二甲双胍抑制G-MDSCs的免疫功能及其在小鼠抗瘤免疫应答的研究

发布时间：2018-01-15 18:19

本文关键词：二甲双胍抑制G-MDSCs的免疫功能及其在小鼠抗瘤免疫应答的研究　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:二甲双胍(metformin)体外处理小鼠粒细胞样髓源抑制性细胞(granulocytic myeloid-derived suppressor cells,G-MDSCs),观察 G-MDSCs 免疫抑制功能的变化,检测G-MDSCs相关效应分子的表达变化,并探讨可能的作用机制;在CT-26荷瘤小鼠体内,研究二甲双胍是否能够通过调控G-MDSCs的功能影响肿瘤的生长。方法:(1)使用皮下注射CT-26细胞的方式构建结肠癌荷瘤小鼠模型,采取MACS分离荷瘤小鼠脾脏中的G-MDSCs。经二甲双胍作用后,使用Western-blot技术检测STAT3磷酸化的表达水平;利用CFSE检测G-MDSCs对CD4+T细胞的免疫抑制功能的变化;流式细胞术(FCM)检测G-MDSCs中的活性氧(reactive oxygen specise,ROS)的表达情况;精氨酸酶(ARG-1)试剂盒检测ARG-1活性。(2)将分离得到的G-MDSCs在体外经二甲双胍刺激后,通过Western-blot检测G-MDSCs中AMPK的磷酸化水平。利用AMPK抑制剂(Compund C)抑制G-MDSCs中AMPK分子的磷酸化,再经二甲双胍作用,Western-blot检测细胞中AMPK以及STAT3分子的磷酸化;CFSE检测G-MDSCs的免疫抑制功能;FCM检测G-MDSCs中的ROS的表达情况;精氨酸酶(ARG-1)试剂盒检测ARG-1活性。(3)对荷瘤小鼠进行腹腔注射二甲双胍。观察小鼠皮下肿瘤的生长情况,观察肿瘤大小并测量其体积;FCM检测总MDSCs、G-MDSCs、CTLs、Th1及Treg细胞的比例。(4)分离经二甲双胍处理的荷瘤小鼠脾脏G-MDSCs,CFSE检测G-MDSCs的免疫抑制功能;FCM检测G-MDSCs中的ROS的表达情况;精氨酸酶(ARG-1)试剂盒检测ARG-1活性;运用Western-blot技术检测G-MDSCs中STAT3分子的磷酸化水平。结果:(1)从荷瘤小鼠脾脏中分选出的G-MDSCs经二甲双胍处理后,结果显示二甲双胍能够下调其STAT3磷酸化水平(P0.05);二甲双胍明显减弱G-MDSCs对CD4+T细胞的免疫抑制功能(P0.01)并显著抑制G-MDSCs精氨酸酶活性以及ROS表达水平(P0.05)。(2)G-MDSCs经二甲双胍处理后AMPK磷酸化水平明显上调(P0.05)。在G-MDSCs培养体系中加入Comound C预处理,再加入二甲双胍处理后,G-MDSCs中AMPK的磷酸化水平与对照组相比明显下调(P0.05),但STAT3的磷酸化水平显著上调(P0.05);G-MDSCs对CD4+T细胞的免疫抑制功能与对照组相比显著增强(P0.001);ROS表达水平较对照组明显升高(P0.05);精氨酸酶活性则无明显变化。(3)与生理盐水对照组相比,二甲双胍处理组肿瘤生长速度减缓(P0.01),肿瘤体积减小,肿瘤重量减轻(P0.01);FCM结果显示:二甲双胍处理后,小鼠体内浸润的总 MDSCs(P0.05)、G-MDSCs(P0.01)、Tregs(P0.001)的比例较对照组明显降低;CTLs、Th1较对照组显著升高(P0.05)。(4)与生理盐水对照组相比,二甲双胍处理组小鼠脾脏G-MDSCs的免疫抑制功能明显下降(P0.001);ROS表达水平明显下降(P0.05);ARG-1活性显著降低(P0.05,P0.01);G-MDSCs中STAT3磷酸化水平明显下调(P0.01)。结论:(1)二甲双胍在体外能够通过增强AMPK的磷酸化,抑制STAT3分子的磷酸化水平,从而下调G-MDSCs的免疫抑制功能。(2)在荷瘤小鼠体内,二甲双胍能通过下调G-MDSCs的免疫抑制功能从而延缓肿瘤的生长。
[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.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965

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