| Esophageal carcinoma is a common malignant tumor throughout the world,about 300,000 persons died of it every year. The disease is very popular in China,where its incidence each year is 20/100,000,nowadays, it ranges the first in the world,and deaths due to esophageal carcinoma nears to a quarter in the malignancies. After therapy with radical excision, combined with chemotherapy and radiotherapy, long-term curative effect was still not satisfactory, and five-year survival rate of post-treatment has kept lingering about 30% for recent years. The main reasons for the deaths are the metastasis and the recurrence of tumors. With the development of immunology and molecular biology studies and further exploration of tumor, esophageal carcinogenesis has a close relation with weak immune function in the patients. Accordingly, the way that stimulates antitumor responses and eradicates residue malignant cells in vivo is a key point to prevent metastasis , recurrence for curative effect. For the past few years, tumor biotherapy, more and more, is becoming a new trend, its essence lies in a full initiation in the response of competent tumor-specific T-cells against tumors.Dendritic cell (DC) is the most potent antigen presenting cell(APC) with the ability to acquire, process, present antigens and to stimulate naive T cell to proliferate and differentiate. DC plays an important role in the interaction of tumor cells and immune responses mediated by T lymphocyte. Mature DC expresses high levels of costimulatory molecules, adhesion molecules,MHC class I and II molecules,and provides secondary signals for stimulation of the naive T cell population so as to induce specific antitumor immune responses mediated by T lymphocyte. DC dysfunction in vivo is one of several immune escape mechanisms. The reduction and hypofunction or defect on DC fails to efficiently present tumor antigen and secondary signals for stimulation so that the specific antitumor immune responses mediated by T lymphocyte can not develop. Hence,it becomes a focal point of tumor immunetherapy to restore and enhance the number and function of DC in cancer patients.Cytokines are important components of immunoregulation system, which could stimulate immune cells to enhance immune function, even directly kill tumors.Thus,cytokine genes carried by appropriate vectors in host can produce cytokines persistently to induce immune responses to tumors in tumor microenvironment. Besides, cytokines can activate DC and induce specific antitumor responses. Interleukin-27(IL-27), which is newly reported by Pflanz et al in 2002,is a new cytokine produced mainly from activated DC and macrophage cells. IL-27 plays an important role in antitumour immune since it possess immunoregulation activity. As it is a newly discovered cytokine, some questions remain yet uncharacterized. We established human DC expressing IL-27 and investigated IL-27-mediated antitumor role and the related mechanisms of tumor-bearing mice in vivo and vitro, which provides the experiment and theory foundation for immunotherapy of esophageal carcinoma.Concrete contents and results are as follows:Part1 CD1a and CD83 expression and their clinical significance of tumor infiltrating DC in esophageal squamous cell carcinoma and tumor-draining lymph nodeObjective: To investigate CD1a and CD83 expression of tumor infiltrating dendritic cell (TIDC) in esophageal squamous cell carcinoma(ESCC) and tumor-draining lymph node tissues and the relationship with clinical pathological characteristics of esophageal carcinoma.Methods: Seventy-eight paraffin samples were obtained from pathologically diagnosed ESCC patients after surgery from 2002 to 2003 in the Thoracic Surgery Department of the Fourth Hospital of Hebei Medical University. Flow cytometry was applied to detect the expression of CD1a and CD83 on TIDC in 78 esophageal carcinoma tissues, 24 normal esophageal mucosa, 35 normal lymph nodes and 32 metastatic lymph nodes.Results:The expression of CD1a and CD83 in ESCC tissue were less than those in normal esophageal mucosa (all P<0.05); CD1a expression was not correlated with tumor infiltration,clinical stage and lymph node metastasis of ESCC(all P>0.05),while the expression of CD83 was correlated with clinical stage and lymph node metastasis(all P<0.05). There was no significant difference in the expression of CD1a between normal lymph node with metastatic lymph node (P>0.05), but the expression of CD83 in metastatic lymph node was less than that in normal lymph node (P<0.05).Conclusion:The expression of CD83 on TIDC reflects local immune status of ESCC and plays important roles in the development and progression of esophageal carcinoma. The low expression level of CD83 reduces the ability of antigen presentation, the organism fails to discriminate ESCC cells, and results in immune escape. The study can provide the theoretical and clinical foundation for exploring the mechanism of immune escape of esophageal carcinoma and immunobiologic therapy of esophageal carcinoma. Part 2 Experimental study on proliferation of DC from peripheral blood of patients with esophageal carcinomaObjective: To study the efficient method to induce and culture DC from peripheral blood of esophageal carcinoma patients.Methods: The peripheral blood mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation from peripheral blood monocytes of esophageal carcinoma patients and cultured to differentiate into DC. The DC were divided into three groups to culture in 37℃,5%CO2 enviroment: cultured with GM-CSF(100ng/ml)+IL-4(50ng/ml),as control group; cultured with GM-CSF(100ng/ml)+IL-4(50ng /ml)+ flt3-L(40ng/ml),as experimental one group; cultured with GM-CSF(100ng/ml)+IL-4(50ng/ml)+flt3-L(40ng/ml) +SCF(100ng/ml),as experimental two group. The number of DC was counted and the morphology of DC was observed under inverted microscope and election microscope. The expression of cell surface molecule like CD1a,CD80,CD83 