【作者】 王海燕；

【导师】 葛银林； 苗志敏；

【作者基本信息】 青岛大学 ， 病原生物学， 2009， 博士

【摘要】 目的血管新生和免疫受抑是肿瘤发生的重要生物学机制。肿瘤患者体内多存在免疫缺陷或功能受损,肿瘤细胞通过多种细胞和分子机制逃避机体的抗肿瘤免疫反应,其中树突状细胞(DC)分化异常和对T细胞刺激能力的下降是导致肿瘤免疫逃逸的重要因素。血管内皮生长因子(VEGF)是主要的促血管新生因子,肿瘤细胞过量分泌的VEGF除了具有促血管新生活性外,还可通过抑制造血祖细胞来源的DC的分化而发挥免疫抑制作用。基于VEGF促血管新生活性和免疫抑制的双重作用,本研究拟应用siRNA阻断VEGF活性达到抗肿瘤血管新生的同时改善肿瘤免疫状态,同时应用体外诱导正常DC的联合抗瘤作用,观察这种联合是否能够增强体外抗瘤效果,为临床抗肿瘤血管新生联合DC免疫治疗提供实验依据。方法以MCF-7乳腺癌细胞为观察对象,体外设计合成靶向VEGF的siRNA,采用siRNA技术靶向抑制MCF-7细胞VEGF基因的表达,观察siRNA基因沉默效果及对靶细胞的影响;分离正常人外周血单个核细胞(PBMC),在一定细胞因子组合下诱导、培养DC;模拟肿瘤体内环境,添加MCF-7乳腺癌细胞培养上清培养DC,观察MCF-7乳腺癌细胞培养上清对正常外周血单个核细胞诱导的DC分化、成熟及功能的影响,探讨肿瘤机体内DC的免疫抑制状态;观察VEGF下调后MCF-7乳腺癌细胞培养上清对正常外周血单个核细胞诱导的DC分化、成熟及功能的影响的变化,探讨阻断VEGF活性对于改善DC功能的作用;进一步探讨靶向MCF-7细胞VEGF基因的siRNA和负载肿瘤抗原的DC二者联合对MCF-7细胞的抗瘤效果。结果设计合成的siRNA转染MCF-7细胞后VEGF mRNA和蛋白表达水平明显减低,同时抑制了靶细胞的增殖;在体外应用粒-巨细胞集落刺激因子(GM-CSF),白介素-4(IL-4)及肿瘤坏死因子α(TNF-α)能有效培养人外周血单个核细胞来源的DC;MCF-7乳腺癌细胞培养上清能够明显抑制正常外周血单个核细胞诱导的树突状细胞的分化成熟及抗原提呈能力,CD80、CD83、CD86和HLA-DR的表达明显降低(p＜0.01),DC致敏的CTL对MCF-7细胞杀伤活性及IL-12分泌和共刺激T淋巴细胞所分泌的IFN-γ明显降低(p＜0.01);干扰VEGF基因后MCF-7乳腺癌细胞培养上清对DCs的影响明显降低,CD80、CD83、CD86和HLA-DR的表达显著升高,DC致敏的CTL对MCF-7细胞杀伤活性及IL-12分泌和共刺激T淋巴细胞所分泌的IFN-γ明显升高(p＜0.01);靶向VEGF基因的siRNA和DC联合应用可显著抑制肿瘤细胞生长,细胞几乎完全溶解,细胞杀伤率达99%。结论体外设计合成的双链siRNA能有效抑制乳腺癌MCF-7细胞VEGF基因的表达,抑制肿瘤细胞的增殖,促进肿瘤细胞的凋亡;MCF-7乳腺癌细胞上清明显抑制外周血单个核细胞诱导培养的树突状细胞的分化、成熟及抗原提呈能力。下调VEGF后的MCF-7细胞上清对DCs的分化、成熟及功能的抑制作用明显降低,从而推测VEGF在肿瘤的发生、发展和免疫抑制方面可能起着重要的作用;应用siRNA联合负载肿瘤抗原DC能有效抑制乳腺癌MCF-7细胞的生长,为该课题进一步的体内实验和临床应用基因治疗联合免疫治疗提供理论基础。更多还原

