| OBJECTIVES:1. Methyl theiazoyl tetrazolin (MTT) colorimetric assay was used to detect the inhibitory effect of various extracted fractions prepaired from Qi-Boosting Toxin-Resolving Formula (QBTRF) on nasopharyngeal carcinoma cell line CNE2in order to screen the most effective fraction against nasopharyngeal carcinoma cells.2. To investigate the intervening effect (tumor-inhibiting rate) of the effective fraction ethyl acetate extract (EAE) of QBTRF, which was screened out based on the first experiment as mentioned above, on the implanted tumor of NPC cell line CEN2among nude mice and its intervening effect on secretion activities of various kinds of tumor-associated-cytokines in the microenviroment of NPC tumor.3. To investigate the regulating effects of EAE made from QBTRF on the differentiation and functions of DCs, which were induced from the differentiated peripheral mononuclear blood cells of NPC patients by rhGM-CSF and rhIL-4, and their activating effect on the function of CTL cells to explore the impact of the EAE on DC in the tumor microenviroment and their antigen presnting action.METHODSPART ONE:Screening of effective fractions extracted from Qi-Boosting Toxin-Resolving FormulaBy using different solvents, three effectivd fractions were extracted from QBTRF. And then, MTT method was used to detect the inhibiary activity of the extracted effective fractions on NPC cell line CHE2in order to screen out the most effective fraction among these extracts.PART TWO:The intervention effects of effective fraction EAE from QBTRF on the implanted tumors in nude mice with NPC cells of cell line CEN2.1. NPC cells of cell line CNE2were cultured by conventional procedures.2. BALB/c nude mice were used to prepare implanted tumor models with CNE2cells.3. Implanted tumor-bearing nude mice were randomly divided into four groups, i.e. modeling group (MG), extract EAE from QBTRF treating group (EAEG), cisplatin treating group (DDPG) and the combined therapy treating group (EAE+DDPG). Then, intervention was lasted for12days for each group by coresponding treating procedure, with their general behavior changes observed and recorded carefully during this course. By the end of intervening period, all mice were sacrificed to take tissue samples for following experiments.4. A comparison on the tumor-inhibiting rate was made among all groups of nude mice to see their differences.5. IL-12and MHC-II protein expressive activities were determined by using SABC immunohistochemical assay among all groups of implanted tumor tissue samples.6. ELISA was used to detect the contents of transplanted tumor tissue IL-4, IL-12and IFN-y in the supernants of tumor tissue homogenate, followed a comparative analysis on these data to see their activiting differences.7. RT-PCR method was taken to detect IL-4,IL-12and IFN-y gene expression levels in implanted tumor tissue samples. Also, a comparative analysis was followed on these data to see their activiting differences.PART THREE:The regulating Effects of effective fraction EAE on the functions of induced DC in vitro from peripheral mononuclear blood cells of patients with NPC1. Sterile separated peripheral blood mononuclear cells (PBMC) was obtained from patients with NPC and cultured regularily at first. Then, recombinant human granulocyte colony stimulating factor (rhGM-CSF) and recombinant human interleukin-4(rhIL-4) were added into the culturing system to induce DC differentiation in vitro from these white blood cells. At the same time, various levels of EAE (at the concentrations of100μg/ml and200μg/ml respectively) were also added into the culture medium, with normal saline (NS) as blank controlling group, to observe changes in morphological characteristics of DCs under inverted microscope following these treatments.2. At7th day of culturing, DCs were collected from the culturing system and their density was adjusted for flow cytometry (FCM) to detect the expressive state of surface markers CD1a, CD86and HLA-DR on DCs.3. Supernant samples were collected periodically from this culturing system to detect the expressive activity changes of cytokine IL-12by ELISA dynamically.4. Cytotoxicity effect of CTL on CNE2cells, induced and activated by cultured DCs following the stimulation of effective fraction EAE, was determined by MTT colorimetric assay.RESULTS:PART ONE:Screening of effective fractions from QBTRFBy solvent extraction procedures, three extracting fractions were prepared from QBTRF, i.e. polysaccharide extract, ethyl acetate extract and ethanol extract. Then, it was determined by the use of MTT colorimetric procedure that the significant inhibiting activity on nasopharyngeal carcinoma cells of CNE2cell line was shown only in the culturing system with the fraction ethyl acetate extract added even at the level of100μg/ml and with a dosage associated relationship, but polysaccharide and ethanol extracts shown no such significant effect on these tumor cells. PART TWO:The inhibiting effects of fraction EAE on implanted tumors of NPC cells among nude mice1. General statous comparisonThe animals in EAE treating group showed no obvious drug toxicity, with a good mental state, sensitive in response to stimuli, higher