and CD86 on DC were examined on the 6th day culture by flow cytometry.Results:Three groups all succeeded to induce DC from peripheral blood mononuclear cells. A larger number of DC were found in experimental group than control group. The DC number in experimental two group was the largest in three groups. On the 6th day after cell culture, the cell phenotype CD1a expression on peripheral blood DC had no significant difference in experimental group compared with that in control group(P>0.05),while CD83,CD80 and CD86 showed significantly higher expressions as compared with those in control group(P<0.01),and the DC cell phenotype expressions in experimental two group were the highest (P<0.01).Conclusion:It is an effective way to cultivate large amount of active DC in peripheral blood from esophageal carcinoma patients by using GM-CSF,IL-4,flt-3L and SCF,together,which will lay a foundation for further clinical trial.Part3 In vitro study on specific antitumor immunity induced by using retrovirus mediated interleukin-27 gene modified DC loaded with esophageal tumor lysateObjective: To investigate the efficacy and mechanisms of interleukin27 (IL-27) gene modified DC in inducing specific antitumor immunity against esophageal carcinoma in vitro.Methods: DC isolated from the peripheral blood of ESCC patients were modified by recombinant IL-27 retrovirus and pulsed with tumor lysate (TuLy) obtained from cooling and thawing of Eca109. Expression of IL-27 gene was detected with RT-PCR. The IL-12,IL-27 and IFN-γlevels in culture supernatant of DC and IFN-γlevel of CTL induced with different DCs were examined by ELISA. The expression of CD1a,CD83,CD80,CD86 molecules on DC surface were measured by flow cytometry. The T-cell proliferation response stimulated by DC was detected by MTT assay. The killing efficacy of cytotoxic T lymphocyte (CTL) activated by DC,against EC109 cells was evaluated by MTT assay in vitro.Results: Human DCIL-27 cells secreting IL-27 are successfully obtained.The cells, which have been confirmed by RT-PCR and ELISA ,can steadily express IL-27 in mRNA and protein level. After being modified by IL-27 gene and pulsed with tumor antigen,DC produced markedly higher level of IL-12(107.85±7.20)pg/ml, IL-27(430.39±10.12)pg/ml and IFN-γ(411.97±17.80) pg/ml as compared with other DC groups,and increased the expression of CD1a, CD83, CD80, CD86 molecules [(55.50±6.62)%, (82.67±7.92)%, (78.33±7.31)% and (85.33±4.32)% respectively)]. Proliferation of autologous T lymphocytes was also enhanced after stimulated by DC vaccine. DC vaccine could induce and activate CTL to produce higher level of IFN-γ(751.50±31.30)pg/ml. DCIL-27+Ag could induce the strongest cytotoxicity of antigen-specific CTL against Eca109,significantly compared with other groups.Conclusion: IL-27 gene modification enhances the capacity of DC in inducing autologous T lymphocytes to generate specific antitumoral immunity against esophageal carcinoma. The second signal of antigen presenting of DC is activated,the functions of IL- 27,IL-12 excretion and T cell activation of DC are promoted,the capacity of CTL excreting IFN-γis enhanced,which are relevant to the immune mechanisms.Part4 Effect of specific CTL activated by interleukin-27 gene modified DC loaded with esophageal tumor lysate on growth of transplantation tumor of human esophageal carcinoma in nude miceObjectives:To investigate the effect of specific CTL activated by IL- 27gene modified DC loaded with esophageal tumor lysate on the growth of transplantation tumor of human esophageal carcinoma and to study the effects of IL-27 on cell apoptosis and its mechanisms.Methods: After the nude mice models of human esophageal carcinoma transplantation tumor were established, thirty mice were randomly divided into five groups (6 per group). The mice were immunized evevy four days , totally five times with PBS(I group), Tnaive(II group), DCnaive+Tnaive(III group), DCIL-27+Tnaive(Ⅳgroup) and DCIL-27+Ag+Tnaive(Ⅴgroup), respectively. Tumor volume, tumor weight of transplantation tumors were measured. Cell apoptosis in tumor tissue was detected by TUNEL.The cell cycle and apoptosis rate of transplantation tumors at G0/G1, S and G2/M stages were assayed by FCM.The protein expressions of Fas and caspase-3 were detected by FCM.Results:The volume and weight of transplantation tumors in nude mice were(0.56±0.07)cm3 and (0.68±0.04)g inⅤgroup, which were smaller obviously than those in other therapy group and control group,the difference was statistically significant(P<0.01);The inhibition rate inⅤgroup was 58.28%, which was higher obviously than that in other therapy group and control group(P<0.01). TUNEL also showed the morphology of cell apoptosis inⅤgroup had typical changes of apoptosis than that in other therapy group and control group,the difference was statistically significant(P<0.01). FCM showed that the proliferation index of transplantation tumor tissue was(23.92±1.60)% inⅤgroup ,which is lower evidently than that in other therapy group and control group,the difference was statistically significant(P<0.01). The ratio of apoptosis cells of transplantation tumors inⅤg roup was(32.78±0.83)%,and it was higher evidently than that in other therapy group and control group,the difference was statistically significant(P<0.01).FCM showed that the expressions of fas and caspase-3 protein of transplantation tumor tissue inⅤgroup were significantly higher than those of other groups,respectively(P<0.01).Conclusions: Specific CTL activated by interleukin27 gene modified DC loaded with esophageal tumor lysate played great cytotoxic effect on esophageal carcinoma of tumor-bearing mice in vivo and could obviously inhibit the proliferation and enhance the apoptosis of esophageal carcinoma cells in vivo, which provides theoretical and experimental foundation for the application of DC in immunotherapy of esophageal carcinoma.
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