【摘要】：體內(nèi)外研究表明,EGCG對(duì)前列腺癌、乳腺癌、肺癌等諸多腫瘤具有一定的防治作用。然而,EGCG的低生物利用度使其在體內(nèi)難以達(dá)到抗腫瘤的有效濃度,導(dǎo)致其抗腫瘤機(jī)理至今尚未明確。腫瘤與免疫息息相關(guān)。腫瘤免疫逃逸,即腫瘤能夠通過(guò)某些途徑使宿主機(jī)體不能產(chǎn)生有效抗腫瘤的免疫應(yīng)答,其作用機(jī)制相當(dāng)復(fù)雜。其中以髓系抑制性細(xì)胞(Myeloid-Derived SuppressorCells,MDSCs)為主的負(fù)向調(diào)節(jié)抗腫瘤免疫應(yīng)答的免疫抑制性細(xì)胞亞群被認(rèn)為是腫瘤免疫逃逸的重要原因之一。然而,EGCG是否能通過(guò)調(diào)控MDSCs這一免疫學(xué)途徑發(fā)揮抗腫瘤作用的研究卻鮮有報(bào)道。在本論文中,我們通過(guò)建立高轉(zhuǎn)移性癌癥經(jīng)典模型(4T1小鼠乳腺癌),評(píng)價(jià)EGCG在體內(nèi)外乳腺癌生長(zhǎng)的抑制作用;考察EGCG對(duì)乳腺癌小鼠體內(nèi)CD11b~+Gr-1~+MDSC、CD4~+ T 和 CD8~+T 及 Treg、Arg-1 和 iNOS 的影響;并在此基礎(chǔ)上,利用磁珠從小鼠脾臟中分選出高純度乳腺癌誘導(dǎo)的MDSCs,進(jìn)一步研究EGCG對(duì)MDSCs生長(zhǎng)、凋亡及其介導(dǎo)免疫抑制的關(guān)鍵效應(yīng)分子的影響。實(shí)驗(yàn)結(jié)果如下:1.在4T1體外實(shí)驗(yàn)中,EGCG能顯著改變4T1細(xì)胞形態(tài),并呈濃度依賴性地抑制4T1細(xì)胞增殖,EGCG純度越高其對(duì)4T1細(xì)胞抑制能力越強(qiáng),在濃度范圍內(nèi)(50μg/ml-250μg/ml),EGCG能顯著抑制4T1乳腺癌細(xì)胞的侵襲和遷徙,并呈濃度依賴性地誘導(dǎo)4T1細(xì)胞凋亡。然而,EGCG對(duì)4T1細(xì)胞的誘導(dǎo)凋亡能力并不強(qiáng),當(dāng)EGCG濃度為250μg/ml時(shí),其誘導(dǎo)4T1細(xì)胞的凋亡率僅有12.5%。2.在乳腺癌小鼠模型中,EGCG能顯著抑制乳腺癌小鼠腫瘤的生長(zhǎng)和脾臟的腫大,能呈劑量依賴效應(yīng)地降低荷瘤小鼠體內(nèi)各部位的CD11b~+Gr-1~+MDSCs含量,能夠緩解荷瘤小鼠體內(nèi)的免疫抑制狀態(tài)。同時(shí),EGCG能通過(guò)顯著降低了Treg、Arg-1和iNOS的表達(dá),提升荷瘤小鼠體內(nèi)CD4~+T和CD8~+T來(lái)抑制腫瘤的免疫逃逸。3.在MDSCs的體外實(shí)驗(yàn)中,EGCG能通過(guò)誘導(dǎo)凋亡和阻滯分裂周期直接作用與MDSCs,降低其免疫抑制能力;并且EGCG下調(diào)Arg-1、iNOS、gp91和p47的表達(dá),減少精氨酸酶和ROS的含量,促進(jìn)T細(xì)胞的活化和增殖,降低T細(xì)胞凋亡;同時(shí)EGCG抑制GM-CSF、IL-6、IL-13的表達(dá),從而阻滯MDSCs的活化;此外,EGCG能夠通過(guò)抑制TGF-β、IL-10表達(dá),降低MDSCs對(duì)Treg細(xì)胞的招募,并抑制巨噬細(xì)胞M2轉(zhuǎn)化,提高T細(xì)胞活性。4.通過(guò)比較發(fā)現(xiàn),MDSCs對(duì)EGCG的敏感度遠(yuǎn)高于4T1乳腺癌細(xì)胞。0.5μg/ml的EGCG處理后,MDSCs的凋亡率從空白的6.8%提升到10.7%,50 μg/ml的EGCG處理后,4T1細(xì)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%.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R730.3

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