| Esophageal carcinoma is one of the most common malignant diseases in our country. At present, operation, chemothreapy and radiotherapy are the main meathods to treat tumors with some limitations. Most of esophageal carcinoma patients are in middle or late stage, the 5 year survival rate is about 10 %. So it is urgent to investigate new therapic method. In recent years, more and more people have pay attention to tumor biotherapy, which is gradually becoming the fourth treatment of tumors, especially tumor immunotherapy can not only enhance the immune function of host, kill tumor cells in specific way, but also has less side-effects on normal tissues.The aim of tumor immunotherapy is to enhance the immunogenicity of tumor cells, elevate the recognition and clearance ability of immune system. Cytokines are main component of immunoregulation system, which could stimulate immune cells to enhance immune function, even directly kill tumors. But there are some effects by administration of cytokines directly in treatment of tumors, including the short half-life, large and lasting usage and strong side-effects, which limit their clinical applications. Cytokine genes carried by vectors in host can produce cytokines to induce immune responses to tumors in tumor microenviroment. Besides, tumor cells and dendritic cells loaded tumor antigens as cytokine gene therapeutic vectors can provide the antigens to induce specific antitumor responses.Interleukin-27 (IL-27), which is newly reported by Pflanz et al in 2002, is produced primarily from activited antigen presenting cells (APCs) like dentritic cells and macrophage cells. IL-27 can induce the proliferation of naive T cells and facilitates the differentiation into T helper type 1 (Th1), synergizes with IL-12 to potentiate interferon-γ(IFN-γ) in the early regulation of Th1 responses, suggesting that IL-27 is involved in the development and control of autoimmune diseases, chronic inflammatory response and some infections. Several reports have shown that expression of IL-27 in murine tumor cells can induce antitumor response by enhancing CTL activity and production of IFN-γ. So, IL-27 can be a candidate of cytokine gene therapy, but some questions remain yet uncharacterized. In this regard, we established human esophageal carcinoma cells expressing IL-27 and investigated IL- 27-mediated antitumor and the related mechanisms to provid basis for clinical application.Part 1 Establishment human esophageal carcinoma Eca109/IL-27 cells expressing IL-27 geneObjective: To obtain human esophageal carcinoma Eca109/IL-27 cells steadily expressing IL-27.Methods: Retrovirus was generated by transfecting retroviral vector (LXSN/IL-27) intoψ2 and then PA317 packaging cells. Retroviruses were used to transduce IL-27 gene into human esophageal carcinoma Eca109 cells and stable clones expressing IL-27 gene (Eca109/IL-27) were obtained by screening with G418. Expression of IL-27 gene was detected with RT-PCR. The level of IL-27 secreted by Eca109/IL-27 cells was determined by ELISA and the expression of IL-27 protein was measured by laser scanning confocal microscope (LSCM). Growth of Eca109/IL-27 cells in vitro was assessed by MTT. Flow cytometry was used to analyze the expression of HLA-A,B,C (MHC I molecule), HLA-DR (MHC II molecule), CD80 and CD86 on the surface of cells.Results: 1 Retrovirus produced by PA317/IL-27 cells was about 4~6×105CFU/ml. IL-27 in the culture supernatants was 157.24±5.9 pg/ml2 Eca109/IL-27 cells which express IL-27p28, IL-27EBI3 mRNA and produce IL-27 (153.73±7.00pg/ml) were obtained and IL-27 gene had been intergrated into the genome of Eca109/IL-27 cells. LSCM results proved expression of IL-27 protein in Eca109/IL-27 cells.3 The morphology and growth of Eca109/IL-27 cells were similar to that of Eca109 cells and Eca109/LXSN cells in vitro (P>0.05). 4 Expression level of HLA-A,B,C, HLA-DR, CD80 and CD86 on Eca109/IL-27 cells was similar to that of Eca109 cells and Eca109/LXSN cells (P>0.05).Conclusion: Human esophageal carcinoma Eca109/IL-27 cells secreting IL-27 was obtained. Transfection of IL-27 gene did not affect cell growth and biological character in vitro.Part 2 Effects on immune cells function of IL-27 secreted from Eca109/IL-27 cells in vitroObjective: To study the effects of IL-27 secreted from Eca109/IL-27 cells on human peripheral blood mononuclear cells (hPBMCs) and spleen cells of nude mice.Methods: The supernaturants of Eca109/IL-27, Eca109/LXSN and Eca109 cells was cultured with hPBMCs and spleen cells of nude mice. ELISA, flow cytometry and LDH releaese assay were used to determine cytokine production (IL-12p70, IFN-γ, TNF-α?and IL-4), percentage of CD4+ and CD8+ T cells, cell surface molecule expression (HLA-A,B,C, HLA-DR, CD80 and CD86) and CTL activity in hPBMCs respectively. IFN-γproduction and cytotoxicity activity in spleen cells of nude mice were measured by ELISA and LDH releaese assay.Results: 1 Effects of IL-27 on hPBMCsThe supernaturants of Eca109/IL-27 cells could induce the production of IL-12p70, IFN-γand TNF-αhigher than