones were cut with sterile scissors. The marrow was flushed out using a 20-gauge needle and syringe with culture medium. The tissue was then suspended in culture medium and pass through nylon mesh to remove any bone or debris. The suspension contain the marrow tissue was centrifuged and then the upper liquid was abandoned. After the erythrocytes were removed, the morrow tissue was washed by PBS and IMDM culture medium without serum . These borrow tissue suspension were then supplement with 20% fetal bovine serum and GM-CSF and IL-4.The mature DC was harvested after 8 days and then was observed through scanning microscope and analyzed by flow cytometric. C6 tumor cell of logarithm growth period were gotten through digestion and calculated. Prepare C6 tumor antigen by freezing-melt method. Take C6 tumor cells at logarithm growth period, digest and beat upon them to equality and calculate the number of the cells. Then take them into freezing deposit tube and freeze it at -70℃ in low temperature refrigeratory.Next day,make it thaw at room temperature and put it into low temperature refrigeratory again.Repeat the action 4 times and centrifugal for 30 minutes. Extract superior liquid and store it at -70℃.And then added it into the culture plant that contained DC and cultured together for 48 hours. Divided 20 female rats into 4 groups, each group had 5 rats: ⑴ tumor vaccine group 1 :the DCs that were pulsed with C6 tumor vaccine were injected through tail veins once a week for 3 weeks. Toke experiment after 1 week following the 3rd injection.⑵ tumor vaccine group 2 :the DCs that were pulsed with C6 tumor vaccine were injected through tail veins. Toke experiment after 1 week following the injection.⑶DC group: the DCs were injected through tail veins once a week for 3 weeks. Toke experiment after 1 week following the 3rd injection.⑷control group: Took experiment after the rats were injected NS through tail veins once a week for 3 weeks. Extract spleen lymphocytes of experiment animals and examine lymphocytes kill activity through MTT, then calculate kill efficiency. Results:Observation through inversion microscope, we could see marrow monocyte and a few infantility red cells on the first day; the share of the red cells was vanished on the 3rd day and the ratio of the marrow monocyte increased obviously. The cells have stick to the wall.The dendritic-like cells appeared in the cultured rat marrow cells in the 5th day and there were lots of DCs which had irregular nuclear and long cell tuber in the 7th or 8th day.Through scanning electronic microscope we could observe that the mature DCs was rotundity or ellipse, and there were lots of dendritic tubers at the cells surface and there were also fine embranchment at the tubers' end. Examining through flow cytometric showed that the expression of OX62 was 90.39% and the expression of OX6 was 85.31%. Calculated the OD data of different experimental groups and different effect-target ratio groups and then disposed the results through statistic method by SPSS. There were statistic difference between the groups of different effect-target ratio in C6 tumor vaccine group 1 and only the difference of the groups of 10:1 and 2.5:1 were obviously in other groups. Comparing between the groups of differ experimental groups and same effect-target ratio showed that there were obviously difference betwe...