| Objective: To induse the proliferation and differentiation of dendritic cell(DC)derived from human cord blood CD34~+ progenitor cells, transfected it with HPV16E6/18E7 gene to prepare the DC vaccine, detect its biological characteristics,observe the anti-esophageal carcinoma immunological effect of cytotoxic Tlymphocytes (CTL) activated by HPV16E6/18E7-DC vaccine in vitro, and alsostudy the immunopreventive and immunotherapeutic effects of HPV16E6/18ET-DC vaccine on human esophageal carcinoma cells EC-109 in SCID mice, whoseimmunologic system have been reconstituted.Methods: 1. To extract the pcDNA3.1-HPV16E6/18E7 plasmid by QiagenPlasmid Mini Kid, detect its density and purity by means of spectrophotometer,transfect it into DC derived from human cord blood CD34+ progenitor cells whichhave been induced by recombined human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombined human tumor necrosis factor(rhTNF)-a for 12 days, after another 48 hour's culture, the mature HPV 16E6/18E7 -DC vaccine was harvested. The change in cell morphology was observed underphase contrast microscope, the expression of E6/E7 protein in HPV16E6/18E7-DCvaccine was analyzed by flow cytometry. In addition, DC phenotypes CDS0, CD86and CD83 pro- and post- transfection were determined as well. 2. The dot blothybridization technique was applied to analyze the HPV type of esophagealcarcinoma cell EC-109. 3. In vitro, the cytotoxic activity of CTL which derivedfrom human peripheral blood and activated by HPV18E7-DC vaccine on esopha-geal carcinoma cells EC-109 was detected by MTT assay. 4. In order to study theoncogenicity of the DC vaccines, five SCID mice were received two vaccinationsby HPV16E6/18E7-DC vaccine at 3-day intervals intraperitoneally, and all themice were observed for 70 days. 5. For studying the immunopreventive andimmunotherapeutic effects of HPV16E6/18E7-DC vaccine on EC-109 cells in vivo,SCID mice were divided into two groups: immunoprotection group and immuno-therapy group according to random numbers. Immunoprotection group is dividedinto C, Ira-T, Im-16D and Im-18D subgroups, immunized by PBS, T cells, HPV 16E6-DC+T cells and HPV 18E7-DC+T cells respectively, and then attacked byEC-109 cells. Immunotherapy group is also divided into four subgroups: C, Tr-T,Tr-16D and Tr-18D, human esophageal carcinoma bearing mice were treated withPBS, T cells, HPV16E6-DC+T cells and HPV18E7-DC+T cells respectively.Tumor latent period, tumor volume, tumor weight and tumor growth velocity wererecorded and statistically analyzed.Results: 1. About 1.0×10~6 CD34~+ progenitor cells were isolated from 50ml cordblood, trypanblau staining showed that 95％were negative; Under the stimulation of rhGM-CSF and rhTNF-a for 2 weeks, cord blood CD34~+ progenitor cells wereamplified about 10-20 folds, and the purity of DC is above 90％. During theproliferation process of DC, the cell number increased and formed cell clones, thecolony augmented day by day and achieved the largest during 8 to 9 days, matureDC with typical long dendritic processes shed from the clones in later period ofculture. DC transfected with HPV16E6/18E7 gene were grown well and had amore irregular shape and longer dendritic processes, the result of flow cytometryshowed that the expression rate of mature transfected DC's surface markers CD80,CD86 and CD83 were 81.6％,80.5％,86.6％, respectively, evidently higher thanthat of mature untransfected DC (65.5％,65.9％,80.7％, respectively), and theexpression rate of E6,E7 gene protein were 38.6％and 47.5％, which suggestedefficient gene transfer. 2. The dot blot hybridization technique showed that theHPV type of esophageal carcinoma cell EC-109 is HPV-18. 3. The result of MTTassay indicated that the cytotoxic activity of HPV18E7-DC vaccine primed Tlymphocytes were significantly higher than that of untransfected DC primed lym-phocytes, T cell group and control group(P＜0.01), and the cytotoxicity enhanced inaccordance with the increasing rate of effector-to-target cells (P＜0.05). What'smore, the cytotoxic activity of untransfected DC primed lymphocytes were alsohigher than T cell group (P＜0.05). 4. No oncogenesis in liver, spleen, lung andkidney after 70 days' observation in SCID mice, which means that the HPV16E6/18E7-DC vaccines are safety. 5. In immunoprotection groups, the tumor growthvelocity was significantly step down, and the latent period of tumor was longer inIm-16D and Im-18D subgroups than that of in Control subgroup and Im-T subgroup (P＜0.01), the tumor volume and tumor weight were less in Im-16D andIm-18D subgroups than that of in Control subgroup and Im-T subgroup (P＜0.01),there were not statistically significant between Im-16D and Im-18D (P＞0.05); Inimmunotherapy groups, the tumor volume and tumor weight in Tr-16D and Tr-18Dwere evidently less than that of in Control subgroup and Tr-T subgroup (P＜0.01),and there were not statistically significant between Tr-16D and Tr-18D (P＞0.05).Conclusions: 1. CD34~+ stem cells were isolated from human cord bloodmononuclear cells by Mini MACS, and could be stimulated into DC with highpurity and mature in morphology and function under the stimulation of rhGM-CSFand rhTNF-a for 14 days, this is a convenient, reliable and practical method toharvest DC in vitro. 2. Cationic liposome could safely and effectively transferHPVEr/E7 gene into DC. Transfected DC grew well and highly expressed Er/E7gene protein, which indicate that DC vaccine has been constructed successfully. Itis a convenient and beneficial way for preparing DC vaccine. 3. In vitro, HPV18E7-DC vaccine could induce high-performance of specific CTL cytotoxicity onesophageal carcinoma cell EC-109 which expressing HPV18. 4. The HPV16E6/18E7-DC vaccine are safe and reliable in vivo. 5. HPV16E6/18E7-DC vaccinehave notably protective effect against EC-109 cells, and can significantly inhibitthe growth of EC-109 cells in SCID mice, which indicate their outstandingimmunopreventive and immunotherapeutic effects on tumor.
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