本文選題：β-胡蘿卜素 + RAW264.7細(xì)胞��； 參考：《吉林農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：炎癥反應(yīng)的發(fā)生發(fā)展與斷奶仔豬的腸道炎癥以及多種疾病的產(chǎn)生密切相關(guān),而甾體和非甾體這兩種傳統(tǒng)的抗炎物質(zhì)在緩解炎癥的同時(shí)存在極大的副作用,所以,尋求一種新型、高效、副作用小的緩解炎癥的物質(zhì)很有意義。作為一種天然類胡蘿卜素,β-胡蘿卜素是重要的維生素A源,具有抗氧化、促進(jìn)細(xì)胞間隙連接通訊、提高免疫力等多種生物學(xué)功能。而NF-κB是炎癥中一個(gè)重要的核轉(zhuǎn)錄因子,在受到脂多糖的刺激時(shí),會(huì)誘導(dǎo)相關(guān)炎癥因子的釋放。所以,本研究以脂多糖誘導(dǎo)的巨噬細(xì)胞為炎癥模型,探討β-胡蘿卜素對(duì)其炎癥因子的作用以及與NF-κB通路的關(guān)系。本研究首先用MTT法檢測(cè)細(xì)胞活性,確定β-胡蘿卜素、LPS的最佳添加濃度和時(shí)間;熒光定量PCR、ELISA檢測(cè)主要炎癥因子IL-1β、IL-6、TNF-α的mRNA相對(duì)表達(dá)量和在細(xì)胞中的含量,建立細(xì)胞炎癥模型,分析β-胡蘿卜素對(duì)炎癥因子的影響。然后,熒光定量PCR、Western Blot檢測(cè)NF-κB p65 mRNA、蛋白的相對(duì)表達(dá)量,分析β-胡蘿卜素對(duì)NF-κB通路的影響。最后,綜合運(yùn)用以上方法,分析LPS+PDTC+β-胡蘿卜素和LPS+PDTC組對(duì)炎癥因子、NF-κB p65的影響。結(jié)果如下:在β-胡蘿卜素對(duì)炎癥因子的研究中,發(fā)現(xiàn)β-胡蘿卜素最佳添加濃度梯度和時(shí)間分別為20、40、80、160μmol/L和3 h,LPS最佳添加濃度和時(shí)間分別為5μg/mL和24 h。5μg/mL的LPS誘導(dǎo)巨噬細(xì)胞24 h后,用不同濃度的β-胡蘿卜素處理細(xì)胞3 h可極顯著降低炎癥因子IL-1β、IL-6、TNF-α的mRNA相對(duì)表達(dá)量(P0.01);對(duì)炎癥因子在細(xì)胞中的含量有顯著降低的趨勢(shì),提示β-胡蘿卜素對(duì)LPS誘導(dǎo)的巨噬細(xì)胞的炎癥因子有抑制作用。在β-胡蘿卜素對(duì)NF-κB通路的研究中,發(fā)現(xiàn)PDTC與LPS共同處理巨噬細(xì)胞24 h后,與LPS組相比,可顯著降低NF-κB p65基因、蛋白的相對(duì)表達(dá)量(P0.05),極顯著降低炎癥因子IL-1β、IL-6、TNF-α的mRNA相對(duì)表達(dá)量(P0.01),減少炎癥因子在細(xì)胞中的含量,提示在LPS誘導(dǎo)的巨噬細(xì)胞中,抑制NF-κB p65的表達(dá)可以抑制炎癥因子。在NF-κB通路的特異性研究中,發(fā)現(xiàn)不同濃度的β-胡蘿卜素可顯著降低LPS誘導(dǎo)的NF-κB p65基因、蛋白的相對(duì)表達(dá)量(P0.05),說(shuō)明在LPS誘導(dǎo)的巨噬細(xì)胞中,β-胡蘿卜素通過(guò)抑制NF-κB p65的表達(dá)可以抑制炎癥因子;LPS+PDTC+β-胡蘿卜素組對(duì)炎癥因子的抑制作用比LPS+PDTC組明顯,但兩組對(duì)NF-κB p65的表達(dá)差異不顯著(P0.05),提示NF-κB通路不是β-胡蘿卜素抑制炎癥因子的特異性的通路。依據(jù)結(jié)果分析歸納總結(jié),本研究發(fā)現(xiàn)β-胡蘿卜素對(duì)LPS誘導(dǎo)的巨噬細(xì)胞的炎癥因子有抑制作用,此抑制作用與NF-κB通路密切相關(guān)。
[Abstract]:The occurrence and development of inflammatory reaction is closely related to the intestinal inflammation of weaning piglets and the production of many diseases, while the two traditional anti-inflammatory substances, steroidal and non-steroidal, have great side effects while alleviating inflammation, so we seek a new type of inflammation. Highly effective, with little side effects, anti-inflammatory substances are significant. As a kind of natural carotenoid, 尾 -carotene is an important vitamin A source, which has many biological functions, such as anti-oxidation, promoting intercellular junctional communication, enhancing immunity and so on. NF- 魏 B is an important nuclear transcription factor in inflammation. When stimulated by lipopolysaccharide, NF- 魏 B induces the release of related inflammatory factors. In this study, macrophages induced by lipopolysaccharide were used as inflammatory model to investigate the effect of 尾 -carotene on inflammatory cytokines and the relationship between 尾 -carotene and NF- 魏 B pathway. In this