Experimental And Clinical Study On The Biologic Characteristic Of Dendritic Cells And Anti-Tumor Immune Efficacy

Objective: To induce and cultivate DC in vitro and investigate the biologiccharacteristic of DC and anti-tumor immune efficacy. Methods: The mononuclear cells isolated from human peripheral blood werecultured with the supplement of rhGM-CSF and rhIL-4 to generate DC. Themorphological characteristics of those cells were observed and the phenotypefeatures and the nucleio-type were analysed. The first experiment in vitro: DC andMacrophage (Mφ)acted as stimulating cells to activate mixed lymphocyte reaction(MLR) respectively, then observed their ability of activating MLR. The secondexperiment in vitro: DC acted as effector cells had co-cultured with BEL-7402 andReh acted as target cells on three kinds of ratio (20∶1; 10∶1; 5∶1) respectivelyfor 18 hours, to detect the DC killing activity to BEL-7402 cells and Reh cells. Thethird experiment in vitro: DCs were pulsed with BEL-7402 tumor cells antigen.The killing activity on BEL-7402 hepatocarcinoma cells of CD3AK cellscombined with the tumor antigen-pulsed DC was measured in vitro using thelactate dehydrogenase (LDH) release assay. The first animal experiment: DC wereisolated and cultured from C57BL／6 mouse's marrow and were stimulated by 7impacting B16 tumor antigen. 30 C57BL／6 mouse were divided into three groupsrandomly. DC stimulated by B16 tumor antigen, DC not stimulated by tumorantigen and HBSS acted as comparison were immunized three-group mouserespectively, then compared with the killing activity of CTL that induced formthree groups mouse's spleen T lymphocytes in vitro which obtained from twomouse's spleen of each group. The second animal experiment: Three-group othermouse were inoculated B16 cells respectively and observed the growth of tumor.The third animal experiment: DC were isolated and cultured from C57BL／6mouse marrow and were stimulated by impacting 3LL tumor antigen. 24 C57BL／6 mouse which were inoculated by 3LL cells and all had come out pulmonarymetastasis were divided to three groups randomly. Three-group mouse were treatedwith DC stimulated by 3LL tumor antigen, DC not stimulated by tumor antigenand HBSS treated respectively, then weighed and counted their lung metastasistumors. Results: We could observe a lot of suspension cells with the typical DCmorphologic characteristics, which observed cell surface being showed rough andwrinkle by inverted microscope and observed cell membrane stretching outdifferent-length apophysis, irregularity cellular nucleus shape, deep karyotin, morechondrosome and less lysosome in intracytoplasm by electron microscope. DCpresented moderately mark such as CD1 and presented highly mark such as CD86,CD40, HLA-DR by flow cytometry. The first experiments in vitro: DC was moreeffective on activating mixed lymphocyte reaction (MLR) than Mφ(P<0.01). The 8second experiments in vitro: DC was not able to kill BEL7402 cells and Reh cellsdirectly on three kind of ratios (20∶1; 10∶1; 5∶1). The third experiments invitro: CD3AK cells combined with DC were more effective on killing BEL-7402cells than CD3AK cells without DC(P<0.01) on two kind of ratios((20∶1; 10∶l).The fist animal experiment: the killing activity of CTL induced by DC whichstimulated by B16 tumor antigen was more effective on killing B16 cells than thatof DC which wasn't stimulated by B16 tumor antigen(P<0.01) and the CTLdisposed by HBSS could not kill B16 cells. The second animal experiments: Thegroup of mice immunized by DC pulsing with B-16 tumor antigen hadn't tumorgrowth after injecting B16 cells on hypodermis for 9 days. The group of miceimmunized by DC not pulsing with B-16 tumor antigen could resist the B-16tumor growth but less effective than the group immunized DC pulsing with B-16tumor antigen(P<0.01). The group treated with HBSS all had grown tumor. Thethird animal experiments: Compared the weight and the quantities of lungmetast...