本文選題：FoxO1 + 炎癥反應　； 參考：《山西醫(yī)科大學》2017年碩士論文

【摘要】：研究目的:1.觀察Fox O1轉(zhuǎn)錄因子在巨噬細胞活化前后的表達情況。2.觀察Fox O1轉(zhuǎn)錄因子敲低后對巨噬細胞炎癥過程的影響。3.檢測Fox O1是否通過TGF-β來影響炎癥反應。研究方法:1.應用脂多糖(Lipopolysaccharide,LPS)激活巨噬細胞,酶聯(lián)免疫吸附測定法(enzyme-linked immunosorbent assay,ELISA)測LPS活化巨噬細胞的最適濃度和時間。收集LPS各個濃度、培養(yǎng)時間的細胞上清液。ELISA試劑盒檢測各個濃度和時間段收集上清液中炎癥因子IL-6和TNF-α的含量,推出LPS活化巨噬細胞的最適時間及濃度。瓊脂糖凝膠電泳檢測Foxo1、Foxo3a、Foxo4、Foxo6四個基因在活化前后巨噬細胞的表達變化。實時熒光定量PCR檢測Foxo1基因的mRNA水平,Western blot檢測FOXO1蛋白表達。2.敲低活化組巨噬細胞Foxo1基因,應用流式細胞術(shù)、實時熒光定量PCR和Western blot檢驗小干擾RNA轉(zhuǎn)染效率。同時使用ELISA試劑盒檢驗兩組細胞上清液IL-6、TNF-α的含量,比較活化組巨噬細胞在轉(zhuǎn)染前后IL-6、TNF-α含量變化情況。3.巨噬細胞敲低Foxo1表達,分兩組。一組不處理,另一組LPS激活細胞。應用Western blot實驗方法比較四組巨噬細胞中TGF-β蛋白的表達情況。研究結(jié)果:1.脂多糖激活巨噬細胞后,上清液中IL-6和TNF-α的含量逐漸升高。在LPS濃度50 ng/ml,培養(yǎng)36 h時測得炎癥因子含量均高于其他濃度和時間的LPS刺激下細胞上清液中炎癥因子IL-6和TNF-α含量,表明此時是巨噬細胞活化的最適條件。采用實時熒光定量PCR和Western blot實驗得出活化組FoxO1轉(zhuǎn)錄因子在m RNA水平和蛋白水平表達均低于未活化組,且差異具有統(tǒng)計學意義(p0.05)。2.活化組敲低Foxo1,應用流式細胞術(shù)、實時熒光定量PCR、Western blot方法檢驗轉(zhuǎn)染效率。結(jié)果表明FoxO1轉(zhuǎn)錄因子在mRNA水平和蛋白水平的表達量是轉(zhuǎn)染前的0.47倍,轉(zhuǎn)染成功。ELISA實驗結(jié)果顯示:活化組巨噬細胞IL-6的含量為151.2 ng/ml±49.1 ng/ml,TNF-α含量為746.2 ng/ml±68.2 ng/ml,轉(zhuǎn)染組IL-6的含量上升到247.2 ng/ml±56 ng/ml、TNF-α的含量上升到1282 ng/ml±26.7 ng/ml。轉(zhuǎn)染組炎癥因子IL-6和TNF-α含量明顯升高,且差異有統(tǒng)計學意義(p0.01)。3.將正常巨噬細胞分為兩組:一組不處理,另一組敲低Foxo1。然后分別將兩組巨噬細胞再分為兩組:一組不做處理,另一組用LPS激活。采用Western blot分析四組細胞中TGF-β蛋白相對表達量。實驗結(jié)果顯示:在活化組、活化組+siRNA組中TGF-β蛋白均有表達�；罨MTGF-β蛋白的相對表達量為0.37,活化組+siRNA組的相對表達量是0.54。說明巨噬細胞敲低Foxo1后活化會引起TGF-β因子的增加,說明Fox O1轉(zhuǎn)錄因子有可能通過TGF-β因子影響炎癥反應。研究結(jié)論:1.Fox O1有可能參與炎癥過程。2.轉(zhuǎn)錄因子Fox O1可能具有負性調(diào)控炎癥反應的作用。3.Fox O1轉(zhuǎn)錄因子可以通過增加TGF-β表達影響炎癥反應。
[Abstract]:Objective: 1. To observe the expression of Fox O 1 transcription factor before and after macrophage activation. To observe the effect of Fox O 1 transcription factor knockdown on macrophage inflammation. Determine whether Fox O 1 affects inflammation through TGF- 尾. Research method: 1. Lipopolysaccharide (LPS) was used to activate macrophages and enzyme linked immunosorbent Assay Elisa was used to determine the optimal concentration and time of LPS activated macrophages. The concentration of inflammatory cytokines IL-6 and TNF- 偽 in supernatant of culture time was determined by Elisa kit. The optimal time and concentration of LPS to activate macrophage were deduced. Agarose gel electrophoresis (agarose) was used to detect the expression of four genes of Foxo1, Foxo3, Foxo4 and Foxo6 in macrophages before and after activation. Real time fluorescence quantitative PCR was used to detect the mRNA level of Foxo1 gene. Western blot was used to detect the expression of FOXO1 protein. The transfection efficiency of small interfering RNA was detected by flow cytometry, real-time fluorescence quantitative PCR and Western blot. At the same time, ELISA kit was used to detect the content of IL-6 TNF- 偽 in the supernatant of the two groups, and to compare the changes of