study, MTT assay was used to determine the optimal concentration and time of 尾 -carotene lipopolysaccharide (尾 -carotene), and the relative expression of mRNA and the content of TNF- 偽 in cells were detected by fluorescence quantitative PCR Elisa to establish a cellular inflammatory model. To analyze the effect of 尾-carotene on inflammatory factors. Then, the relative expression of NF- 魏 B p65 mRNAs and the effect of 尾 -carotene on NF- 魏 B pathway were analyzed by fluorescence quantitative Blot. Finally, the effects of LPS PDTC 尾 -carotene and LPS PDTC on the inflammatory factor NF- 魏 B p65 were analyzed. The results were as follows: in the study of inflammatory factors induced by 尾 -carotene, it was found that the optimal concentration gradient and time of 尾 -carotene were 2040 mol/L and 80160 渭 mol/L, respectively, and the optimal concentration and time of LPS were 5 渭 g/mL and 24h.5 渭 g/mL, respectively. Treated with different concentrations of 尾 -carotene for 3 h, the relative expression of IL-1 尾 -IL-6TNF- 偽 mRNA was significantly decreased, and the content of inflammatory factors in the cells was significantly decreased. These results suggest that 尾-carotene inhibits the inflammatory cytokines of macrophages induced by LPS. In the study of 尾 -carotene on NF- 魏 B pathway, it was found that after co-treatment of PDTC and LPS for 24 h, NF- 魏 B p65 gene was significantly decreased compared with LPS group. The relative expression of P0.05 protein significantly decreased the relative expression of IL-1 尾 -IL-6TNF- 偽 mRNA and decreased the content of inflammatory factors in the cells. It suggested that the inhibition of NF- 魏 B p65 expression in macrophages induced by LPS could inhibit the expression of inflammatory factors. In the specific study of NF- 魏 B pathway, it was found that 尾 -carotene at different concentrations could significantly reduce the NF- 魏 B p65 gene induced by LPS. The relative expression of P0.05 protein indicated that 尾 -carotene could inhibit the expression of NF- 魏 B p65 by inhibiting the expression of NF- 魏 B p65 in macrophages induced by LPS. The inhibitory effect of 尾 -carotene on inflammatory cytokines in LPs PDTC 尾 -carotene group was more obvious than that in LPS PDTC group. However, there was no significant difference in the expression of NF- 魏 B p65 between the two groups, suggesting that the NF- 魏 B pathway was not a specific pathway for 尾 -carotene to inhibit inflammatory factors. According to the results, it was found that 尾 -carotene inhibited the inflammatory cytokines of macrophages induced by LPS, which was closely related to the NF- 魏 B pathway.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.28

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本文編號(hào)：1785505