本文選題：環(huán)狀RNA + 肺癌��； 參考：《江蘇大學》2017年碩士論文

【摘要】：目的研究小鼠miR-223相關的環(huán)狀RNA分子circ-012559(mmu-circ-012559)對髓源性抑制細胞(myeloid-derived suppressor cells,MDSCs)免疫抑制功能的影響,并分析其可能的作用機制。方法(1)構建小鼠Lewis肺癌移植瘤模型,免疫磁珠法分離荷瘤小鼠脾臟及腫瘤組織來源的MDSCs,流式細胞術(flow cytometry,FCM)鑒定MDSCs分選純度。通過實時熒光定量PCR(quantitative real-time polymerase chain reaction,qRT-PCR)檢測MDSCs中mmu-circ-012559及miR-223的相對表達水平。(2)免疫磁珠法分離荷瘤小鼠脾臟MDSCs,給予Lewis肺癌細胞培養(yǎng)上清(tumor culture conditioned medium,TCCM)處理24h。qRT-PCR檢測TCCM處理24h后MDSCs中mmu-circ-012559及miR-223的相對表達水平。將處理過的MDSCs與正常小鼠脾臟來源的CD4+T細胞進行共培養(yǎng),通過3H-胸腺嘧啶核苷([3H]-thymidine,3H-TdR)摻入法檢測培養(yǎng)體系中每分鐘脈沖數(shù)(counts per minute,CPM),反映CD4+T細胞增殖能力。Western-Blot檢測MDSCs中總STAT3蛋白表達及STAT3蛋白磷酸化水平變化。(3)將si-NC+miR-inhibitor NC、si-mmu-circ-012559+miR-inhibitor NC以及si-mmu-circ-012559+miR-223 inhibitor轉染至TCCM處理的MDSCs中。培養(yǎng)24h后,將MDSCs與正常小鼠脾臟來源的CD4+T細胞進行共培養(yǎng),3H-TdR摻入法檢測CD4+T細胞增殖能力。FCM檢測MDSCs免疫抑制相關效應分子活性氧(reactive oxygen species,ROS)的表達,試劑盒檢測MDSCs精氨酸酶1(arginase 1,Arg1)活性。Western-Blot檢測MDSCs中總STAT3蛋白表達及STAT3蛋白磷酸化水平變化。結果(1)荷瘤小鼠腫瘤組織來源MDSCs中mmu-circ-012559的表達量顯著高于脾臟MDSCs(3.59±0.04 vs 0.80±0.08,p0.01),而miR-223的表達量顯著低于脾臟MDSCs(0.21±0.05 vs 2.57±0.29,p0.01)。(2)TCCM處理組MDSCs中mmu-circ-012559的表達水平顯著高于對照組(1.95±0.08 vs 1.01±0.13,p0.01),而miR-223顯著低于對照組(0.50±0.12vs 2.13±0.75,p0.05)。MDSCs經(jīng)TCCM處理后,其CPM值明顯低于對照組(p0.01)。此外,MDSCs中STAT3及磷酸化STAT3蛋白表達量顯著高于對照組(p0.05)。(3)轉染si-mmu-circ-012559至TCCM處理后的MDSCs,再加入CD4+T細胞增殖體系,其CPM值高于轉染對照組(p0.05)。而相比si-mmu-circ-012559+miR-inhibitor NC組,si-mmu-circ-012559與miR-223 inhibitor共轉染組CPM值降低(p0.05)。另外,MDSCs給予si-mmu-circ-012559+miR-inhibitor NC處理后,ROS表達量下降(p0.05),Arg1活性也降低(8.96±1.44 vs 12.75±0.36,p0.05),相應的,STAT3及磷酸化STAT3蛋白表達量也減少。而si-mmu-circ-012559與miR-223 inhibitor共轉染組ROS表達量(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蛋白表達量也有所恢復。以上結果顯示敲減mmu-circ-012559后,MDSCs免疫抑制能力明顯減弱,而miR-223 inhibitor與si-mmu-circ-012559共轉染后,MDSCs免疫抑制能力可得到恢復。結論腫瘤微環(huán)境MDSCs中的mmu-circ-012559表達顯著增多,而miR-223的表達水平顯著下降。體外敲減mmu-circ-012559能夠下調MDSCs免疫抑制相關效應分子的表達,減弱MDSCs的免疫抑制功能,而共敲減miR-223后,MDSCs免疫抑制能力恢復。提示mmu-circ-012559可能通過靶向miR-223,上調STAT3活化,進而調控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.

【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R392

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