mmu-circ-012559调控髓源性抑制细胞功能的初步实验研究

发布时间：2018-05-09 02:33

  本文选题：环状RNA + 肺癌　； 参考：《江苏大学》2017年硕士论文

【摘要】：目的研究小鼠miR-223相关的环状RNA分子circ-012559(mmu-circ-012559)对髓源性抑制细胞(myeloid-derived suppressor cells,MDSCs)免疫抑制功能的影响,并分析其可能的作用机制。方法(1)构建小鼠Lewis肺癌移植瘤模型,免疫磁珠法分离荷瘤小鼠脾脏及肿瘤组织来源的MDSCs,流式细胞术(flow cytometry,FCM)鉴定MDSCs分选纯度。通过实时荧光定量PCR(quantitative real-time polymerase chain reaction,qRT-PCR)检测MDSCs中mmu-circ-012559及miR-223的相对表达水平。(2)免疫磁珠法分离荷瘤小鼠脾脏MDSCs,给予Lewis肺癌细胞培养上清(tumor culture conditioned medium,TCCM)处理24h。qRT-PCR检测TCCM处理24h后MDSCs中mmu-circ-012559及miR-223的相对表达水平。将处理过的MDSCs与正常小鼠脾脏来源的CD4+T细胞进行共培养,通过3H-胸腺嘧啶核苷([3H]-thymidine,3H-TdR)掺入法检测培养体系中每分钟脉冲数(counts per minute,CPM),反映CD4+T细胞增殖能力。Western-Blot检测MDSCs中总STAT3蛋白表达及STAT3蛋白磷酸化水平变化。(3)将si-NC+miR-inhibitor NC、si-mmu-circ-012559+miR-inhibitor NC以及si-mmu-circ-012559+miR-223 inhibitor转染至TCCM处理的MDSCs中。培养24h后,将MDSCs与正常小鼠脾脏来源的CD4+T细胞进行共培养,3H-TdR掺入法检测CD4+T细胞增殖能力。FCM检测MDSCs免疫抑制相关效应分子活性氧(reactive oxygen species,ROS)的表达,试剂盒检测MDSCs精氨酸酶1(arginase 1,Arg1)活性。Western-Blot检测MDSCs中总STAT3蛋白表达及STAT3蛋白磷酸化水平变化。结果(1)荷瘤小鼠肿瘤组织来源MDSCs中mmu-circ-012559的表达量显著高于脾脏MDSCs(3.59±0.04 vs 0.80±0.08,p0.01),而miR-223的表达量显著低于脾脏MDSCs(0.21±0.05 vs 2.57±0.29,p0.01)。(2)TCCM处理组MDSCs中mmu-circ-012559的表达水平显著高于对照组(1.95±0.08 vs 1.01±0.13,p0.01),而miR-223显著低于对照组(0.50±0.12vs 2.13±0.75,p0.05)。MDSCs经TCCM处理后,其CPM值明显低于对照组(p0.01)。此外,MDSCs中STAT3及磷酸化STAT3蛋白表达量显著高于对照组(p0.05)。(3)转染si-mmu-circ-012559至TCCM处理后的MDSCs,再加入CD4+T细胞增殖体系,其CPM值高于转染对照组(p0.05)。而相比si-mmu-circ-012559+miR-inhibitor NC组,si-mmu-circ-012559与miR-223 inhibitor共转染组CPM值降低(p0.05)。另外,MDSCs给予si-mmu-circ-012559+miR-inhibitor NC处理后,ROS表达量下降(p0.05),Arg1活性也降低(8.96±1.44 vs 12.75±0.36,p0.05),相应的,STAT3及磷酸化STAT3蛋白表达量也减少。而si-mmu-circ-012559与miR-223 inhibitor共转染组ROS表达量(p0.05)及Arg1活性(13.43±1.03 vs 8.96±1.44,p0.05)回升,STAT3及磷酸化STAT3蛋白表达量也有所恢复。以上结果显示敲减mmu-circ-012559后,MDSCs免疫抑制能力明显减弱,而miR-223 inhibitor与si-mmu-circ-012559共转染后,MDSCs免疫抑制能力可得到恢复。结论肿瘤微环境MDSCs中的mmu-circ-012559表达显著增多,而miR-223的表达水平显著下降。体外敲减mmu-circ-012559能够下调MDSCs免疫抑制相关效应分子的表达,减弱MDSCs的免疫抑制功能,而共敲减miR-223后,MDSCs免疫抑制能力恢复。提示mmu-circ-012559可能通过靶向miR-223,上调STAT3活化,进而调控MDSCs的免疫抑制功能。
[Abstract]:Objective to study the effect of miR-223 related cyclic RNA molecule circ-012559 (mmu-circ-012559) on the immunosuppressive function of myelinated inhibitory cells (myeloid-derived suppressor cells, MDSCs) and to analyze the possible mechanism of action. Method (1) the model of mice's Lewis lung cancer transplant tumor was constructed, and the immunomagnetic bead method was used to separate the spleen and tumor of the tumor bearing mice. The purity of MDSCs was identified by MDSCs, flow cytometry (FCM), and the relative expression level of MDSCs was detected by real-time quantitative fluorescence quantitative PCR (quantitative real-time polymerase chain reaction, qRT-PCR). (2) immunomagnetic beads were used to separate the spleen of tumor bearing mice. Tumor culture conditioned medium (TCCM) was used to treat 24h.qRT-PCR to detect the relative expression level of mmu-circ-012559 and miR-223 in MDSCs after TCCM treatment. The treated MDSCs was co cultured with the normal mouse spleen derived cells, and the culture system was detected by the incorporation of thymidine nucleoside. The number of pulses per minute (counts per minute, CPM) reflected the changes in the expression of total STAT3 protein and the level of STAT3 protein phosphorylation in MDSCs by CD4+T cell proliferation ability.Western-Blot. (3) si-NC+miR-inhibitor NC, si-mmu-circ-012559+miR-inhibitor, and transfected to the treatment of MDSCs. After 24h, both MDSCs and CD4+T cells from normal mice were co cultured. 