【Abstract】 Objective Angiogenesis and immunosuppression are the main biological mechanisms responsible for cancer progression.It has been reported that immune deficiency or impairment is a common phenomenon in patients with cancer,and tumor cells have developed a variety of cellular and molecular mechanisms to escape from antitumor immune responses.Dendritic cells(DC) play a central role in antitumor immune responses.Abnormal differentiation of DC and its inability to stimulate T cells are the important factors in tumor escaping from immune-system supervision. Vascular endothelial growth factor(VEGF) is the most important pro-angiogenic factor,produced by many human malignancies.Recent research has shown that VEGF may also play an immunosuppressant role by inhibiting the maturation of DC from hematopoietic progenitors in addition to its angiogenic activity.Based on VEGF dual roles of angiogenic activity and immune inhibition,we expected therapeutic blockade of VEGF action using small interfering RNA might inhibit cancer angiogenesis as well as improve prospects for immunotherapy.In this study,we observed the effect of dendritic cells loaded tumor antigen combinated siRNA targeting VEGF gene on MCF-7 cells,investigated whether this combination could improve anticancer immunity in vitro to provide the experimental foundation for combination of antiangiogenic and immunotherapy in cancer.Medhods Small interfering RNA(siRNA) targeting VEGF gene was designed and synthesized,then transfected cells with Lipofectimine to observe the effect of siRNA on VEGF gene and MCF-7 cells;The peripheral blood were drawn from healthy adult persons,then cultured DC with granulocyte-macrophage colony stimulating factor(GM-CSF),interleukin 4(IL-4) and tumor necrosis factor-a(TNF-a).Mononuclear cells were cultured with the culture supernatants from primary MCF-7 cells and cytokines to analyse the effect of the culture supernatants on the differentiation,maturation and function of DCs;Then siRNA directed against VEGF gene was designed and transfected into MCF-7 cells,so as to invested the change of the effect of the culture supernatants on the differentiation, maturation and function of DCs;Dendritic cells loaded tumor antigen combinated siRNA targeting VEGF gene on MCF-7 cells,from which we could investigate whether this combination could improve anticancer immunity.Results The siRNA targeting human VEGF effectively silenced VEGF gene and inhibited the secretion of VEGF in MCF-7,whereas the control scramble siRNA showed no effects;MCF-7 cell supernatant could inhibit the differentiation,maturation and function of DC induced from PBMC,the cultured DC cultured with MCF-7 cell supernatant showed low expression of CD80,CD83,CD86 and HLA-DR,and IL-12 secreted by DC and IFN-γproduced by PBMNC all reduced(p＜0.01);After silenced VEGF gene DCs cultured in the culture supernatants from MCF-7 cells had significantly higher expression of CD86,CD80,CD83 and HLA-DR than the control group(p＜0.01),IL-12 secreted by DCs and IFN-γproduced by PBMNC were improved significantly(p＜0.01);The activity of antitumor of dendritic cells loaded tumor antigen combinated siRNA targeting VEGF gene was markly,the target cells were lysised,and the inhibition rate was 99%.Conclutions siRNA designed and synthesized in vitro could reduce VEGF gene expression, inhibit proliferation and induce apoptosis of MCF-7 cells;the culture supernatants from MCF-7 breast cancer cells could inhibit the development and functions of DCs;siRNA specifically inhibited VEGF expression in MCF-7 cells,the inhibition of DC maturation and function in culture supernatants from siRNA transfected cells was significantly decreased.It suggested that VEGF might play an important role in tumor developmemt,progress and immunorepression;Dendritic cells loaded tumor antigen combinated siRNA targeting VEGF gene could markedly inhibit the proliferation of MCF-7 cells,this provided rationale for breast cancer clinical practice.更多还原