【作者】 刘卉；

【导师】 周瑞祥；

【作者基本信息】 福建医科大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 胃癌是我国最常见的恶性肿瘤之一,其发生发展机制尚不明确,提高机体对肿瘤的免疫效应是目前生命科学领域的研究热点之一。CD4~+CD25~+调节性T(Treg)细胞具有免疫无能和免疫抑制两大特征,对肿瘤发生发展有负调节作用,与胃癌预后呈负相关。前期大量研究证实褪黑素有抗肿瘤及免疫调节作用,而褪黑素与胃癌、Treg细胞三者之间直接联系的研究尚未见公开报道。本研究旨在探讨褪黑素对小鼠胃癌抑制作用及其与CD4~+CD25~+ Treg细胞和TGF-β信号通路的关系及相关机制,为胃癌寻找一条基于神经-内分泌-免疫-肿瘤网络的综合治疗模式的新思路。1.褪黑素体内抗小鼠胃癌作用及其与CD4~+CD25~+ Treg细胞的关系为研究褪黑素体内抗小鼠胃癌作用及其与CD4~+CD25~+调节性T细胞的关系,本部分研究建立荷胃癌小鼠模型,不同浓度褪黑素干预后,运用流式细胞术、实时荧光定量RT-PCR、免疫荧光双染色法、免疫印迹法、ELISA等方法检测CD4~+CD25~+ Treg细胞及相关因子的表达变化。结果表明褪黑素能够缩小荷胃癌小鼠的肿瘤体积,减轻肿瘤重量,达到抗肿瘤作用;褪黑素在抑制肿瘤过程中下调肿瘤组织的Treg细胞及Foxp3表达,且能使荷胃癌小鼠的胸腺、脾增加的Treg细胞数及增高表达的Foxp3蛋白恢复到正常水平,但对脾Foxp3mRNA有上调作用;褪黑素还使荷胃癌小鼠外周血清升高表达的的TGF-β1下降到正常水平。表明褪黑素抗肿瘤的途径之一是降低体内Treg细胞数。2. CD4~+CD25~+ Treg细胞在褪黑素体外抗小鼠胃癌细胞增殖中作用为研究褪黑素体外抗小鼠胃癌细胞增殖作用及其与CD4~+CD25~+ Treg细胞的关系,本部分建立不同浓度褪黑素干预胃癌细胞模型,并选择性加入Treg细胞或CD4~+CD25- T细胞共培养,运用细胞免疫磁珠分选、流式细胞术、荧光双标染色、CCK-8、ELISA等方法检测胃癌细胞增殖活性及细胞周期变化。结果表明褪黑素抑制胃癌细胞的增殖,呈剂量依赖性,并与细胞周期G2/M期阻滞有关;CD4~+CD25~+ Treg细胞在体外对褪黑素抑制肿瘤细胞增殖过程中未起拮抗作用。3.褪黑素对胃癌细胞与CD4~+CD25~+ Treg细胞共培养体系相关因子的影响为进一步研究褪黑素对细胞共培养体系影响的可能机制,本部分沿用小鼠胃癌细胞与Treg细胞或CD4~+CD25- T细胞共培养体系,褪黑素进行干预后,运用实时荧光定量RT-PCR、双荧光素酶报告基因系统、免疫组化、免疫印迹法等方法探讨褪黑素对共培养体系中相关因子Foxp3及survivin、caspase-3的影响。结果发现褪黑素在抑制胃癌细胞增殖过程中对共培养的Treg细胞的Foxp3mRNA表达有明显上调作用,并呈剂量依赖性,但褪黑素未能明显增高Foxp3启动子的转录活性。褪黑素降低胃癌细胞survivin的表达,进而激活caspase-3的活性,是褪黑素体外抑制胃癌细胞增殖的作用机制之一;而与胃癌细胞共培养的Treg细胞对胃癌细胞survivin的表达有上调作用,CD4~+CD25- T细胞则明显增敏了褪黑素对胃癌细胞的survivin及caspase-3的影响。4. TGF-β信号通路在褪黑素体内外抑制小鼠胃癌细胞增殖中的作用为研究TGF-β1在褪黑素抑制胃癌细胞增殖过程中的作用,本部分研究体内实验沿用第一部分动物模型,探讨褪黑素对荷胃癌小鼠胸腺、脾、肿瘤组织TGF-β1基因转录与翻译水平表达的影响;体外实验则建立不同浓度褪黑素在不同时间点干预胃癌细胞模型,利用TGF-β1细胞因子刺激、抗TGF-β1中和抗体阻断、反义核酸技术沉默TGF-β1表达等手段探讨TGF-β信号通路在褪黑素抑制胃癌细胞增殖中的作用。结果发现褪黑素在体内抑制胃癌过程中上调肿瘤组织的TGF-β1表达,且使荷胃癌小鼠胸腺TGF-β1表达明显下调,同时使脾下调的TGF-β1表达恢复到正常水平。褪黑素体外抑制胃癌细胞增殖过程中,伴有TGF-β1表达增高,呈时间依赖性。SiRNA介导的TGF-β1沉默和抗TGF-β1中和抗体联合应用彻底阻断TGF-β1通路,能显著拮抗褪黑素对胃癌细胞生长增殖的抑制,同时促使MFC细胞的G1期向S期转变,提示TGF-β1参与细胞正常生长增殖的调控,褪黑素抑制胃癌细胞增殖的途经之一是依赖TGF-β1信号通路。综上所述,褪黑素体内外能够抑制小鼠胃癌细胞增殖,其机制包括下调体内CD4~+CD25~+ Treg细胞数及Foxp3表达和改变TGF-β信号通路。更多还原