that of Eca109/LXSN cells and Eca109 cells (P<0.01), however there was no significant difference in three groups about IL-4 production (P>0.05); Compared to that of Eca109/LXSN and Eca109 groups, with the supernaturants of Eca109/IL-27 cells, the percentage of CD4+ T and CD8+ T lymphocyte was upregulated, CTL activity was enhanced and cell surface molecule expression (HLA-A,B,C, HLA-DR, CD80 and CD86) was upregulated, too (P<0.01).2 Effects of IL-27 on spleen cells of nude miceThe supernaturants of Eca109/IL-27 cells could induce spleen cells of nude mice produce IFN-γhigher than that of Eca109/LXSN and Eca109 groups (48.7±4.3 pg/ml, 6.3±1.5pg/ml and 4.9±1.4 pg/ml, respectively) and enhance the cytotoxicity activity to tumor cells (P<0.01).Conclusion: IL-27 could upregulate the immunomodulation function of immune cells and enhance cytotoxicity activity to tumor cells.Part 3 Antitumor activity of IL-27 in nude mice and its mechanisms1 Antitumor activity of IL-27 in nude mice and its immunomodulation activityObjective: To study antitumor activity of IL-27 secreted from Eca109/ IL-27 cells and its immunomodulation activity.Methods: Eca109/IL-27, Eca109/LXSN and Eca109 cells were subcuta- neously or intraperitoneally inoculated into nude mice respectively and the tumor volumn and survival time were observed. Expression of IL-27 gene in tumor tissues was detected by RT-PCR; Expression of NK cells, IFN-γproduction, cytotoxicity activity and cytokines production were detected by immunohistochemistry method, ELISA, LDH releaese assay and RT-PCR respectively.Results: 1 Antitumor activity of IL-27 in vivo: The growth of tumor in mice inoculted with Eca109/IL-27 cells was significantly retarded (P<0.05) and the survival time was longer compared to that of two control groups (P<0.01).2 RT-PCR proved the expression of IL-27p28 and IL-27EBI3 mRNA in the tumor of Eca109/IL-27 inoculted mice.3 Infiltration of NK cells in the tumor of Eca109/IL-27 inoculted mice was higher than that of two control groups (P<0.01).4 Production of IFN-γi?n spleen cells of Eca109/IL-27 inoculted mice was higher than that of control groups (P<0.01), independence of re- stimulation with irradiated wide type cells (P>0.05).5 Splenic cytotoxicity activity to tumor cells in Eca109/IL-27 inoculted mice was significantly higher than that of control groups (P<0.01).6 Gene expression of T-bet, IL-12Rβ2, TNF-α, IL-1βand IL-6 mRNA in Eca109/IL-27 inoculated mice was significantly higher than that of two control groups (P<0.01).Conclusion: IL-27 secreted from Eca109/IL-27 cells enhanced immune function and induced antitumor activity.2 Effects of IL-27 on tumor cell apoptosisObjective: To study the effects of IL-27 on cell apoptosis and its mechanisms.Methods: Cell apoptosis in vitro and in vivo were analyzed by flow cytometry. Cell apoptosis in tumor tissue was detected by TUNEL and the ultramicrostructure of apoptosis was observed by electron microscope; RT-PCR was used to investigate gene expression of Fas and Survivin in tumor tissues, protein expression of Fas and Survivin was detected by Western-blot.Results: 1 There was no difference of cell apoptosis in Eca109/IL-27, Eca109/LXSN and Eca109 cells in vitro (P>0.05). But, after the inoculation of nude mice, cell apoptosis in Eca109/IL-27 cells inoculated mice was higher than that of Eca109/LXSN and Eca109 groups (39.5±2.4%, 12.5±0.3% and 13.1±0.8%, respectively)(P<0.01).2 TUNEL and electron microscope also showed the morphology of cell apoptosis in Eca109/IL-27 cells inoculted mice, which had typical changes of apoptosis.3 Compared to Eca109/LXSN and Eca109 cells inoculted mice, the expression of Fas was increased (P<0.01) and Survivin is decreased (P<0.01) in the tumor tissue of mice inculated Eca109/IL-27 cells detected in gene and protein level, the Survivin: Fas rate was downregulated (P<0.01). Conclusion: There were no direct effects of IL-27 on cell apoptosis of tumor cells in vitro. But in vivo, IL-27 could increase cell apoptosis.Increasing of apoptosis was through Fas/FasL pathway related to reduction of expression of Suvivin.3 Antiangiogenic activity of IL-27Objective: To study the effects of IL-27 on antiangiogenesis and its mechanisms.Methods: Immunohistochemistry method was used to detect the expression of VEGF and CD34 in tumor tissue; MIG and IP-10 mRNA expression was measured by RT-PCR.Results: 1 CD34 was used to measure micro- vessle count, which expressed in blood vessel endothelium. MVD in Eca109/IL-27 inculated mice was lower than that of Eca109/LXSN and Eca109 groups (15.56±5.32, 34.27±7.34 and 35.69±4.72, respectively). Expression of VEGF was decreased in the tumor tissue of mice inculated Eca109/IL-27 cells compared to Eca109/LXSN and Eca109 cells inoculated groups (P<0.01).2 The gene expression of MIG and IP-10 mRNA in Eca109/IL-27 cells inoculated mice was significantly higher than that of Eca109/LXSN and Eca109 groups (P<0.01).Conclusion: IL-27 secreted from Eca109/IL-27 cells could induce antitumor activity through antiangiogenic activity. |
PDF Full Text Request