【作者】 邸军；

【导师】 张桂珍；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 本研究以肺癌为DCs疫苗治疗的靶细胞,利用慢病毒载体介导人IL-12基因修饰DCs,利用化疗药物作用的肺癌细胞冲击基因修饰的DCs,使DCs负载肺癌细胞的全部抗原信息。通过观察慢病毒载体介导人IL-12基因的DCs转移效率、IL-12基因在DCs内表达,以及DCs成熟与诱发的针对肺癌细胞的细胞毒作用等几个方面来对慢病毒载体介导的人IL-12基因修饰的DCs疫苗体外抗肺癌的效果进行评价,从而为抗肿瘤的基因修饰的DCs疫苗制备提供新的方法。经过实验研究取得如下研究成果:1成功构建了人IL-12慢病毒载体,该病毒载体被包装后,可有效整合到293FT细胞基因组中并成功表达人IL-12蛋白。2携带人IL-12基因的慢病毒成功进行了DCs细胞的感染,并有较高的感染效率,IL-12基因在DCs内的表达,提高了其刺激淋巴细胞增殖的能力,并促进了DCs表面分子CD83的上调。3应用化疗药物作用的肺癌A549细胞全抗原负载人IL-12基因修饰DCs可诱发高效抗肿瘤免疫反应。更多还原

【Abstract】 Dendritic cells (DCs) play important roles in antitumor immunity ,gene delivery to DCs could represent more powerful, longer-lasting immunity. Interleukin12 (IL-12) is a multifunction cytokine that plays a important role in antitumor immunity . IL-12 gene delivery to DCs by gene transduction may promote DCs mature and enhance the antitumor responses of cytotoxic T cells induced by DCs,While efficient gene transfer methods still need to be improved.Lentivirus vectors are now being widely investigated as useful gene transfer vehicles that can efficiently target many types of cells including DCs . Comparing with retroviral vector and adenovirus vector ,lentivirus vectors offer the unique opportunity to investigate genetically modified monocyte-derived DCs because they do not require cell division for efficient and stable intergration into the host cell genome with little to no cytotoxicity, furthermore,the level of transgene is preserved ,so they are ideal vectors for gene transfer of DCs .Chemotherapy is one of the most important ways in the treatment of cancer.Cancer cells are killed after chemotherapy, and these necrotic cells can be captured by dendritic cells as antigen in vivo,while unlike infectious pathogens, these cells do not induce an effective inflammatory response ,for this reason DCs are immature and they can stimulate regulatory T cell that will evade antitumor immune response. Then ,by channeling tumor antigens into DCs ex vivo and providing the conditions for their optimal maturation can they induce the dendritic cells maturation? Does viral transduction promote antigen-loaded DCs maturation and therefore enhance antitumor immunity effectively?All these question need to be answered.This study was aimed to investigate the effects of dendritic cells transfected with lentivirus vector encoding IL-12 , sensitized with lung cancer cells A549 killed by chemotherapy.We carefully analyze the efficiency of antitumor effect of Lentivirus-mediated interleukin12 gene modified dendritic cells by evaluating the maturation of DCs and the express of IL-12 gene and cytotoxicic T cell reaction to cancer cells A549. The rational approach of genetically modified DCs described should advance DCs vaccination towards a clinical reality.1.Construction and preparation of lentiviral vector encoding interleukin-12 gene.The second generation lentiviral vectors were used: Carrier plasmid, packaging plasmid and pMD2.G were used as transfer vector of interleukin-12 gene. Interleukin-12 was extracted from plasmid and amplified by PCR technique. Introduced enzyme cutting site IL-12 gene was inserted into the carrier plasmid pWPXLd to construct plasmid pWPXLd-12.Plasmid pWPXLd-12、packaging plasmid psPAX2 and plasmid pMD2.G were tansfected 293FT cells together by liposome, we collected supematant of the cultured cell after packaging .Then we gained purificated packaged viral particle by ultracentrifagation.293FT cells were infected by diluted viral particle in different concentration. Extracted the viral gnome of the cell, the effect of IL-12 integrated in genome was evaluated by PCR to assess infection capability of the viral. According to the ability of infection adjust the strength of viral and infected the 293FT cells again. Detecting the level of IL-12 mRNA by RT-PCR .IL-12 was measured in the medium by ELISA.Plasmid pWPXLd-12 was identified by different restricting endonuclease reactions and different strength showed corresponding ability of infecting 293FT cells,that provided the evidence of viral strength .Result of RT-PCR showed the express of IL-12 only in the infected 293FT cells, and the contents of IL-12 was higher in supernatant of viral infected cells than that of non-viral infected.what we have observed indicates that lentiviral vector encoding IL-12 gene can be constructed by molecular cloning technique.Lentrivial vector can be packaged successfully with 293FT cells.2.DCs were induced by peripheral blood monouclear cells with GM-CSF、 IL-4 , and were infected by viral particles at the seventh day.Culture supernatant was collected respectively at the 7d、10d、14d. We assessed contents of IL-12 using ELISA. The expression of cell- surface marker CD83 was analyzed by flow cytometry. Proliferation of lymphocyte sensitized by DCs was assessed with MTT.With inverted microscope,we obsserved that monocyte changed gradually from spindle to round, from adherent cells to suspension cells,demonstrating that monocyte were induced to DCs.In lentiviral vector encoding interleukin-12 gene transfected DCs,content of IL-12 increased with time, while the content of IL-12 was lower and showed no change with time in DCs without modification of LVs. At the same time ,cell-surface marker CD83 and the capability of sensitize lymphocyte was higher than that of non-modification DCs. These changes demonstrated that lentiviral vector encoding interleukin-12 gene transfection can enhance DCs effectively and promote DCs maturation.3.Transfected by lentiviral vector encoding interleukin-12 gene ,DCs loaded with chemotherapeutics effected lung cancer cells A549 could induce far more effective specific CTL .We collected killed lung cancer cells A549 exposed to chemotherapeutics, modified normal DCs and DCs transfected with sentiviral vector encoding interleukin-12 gene with these A549 cells seperately ,DCs were collected and we analyze CD83 with flow cytometry.The proliferation and immunostimulatory capacity of T lymphocytes was assessed according to the results of MTT assay.The maturation of DCs was not induced by loading chemo effected lung cancer cells A549 and their ability of sensitizing lymphocyte is lower than that of viral infected DCs, their capacity to induce antien-specific cytotoxic T lymphocyte (CTL) was also lower comparing with viral infected DCs. In this study we demonstrated that lentiviral vector encoding interleukin- 12 effectivly induced DCs maturation without imparing their biological activity ,including their capacity to induce effective CTL.Conclusion: (1)Lentiviral vectors encoding interleukin-12 can infect DCs effectively, and the level of transgene expression is preserved in the DCs ,and the maturation of DCs can be promoted .(2) Lung cancer cells A549 exposed to chemotherapy can’t induce DCs maturation directly without the help of immune modulatory factors .Tumor cells will be killed by chemo ,and these necrotic cells will be captured by DCs as antigen to load .Our reasearch portends bright prospects for this approach to tumor immune therapy, either alone or in conjunction with other therapies.更多还原