【Abstract】 Gastric cancer is one of the most common malignant tumors in our country, the development mechanism of which is not clear. Augment the host anti-tumor immunological response have received increasingly attention in life science at present. CD4~+CD25~+ regulatory T (Treg) cells have anergic and immunosuppressive characteristics, which implying their negative regulations effect to the development and progression of tumor and were negative correlated with gastric cancer prognosis. Preliminary many study indicated melatonin has the antineoplastic activity and immunomodulating effects, It is not reported that the direct effects of melatonin on Treg cells and gastric cancer. This research will explore inhibitory effect of melatonin on murine gastric cancer and its relation to CD4~+CD25~+ regulatory T cells and TGF-βsignal pathway and clarify the relative mechanism in order to offer a new thought to find a combined therapy model based on neuroendocrine-immune-tumor network for gastric cancer.1. Inhibitory effect of melatonin on gastric cancer and its relation to CD4~+CD25~+ regulatory T cells in vivoTo explore inhibitory effect of melatonin on gastric cancer and its relation to CD4~+CD25~+ regulatory T cells in vivo, we performed an animal model by inoculating the murine foregastric carcinoma (MFC) cell line in mice and treated them with different doses of melatonin. Then the effect of MLT on the expression of CD4~+CD25~+ Tregs and Foxp3 in mice was detected by using flow cytometry, real-time fluorescence quantitative PCR, double immunofluorescence staining, Western blot, ELISA and other methods. This research showed that MLT could reduce the weight and volum of tumor tissue, decrease Tregs numbers and its transcription factor Foxp3 expression in the tumor tissue. Melatonin also could down-regulate Tregs numbers and scurfin in thymus and spleen, then up-regulate Foxp3mRNA expression in spleen, and return the TGF-β1 expression to the normal levels in peripheral serum. The data indicate that one of melatonin anti-tumor way via inhibition of Tregs in vivo. 2. Role of CD4~+CD25~+ Tregs in melatonin-mediated inhibition of murine gastric cancer cell growth in vitroTo explore inhibitory effect of melatonin on gastric cancer cell proliferation and its relation to CD4~+CD25~+ regulatory T cells in vitro, we performed a cell model by MFC cells treatment with different concentrations of melatonin, then with or without Tregs or CD4~+CD25~- T cells co-culture. The effect of MLT on MFC cell proliferation and cycle change was detected by using MACS, flow cytometry, double immunofluorescence staining, CCK-8, ELISA and other methods. We clearly demonstrated that a concentration-dependent effect of melatonin-induced apoptosis in the MFC gastric cancer cells in vitro. This apoptosis was also related to the arrest of the cell cycle at the G2/M phase, but CD4~+CD25~+ Tregs have not antagonistic effect on melatonin-mediated inhibition of murine gastric cancer cell growth in vitro.3. Effect of melatonin on the related cytokines in MFC cells with or without Tregs or CD4~+CD25~- T cells co-culture systemIn order to further investigate the effect mechanism of melatonin on MFC cells with Tregs co-culture system, we performed cells co-culture system with different concentrations of melatonin treatment, which was the same as the second part research. The effect of MLT on the related cytokines of Foxp3, survivin and caspase-3 in co-culture system was detected by using real-time fluorescence quantitative PCR, immunocytochemical reaction, Dual-Luciferase Reporter Assay System, Western blot and other methods. The result showed a certain concentration of MLT increased Foxp3mRNA expression of Tregs with MFC cell co-culture in a dose-dependent manner. But melatonin itself treatments did not obviously enhance the transcriptional activity of the Foxp3 gene promoter. Melatonin also could down-regulate the expression of survivin and activate the activity of caspase-3 protein, which is one of melatonin anti-tumor way in vitro.Tregs with MFC cell co-culture could up-regulate survivin expression in tumor cell, however, CD4~+CD25~- T cells in cell co-culture system obviously enhance the effect of melatonin on survivin and caspase-3 expression in MFC cells.4. Role of TGF-βsignal pathway in melatonin-mediated inhibition of murine gastric cancer cell proliferation in vivo and in vitroIn order to investigate the role of TGF-β1 in melatonin-mediated inhibition of cancer cell proliferation. In vivo study, we performed the animal model which was the same as the first part research to detect the effect of MLT on the expression of TGF-β1 in the thymus, spleen and tumor tissue of mice. In vitro study, we performed a cell model by MFC cells treatment with different concentrations of melatonin in different time, utilizated external conditions change such as using TGF-β1 cytokine to stimulate, anti-TGFβantibody to counteract TGF-β1, or using antisense nucleic acid technique to block TGF-β1, and so on to explore TGF-βsignal pathway in melatonin-mediated inhibition of the proliferation of gastric cancer cell. This research showed that in the melatonin anti-gastric cancer process in vivo where TGF-β1 was up-regulated in the tumor tissue and down-regulated in thymus, at the same time down-regulated TGF-β1 expression was returned to normal levels in spleen. Furthermore, in the melatonin anti-gastric cancer proliferation process in vitro where TGF-β1 was up-regulated in the time-dependent manner. SiRNA mediated TGF-β1 silence, combined with anti-TGFβ1 antibody to counteract TGF-βsignal pathway thoroughly could rivalry the inhibit effect of melatonin on MFC cells proliferation and urge cell course from G1 to S at the same time. The data indicate that TGF-β1 participates in regulation of the normal growth and differentiation of cells, one of melatonin anti-tumor way via TGF-βsignal pathway-dependent manner.In conclusion, melatonin has a potent anti-gastric cancer effect in vivo and in vitro, and the mechanism is associated with down-regulation of CD4~+CD25~+ Tregs and Foxp3 expression in vivo and the change of TGF-βsignal pathway.更多还原