survival rate and good life quality. Then, followed was the better general condition among the mice treated by combined treating group with EAE plus DDP. Not so better were the mice in modeling group. Animals in DDP treating group showed significant drug toxicity, with spandrel shrink back to stimulate the response, slow movement, body tired perfoumance, quality of life is the worse.2. The use of EAE, DDP and combination therapy all showed obvious inhibition rate on tumor, but EAE treatment combined with DDP was shown with a better synergistic anti-tumor effect on implanted tumors.3. Immunohistochemistry results showed that, when compared with that as seen in model group, the protein levels of IL-12and MHC-Ⅱ were obviously elevated in EAE, DDP and combined treating groups respectively, with statistical significance (P<0.01). When compared with the group of EAE, IL-12and MHC-Ⅱ protein expressive levels were lower in the group of DDP, while their levels were the highest in the combined therapy group, with statistical significance (P<0.01).4. IL-4, IL-12and IFN-γ level determination in implanted tumor tissue samples showed that IL-12and IFN-γ levels were increased in EAE group, DDP group and combined treatment group in the supernant, while IL-4content level was decreased, when compared with that of model group, with statistical significance (P<0.01). When compared with EAE group, IL-12and IFN-y levels were lower in DDP group in the supernant, IL-4content was higher, while IL-12and IFN-y were elevated in the combined treatment group in the supernatant and IL-4content was decreased, with statistical significance (P<0.01).5. IL-4, IL-12and IFN-y gene espression levels in the implanted tumor tissueWhen compared with model group, gene expression levels of IL-12and IFN-y were increased in EAE group and combined treatment group, while IL-4was reduced, with statistical significance (P<0.01). Gene expression levels of IL-12and IFN-y in EAE group were increased while IL-4reduced, when compared with that of DDP group. When compared between groups of EAE and combined therapy, the latter showed increased IL-12and IFN-y gene expression level and decreased expression level of IL-4more significantly, with statistical significance (P<0.01).PART THREE:Intervention effects of EAE on the function of induced DCs from peripheral mononuclear blood cells of patients with NPC in vitro1. EAE could promote the differentiation and maturation in vitro of DCs induced from PMBC of NPC patients. It was shown that majority of adherent cells, many cell aggregation and uniforuly dispersed cell colony on the first day. Some cells were seen irregularly with a few short protrusions and some cells started to present semi-suspension and suspended state. Many cells in the high dose group had protrusions extend interwoven, while there was few protrusions in the low dose group and NS controlling group on the fourth day. Cells in the high dose group changed into circular and many of DC from the wall into the suspension, while in low cell density and suspended cells less in the NS group on the seventh day. 2. EAE could increase the expression of CD la, CD86and HLA-DR. It was also shown that the group of high dosage had significant effects, when compared with that of controlling group (P<0.01).3. DCs secrected IL-12in a time-dependen manner, the levels of IL-12in the low and high doseges of EAE were significantly higher than that in NS group (P<0.01). The level of IL-12in the high dose of group was higher than that in the low douse group, with statistical significance (P<0.01).4. EAE could promote differentiation and maturation of DCs in vitro induced from PMBC of NPC patients. It could also enhance the cytotoxic activity of CTL induced by DCs on CNE2cells, with a dosage dependent way. There was statistical significance when compared with that of NS control group (P<0.01).CONCLUSIONS:1. It is determined that the fraction of acetic acid ethylester extract is the anti nasopharyngeal carcinoma effective fraction of QBTRF.2. EAE holds a strong inhibititory effect on implanted tumor of NPC cells in nude mice. When combined with cisplatin, this kind of effect can be further enhanced to show an higher inhibition rate and improved quality of life following treatment on nude mice.3. EAE in a way of combination with cisplatin can effectively increase the protein expression level of MHC-Ⅱ, improve the immune activities of IL-12and IFN-y in the expressive activities at both levels of gene and protein expression, lower IL-4protein content and gene expression level, prompting Th1/Th2drift into the direction of Th1in the tumor microenvironment, thereby enhancing the host's anti-tumor effect.4. EAE can effectively stimulate the differentiation and maturation of DCs in PMBC in patients with NPC, increase theis expression of CDla, CD86and HLA-DR on cellular surface of DCs, improve DC's secretion of IL-12and increase the cytotoxic activity of CTL on NPC CNE2cells.5. EAE can improve cellular immunity within host's body bearing a tumor and restrain tumor growth by regulating DC's function, especially in presenting tumor antigen to immunocompetent cells.
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