3H-TdR incorporation assay was used to detect the proliferation of CD4+T cells by.FCM to detect the expression of reactive oxygen species (reactive oxygen species, ROS) of MDSCs immunosuppressive effect, and the kit was used to detect the activity of MDSCs arginase 1 (1). The expression of TAT3 protein and the level of phosphorylation of STAT3 protein were changed. Results (1) the expression of mmu-circ-012559 in tumor tissue of tumor bearing mice was significantly higher than that of MDSCs in spleen (3.59 + 0.04 vs 0.80 + 0.08, P0.01), and the expression of miR-223 was significantly lower than that of spleen MDSCs (0.21 + 0.05 vs 2.57 + 0.29, P0.01). (2) TCCM treatment group The level of expression was significantly higher than that in the control group (1.95 + 0.08 vs 1.01 + 0.13, P0.01), while miR-223 was significantly lower than the control group (0.50 + 0.12vs 2.13 + 0.75, P0.05).MDSCs after TCCM treatment, and its CPM value was significantly lower than that of the control group (P0.01). Furthermore, STAT3 and phosphorylated STAT3 egg white expression in MDSCs was significantly higher than that of the control group. (3) transfected. The CPM value of the CD4+T cell proliferation system after 9 to TCCM treatment was higher than that of the transfected control group (P0.05). Compared to the si-mmu-circ-012559+miR-inhibitor NC group, the CPM value of the co transfected group of si-mmu-circ-012559 and miR-223 inhibitor decreased (P0.05). .05), the activity of Arg1 was also reduced (8.96 + 1.44 vs 12.75 + 0.36, P0.05), correspondingly, the expression of STAT3 and phosphorylated STAT3 protein decreased, while ROS expression (P0.05) and Arg1 activity (13.43 + 1.03 8.96 + 1.44) in si-mmu-circ-012559 and miR-223 inhibitor co transfection group were recovered, and the expression of phosphorylated proteins was also recovered. The results showed that the immunosuppressive ability of MDSCs decreased significantly after mmu-circ-012559 knockout, and the immunosuppressive ability of MDSCs could be recovered after miR-223 inhibitor was co transfected with si-mmu-circ-012559. Conclusion the expression of mmu-circ-012559 in the tumor microenvironment MDSCs increased significantly, while the expression level of miR-223 decreased significantly. In vitro knockout mmu-circ-012559. It can reduce the expression of MDSCs related immunosuppressive molecules and weaken the immunosuppressive function of MDSCs, and after knocking down miR-223, the immune inhibition ability of MDSCs is restored. It suggests that mmu-circ-012559 may increase the activation of STAT3 through target miR-223, and then regulate the immune function energy of 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】：R392

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