IL-6 TNF- 偽 content in macrophages of activated group before and after transfection. Macrophages knocked down Foxo1 expression and were divided into two groups. One group was not treated, the other group LPS activated cells. The expression of TGF- 尾 protein in macrophages was compared by Western blot assay. The result of the study was: 1. After lipopolysaccharide activated macrophages, the content of IL-6 and TNF- 偽 in supernatant increased gradually. At the concentration of 50 ng / ml of LPS, the levels of inflammatory factors IL-6 and TNF- 偽 in the supernatant stimulated by other concentrations and time of LPS were higher than those in the supernatants stimulated by LPS for 36 h, which indicated that this was the best condition for the activation of macrophages. The results of real-time quantitative PCR and Western blot showed that the expression of FoxO1 transcription factors in activated group was lower than that in non-activated group at m RNA level and protein level, and the difference was statistically significant. The activation group knocked down Foxo1 and the transfection efficiency was detected by flow cytometry and real-time fluorescence quantitative PCR blot method. The results showed that the expression of FoxO1 transcription factors at mRNA and protein levels was 0.47 times higher than that before transfection. The results of successful transfection and Elisa showed that the IL-6 content of activated macrophages was 151.2 ng/ml 鹵49.1 ng / ml, the content of TNF- 偽 was 746.2 ng/ml 鹵68.2 ng / ml, and the content of IL-6 in transfection group increased to 247.2 ng/ml 鹵56 ng / ml TNF- 偽 to 1282 ng/ml 鹵26.7 ng / ml. The levels of inflammatory factor IL-6 and TNF- 偽 in transfection group were significantly higher than those in control group (P < 0.01). Normal macrophages were divided into two groups: one without treatment and the other with low Foxo 1. Then two groups of macrophages were divided into two groups: one group was not treated, the other group was activated by LPS. The relative expression of TGF- 尾 protein in four groups of cells was analyzed by Western blot. The results showed that TGF- 尾 protein was expressed in activated group and siRNA group. The relative expression of TGF- 尾 protein was 0.37 in activated group and 0.54 in siRNA group. It was suggested that activation of macrophages after knocking down Foxo1 could induce the increase of TGF- 尾 factor, which suggested that Fox O1 transcription factor might affect the inflammatory response through TGF- 尾 factor. Conclusion: 1. Fox O1 may be involved in the inflammatory process. 2. The transcription factor Fox O1 may play a negative role in the regulation of inflammatory response. 3. Fox O1 transcription factor may influence the inflammatory response by increasing the expression of TGF- 尾.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R363

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