本文關(guān)鍵詞：二甲雙胍抑制G-MDSCs的免疫功能及其在小鼠抗瘤免疫應(yīng)答的研究　出處：《江蘇大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

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【摘要】：目的:二甲雙胍(metformin)體外處理小鼠粒細(xì)胞樣髓源抑制性細(xì)胞(granulocytic myeloid-derived suppressor cells,G-MDSCs),觀(guān)察 G-MDSCs 免疫抑制功能的變化,檢測(cè)G-MDSCs相關(guān)效應(yīng)分子的表達(dá)變化,并探討可能的作用機(jī)制;在CT-26荷瘤小鼠體內(nèi),研究二甲雙胍是否能夠通過(guò)調(diào)控G-MDSCs的功能影響腫瘤的生長(zhǎng)。方法:(1)使用皮下注射CT-26細(xì)胞的方式構(gòu)建結(jié)腸癌荷瘤小鼠模型,采取MACS分離荷瘤小鼠脾臟中的G-MDSCs。經(jīng)二甲雙胍作用后,使用Western-blot技術(shù)檢測(cè)STAT3磷酸化的表達(dá)水平;利用CFSE檢測(cè)G-MDSCs對(duì)CD4+T細(xì)胞的免疫抑制功能的變化;流式細(xì)胞術(shù)(FCM)檢測(cè)G-MDSCs中的活性氧(reactive oxygen specise,ROS)的表達(dá)情況;精氨酸酶(ARG-1)試劑盒檢測(cè)ARG-1活性。(2)將分離得到的G-MDSCs在體外經(jīng)二甲雙胍刺激后,通過(guò)Western-blot檢測(cè)G-MDSCs中AMPK的磷酸化水平。利用AMPK抑制劑(Compund C)抑制G-MDSCs中AMPK分子的磷酸化,再經(jīng)二甲雙胍作用,Western-blot檢測(cè)細(xì)胞中AMPK以及STAT3分子的磷酸化;CFSE檢測(cè)G-MDSCs的免疫抑制功能;FCM檢測(cè)G-MDSCs中的ROS的表達(dá)情況;精氨酸酶(ARG-1)試劑盒檢測(cè)ARG-1活性。(3)對(duì)荷瘤小鼠進(jìn)行腹腔注射二甲雙胍。觀(guān)察小鼠皮下腫瘤的生長(zhǎng)情況,觀(guān)察腫瘤大小并測(cè)量其體積;FCM檢測(cè)總MDSCs、G-MDSCs、CTLs、Th1及Treg細(xì)胞的比例。(4)分離經(jīng)二甲雙胍處理的荷瘤小鼠脾臟G-MDSCs,CFSE檢測(cè)G-MDSCs的免疫抑制功能;FCM檢測(cè)G-MDSCs中的ROS的表達(dá)情況;精氨酸酶(ARG-1)試劑盒檢測(cè)ARG-1活性;運(yùn)用Western-blot技術(shù)檢測(cè)G-MDSCs中STAT3分子的磷酸化水平。結(jié)果:(1)從荷瘤小鼠脾臟中分選出的G-MDSCs經(jīng)二甲雙胍處理后,結(jié)果顯示二甲雙胍能夠下調(diào)其STAT3磷酸化水平(P0.05);二甲雙胍明顯減弱G-MDSCs對(duì)CD4+T細(xì)胞的免疫抑制功能(P0.01)并顯著抑制G-MDSCs精氨酸酶活性以及ROS表達(dá)水平(P0.05)。(2)G-MDSCs經(jīng)二甲雙胍處理后AMPK磷酸化水平明顯上調(diào)(P0.05)。在G-MDSCs培養(yǎng)體系中加入Comound C預(yù)處理,再加入二甲雙胍處理后,G-MDSCs中AMPK的磷酸化水平與對(duì)照組相比明顯下調(diào)(P0.05),但STAT3的磷酸化水平顯著上調(diào)(P0.05);G-MDSCs對(duì)CD4+T細(xì)胞的免疫抑制功能與對(duì)照組相比顯著增強(qiáng)(P0.001);ROS表達(dá)水平較對(duì)照組明顯升高(P0.05);精氨酸酶活性則無(wú)明顯變化。(3)與生理鹽水對(duì)照組相比,二甲雙胍處理組腫瘤生長(zhǎng)速度減緩(P0.01),腫瘤體積減小,腫瘤重量減輕(P0.01);FCM結(jié)果顯示:二甲雙胍處理后,小鼠體內(nèi)浸潤(rùn)的總 MDSCs(P0.05)、G-MDSCs(P0.01)、Tregs(P0.001)的比例較對(duì)照組明顯降低;CTLs、Th1較對(duì)照組顯著升高(P0.05)。(4)與生理鹽水對(duì)照組相比,二甲雙胍處理組小鼠脾臟G-MDSCs的免疫抑制功能明顯下降(P0.001);ROS表達(dá)水平明顯下降(P0.05);ARG-1活性顯著降低(P0.05,P0.01);G-MDSCs中STAT3磷酸化水平明顯下調(diào)(P0.01)。結(jié)論:(1)二甲雙胍在體外能夠通過(guò)增強(qiáng)AMPK的磷酸化,抑制STAT3分子的磷酸化水平,從而下調(diào)G-MDSCs的免疫抑制功能。(2)在荷瘤小鼠體內(nèi),二甲雙胍能通過(guò)下調(diào)G-MDSCs的免疫抑制功能從而延緩腫瘤的生長(zhǎng)。
[Abstract]:Objective: to treat mouse granulocyte-like medullary inhibitory cells in vitro with metformin metformin (metformin). Granulocytic myeloid-derived suppressor cells. The changes of immunosuppressive function of G-MDSCs were observed and the expression of related effector molecules of G-MDSCs were detected and the possible mechanism was discussed. In CT-26 tumor-bearing mice. To study whether metformin can affect the growth of tumor by regulating the function of G-MDSCs. Methods CT-26 cells were injected subcutaneously to establish the model of colon cancer bearing mice. G-MDSCs were isolated from the spleen of tumor-bearing mice by MACS. After treated with metformin, the expression of STAT3 phosphorylation was detected by Western-blot technique. The immunosuppressive effect of G-MDSCs on CD4 T cells was detected by CFSE. Flow cytometry (FCM) was used to detect the expression of reactive oxygen (Ros) in G-MDSCs. Arginase arginase ARG-1) kit was used to detect the activity of ARG-1. 