EGCG通过调控MDSCs介导免疫抑制阻滞肿瘤免疫逃逸研究

发布时间：2019-04-15 17:20

【摘要】：体内外研究表明,EGCG对前列腺癌、乳腺癌、肺癌等诸多肿瘤具有一定的防治作用。然而,EGCG的低生物利用度使其在体内难以达到抗肿瘤的有效浓度,导致其抗肿瘤机理至今尚未明确。肿瘤与免疫息息相关。肿瘤免疫逃逸,即肿瘤能够通过某些途径使宿主机体不能产生有效抗肿瘤的免疫应答,其作用机制相当复杂。其中以髓系抑制性细胞(Myeloid-Derived SuppressorCells,MDSCs)为主的负向调节抗肿瘤免疫应答的免疫抑制性细胞亚群被认为是肿瘤免疫逃逸的重要原因之一。然而,EGCG是否能通过调控MDSCs这一免疫学途径发挥抗肿瘤作用的研究却鲜有报道。在本论文中,我们通过建立高转移性癌症经典模型(4T1小鼠乳腺癌),评价EGCG在体内外乳腺癌生长的抑制作用;考察EGCG对乳腺癌小鼠体内CD11b~+Gr-1~+MDSC、CD4~+ T 和 CD8~+T 及 Treg、Arg-1 和 iNOS 的影响;并在此基础上,利用磁珠从小鼠脾脏中分选出高纯度乳腺癌诱导的MDSCs,进一步研究EGCG对MDSCs生长、凋亡及其介导免疫抑制的关键效应分子的影响。实验结果如下:1.在4T1体外实验中,EGCG能显著改变4T1细胞形态,并呈浓度依赖性地抑制4T1细胞增殖,EGCG纯度越高其对4T1细胞抑制能力越强,在浓度范围内(50μg/ml-250μg/ml),EGCG能显著抑制4T1乳腺癌细胞的侵袭和迁徙,并呈浓度依赖性地诱导4T1细胞凋亡。然而,EGCG对4T1细胞的诱导凋亡能力并不强,当EGCG浓度为250μg/ml时,其诱导4T1细胞的凋亡率仅有12.5%。2.在乳腺癌小鼠模型中,EGCG能显著抑制乳腺癌小鼠肿瘤的生长和脾脏的肿大,能呈剂量依赖效应地降低荷瘤小鼠体内各部位的CD11b~+Gr-1~+MDSCs含量,能够缓解荷瘤小鼠体内的免疫抑制状态。同时,EGCG能通过显著降低了Treg、Arg-1和iNOS的表达,提升荷瘤小鼠体内CD4~+T和CD8~+T来抑制肿瘤的免疫逃逸。3.在MDSCs的体外实验中,EGCG能通过诱导凋亡和阻滞分裂周期直接作用与MDSCs,降低其免疫抑制能力;并且EGCG下调Arg-1、iNOS、gp91和p47的表达,减少精氨酸酶和ROS的含量,促进T细胞的活化和增殖,降低T细胞凋亡;同时EGCG抑制GM-CSF、IL-6、IL-13的表达,从而阻滞MDSCs的活化;此外,EGCG能够通过抑制TGF-β、IL-10表达,降低MDSCs对Treg细胞的招募,并抑制巨噬细胞M2转化,提高T细胞活性。4.通过比较发现,MDSCs对EGCG的敏感度远高于4T1乳腺癌细胞。0.5μg/ml的EGCG处理后,MDSCs的凋亡率从空白的6.8%提升到10.7%,50 μg/ml的EGCG处理后,4T1细胞的凋亡率只有2.8%。
[Abstract]:In vitro and in vivo studies have shown that EGCG has certain preventive and therapeutic effects on prostate cancer, breast cancer, lung cancer and many other tumors. However, the low bioavailability of EGCG makes it difficult to reach the effective anti-tumor concentration in vivo, which leads to its anti-tumor mechanism is still unclear. Tumors are closely related to immunity. Tumor immune escape, that is, the tumor can make the host body unable to produce an effective anti-tumor immune response through some ways, its mechanism is quite complex. One of the important reasons for tumor immune escape is that myeloid inhibitory cells (Myeloid-Derived SuppressorCells,MDSCs), which negatively regulate anti-tumor immune response, play an important role in tumor immune escape. However, there is little report on whether EGCG can play an anti-tumor role by regulating the immunological pathway of MDSCs. In this study, we established a classical model of highly metastatic cancer (4T1 mouse breast cancer) to evaluate the inhibitory effect of EGCG on the growth of breast cancer in vivo and in vitro. To investigate the effects of EGCG on CD11b~ Gr-1~ MDSC,CD4~ T, CD8~ T, Treg,Arg-1 and iNOS in mice with breast cancer. On this basis, magnetic beads were used to isolate high-purity breast cancer-induced MDSCs, from the spleen of mice. The effects of EGCG on the growth, apoptosis and the key effectors of immunosuppression mediated by MDSCs were further studied. The experimental results are as follows: 1. In 4T1 in vitro, EGCG could significantly change the morphology of 4T1 cells and inhibit the proliferation of 4T1 cells in a concentration-dependent manner. The higher the purity of EGCG, the stronger the inhibitory ability to 4T1 cells, within the concentration range (50 渭 g / ml- 250 渭 g / ml),). EGCG significantly inhibited the invasion and migration of 4T1 breast cancer cells and induced apoptosis of 4T1 cells in a concentration-dependent manner. However, the ability of EGCG to induce apoptosis of 4T1 cells was not strong. When the concentration of EGCG was 250 渭 g / ml, the apoptosis rate of 4T1 cells was only 12.5%. In the model of breast cancer mice, EGCG significantly inhibited the growth of tumor and the swelling of spleen in breast cancer mice, and decreased the content of CD11b~ Gr-1~ MDSCs in various parts of tumor-bearing mice in a dose-dependent manner. It can relieve the immunosuppressive state of tumor-bearing mice. At the same time, EGCG can inhibit the immune escape of tumor by significantly reducing the expression of Treg,Arg-1 and iNOS and increasing the expression of CD4~ T and CD8~ T in tumor bearing mice. 3. In the in vitro experiment of MDSCs, EGCG could directly inhibit the immunosuppressive ability of MDSCs, by inducing apoptosis and blocking the cycle of division. In addition, EGCG down-regulated the expression of Arg-1,iNOS,gp91 and p47, decreased the contents of arginine enzyme and ROS, promoted the activation and proliferation of T cells, and decreased the apoptosis of T cells. At the same time, EGCG inhibited the expression of GM-CSF,IL-6,IL-13 and blocked the activation of MDSCs. In addition, EGCG can inhibit the expression of TGF- 尾 and IL-10, decrease the recruitment of Treg cells by MDSCs, inhibit M 2 transformation of macrophages, and increase the activity of T cells. 4. It was found that the sensitivity of MDSCs to EGCG was much higher than that of 4T1 breast cancer cells. After EGCG treatment with 0.5 渭 g / ml, the apoptosis rate of MDSCs increased from 6.8% of blank to 10.7%. After treatment with EGCG of 50 渭 g/ml, the apoptosis rate of MDSCs increased from 6.8% to 10.7%. The apoptosis rate of 4T1 cells was only 2.8%.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.3

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