2) the isolated G-MDSCs were stimulated by metformin in vitro. The phosphorylation level of AMPK in G-MDSCs was detected by Western-blot. AMPK inhibitor, Compund C, was used to detect the phosphorylation of AMPK in G-MDSCs. Inhibition of phosphorylation of AMPK molecules in G-MDSCs. The phosphorylation of AMPK and STAT3 molecules in the cells was detected by Western-blot with metformin. The immunosuppressive function of G-MDSCs was detected by CFSE. The expression of ROS in G-MDSCs was detected by FCM. Arginase arginase ARG-1 kit was used to detect the activity of ARG-1 in tumor-bearing mice. Metformin was injected intraperitoneally to observe the growth, size and volume of subcutaneous tumor in mice. FCM was used to detect the proportion of CTLs1 and Treg cells in the total MDSCS cell line G-MDSCs1 and the proportion of Treg cells. The spleen G-MDSCs treated with metformin were isolated from the spleen of the tumor-bearing mice. The immunosuppressive function of G-MDSCs was detected by CFSE. The expression of ROS in G-MDSCs was detected by FCM. The activity of ARG-1 was detected by arginase ARG-1 kit. Western-blot technique was used to detect the phosphorylation level of STAT3 molecules in G-MDSCs. G-MDSCs isolated from the spleen of tumor-bearing mice were treated with metformin. The results showed that metformin could down-regulate the phosphorylation level of STAT3. Metformin significantly reduced the immunosuppressive function of G-MDSCs to CD4 T cells (P0.01) and significantly inhibited the argininase activity and ROS expression of G-MDSCs (P < 0.05). The phosphorylation level of AMPK increased significantly (P0.05) after treatment with metformin. To add Comound C pretreatment in G-MDSCs culture system. The phosphorylation level of AMPK in G-MDSCs treated with metformin was significantly lower than that in control group (P 0.05). However, the phosphorylation level of STAT3 was significantly up-regulated (P 0.05). The immunosuppressive function of G-MDSCs on CD4 T cells was significantly increased compared with the control group (P 0.001). The expression of ROS was significantly higher than that of control group (P 0.05). Argininase activity did not change significantly.) compared with normal saline control group, metformin treated group decreased tumor growth rate and tumor volume. The weight of tumor was reduced by P0.01; FCM results showed that after metformin treatment, the infiltrated total MDSCs in mice was P0.05 and G-MDSCsP 0.01). The ratio of Tregsl P 0.001) was significantly lower than that of the control group. Compared with the control group, the level of CTLsTh _ 1 was significantly higher than that of the control group (P 0.05, P < 0.05, P < 0.05, P < 0.05, P < 0.05). The immunosuppressive function of G-MDSCs in the spleen of metformin treated mice decreased significantly (P0.001). The expression of ROS was significantly decreased in P0.05; The activity of ARG-1 decreased significantly (P0.05, P0.01C). The phosphorylation level of STAT3 in G-MDSCs was significantly down-regulated P0.01.Conclusion metformin can enhance the phosphorylation of AMPK in vitro. Inhibition of phosphorylation of STAT3 molecules, thus down-regulating the immunosuppressive function of G-MDSCs in tumor-bearing mice. Metformin can delay tumor growth by down-regulating the immunosuppressive function of G-MDSCs.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R965

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相關(guān)期刊論文 前1條

1 Peter Laszlo Lakatos;Laszlo Lakatos;;Risk for colorectal cancer in ulcerative colitis:Changes,causes and management strategies[J];World Journal of Gastroenterology;2008年25期

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本文編號(hào)：1429512