淫羊藿和巴戟天中药提取物对中国恒河猴不同极化条件下原代巨噬细胞基因表达影响的研究

发布时间：2018-06-02 00:15

本文选题：巨噬细胞 + 淫羊藿　；参考：《广州中医药大学》2016年硕士论文

【摘要】：目的：探讨中药淫羊藿、巴戟天提取物对恒河猴单核衍生的巨噬细胞在不同极化条件下基因表达的影响,分析这两种中药可能的免疫调节的作用机制。方法：密度梯度离心法分离中国恒河猴外周血单个核细胞(PBMCs),采用贴壁法分离出单核细胞,并以粒细胞巨噬细胞集落刺激因子(GM-CSF)刺激5天以使单核细胞分化成巨噬细胞,随后不加入极化因子形成MO型巨噬细胞,以干扰素γ (IFN-γ)或地塞米松继续培养2天诱导巨噬细胞极化为M1和M2c表型,同时分别加入淫羊藿、巴戟天提取物进行干预,随后提取mRNA并逆转录后,以实时荧光定量PCR检测CD4、 FoxP3、CTLA-4、CCR5、IDO、IL-10、TGF-β等基因表达量,以GAPDH为参照基因,与空白组进行对比。结果：与空白对照组相比,加入巴戟天提取物培养的MO巨噬细胞基因表达上调不明显；经淫羊藿提取物培养的MO巨噬细胞,CCR5基因表达量上调4.21倍,TGF-β上调7.66倍,而CD4、CTLA-4、FoxP3、IL-10、IDO的基因表达无明显变化。经淫羊藿提取物刺激的M1型巨噬细胞,IL-10的表达量上调11.83倍,FoxP3上调4.55倍,IDO、CCR5、 CD4、CTLA-4无明显变化, TGF-β未测出；而由巴戟天提取物刺激的M1型巨噬细胞,CTLA-4基因表达量上调3.22倍,FoxP3上调3.69倍,CCR5、CD4、IL-10、IDO均无明显变化,TGF-β未测出。经淫羊藿提取物培养刺激的M2c型巨噬细胞,CCR5基因增加5.94倍,TGF-β基因增加3.53倍,CTLA-4、FoxP3、IDO、IL-10等基因无明显变化,经巴戟天提取物刺激的M2c型巨噬细胞,CCR5基因增加6.43倍,CTLA-4基因增加3.28倍,IL-10基因增加3.76倍,TGF-β基因增加11.05倍,CD4基因增加3.77倍,FoxP3和IDO两个基因无明显变化。结论：经淫羊藿提取物培养的MO型巨噬细胞,则能够上调CCR5和TGF-β的基因表达,呈现出促炎、促纤维化的作用。经淫羊藿和巴戟天提取物培养后,M1型巨噬细胞能上调IL-10、FoxP3、CTLA-4等抑炎、抑纤维化基因表达,减轻炎症反应和组织纤维化,有利于组织结构的维持和保护。通过淫羊藿和巴戟天提取物培养后,M2c型巨噬细胞的CCR5、CD4和TGF-β基因表达大量增加。表现出促炎和促纤维化的免疫调节作用。总结本实验的结果,淫羊藿和巴戟天提取物通过对不同极化条件下的巨噬细胞基因表达影响的不同,发挥促炎和抑炎、促纤维化和抑制纤维化的双向调节作用。
[Abstract]:Aim: to investigate the effect of Herba Epimedii and Morinda officinalis extract on gene expression of monocyte derived macrophages in rhesus monkey under different polarization conditions and to analyze the possible immunomodulatory mechanism of these two herbs. Methods: PBMCs were isolated from the peripheral blood mononuclear cells of Chinese rhesus monkeys by density gradient centrifugation. Monocytes were isolated by adherent method and stimulated with granulocyte macrophage colony stimulating factor (GM-CSF) for 5 days to differentiate monocytes into macrophages. No polarizing factor was added to form MO type macrophages, and the macrophages were further cultured with interferon 纬 (IFN- 纬) or dexamethasone for 2 days to induce the polarization of macrophages into M1 and M2c phenotypes, and the extracts of Herba Epimedii and Morinda officinalis were added for intervention, respectively. Then mRNA was extracted and reverse transcripted, and the expression of CD4, FoxP3 CTLA-4 and CCR5 IDO5 IL-10TGF- 尾 was detected by real-time fluorescence quantitative PCR. GAPDH was used as the reference gene and compared with the control group. Results: compared with the blank control group, the gene expression of MO macrophages cultured with the extract of Morinda officinalis was not significantly up-regulated, and the expression of CCR5 gene was increased by 4.21 times and 7.66 times by TGF- 尾 in the MO macrophages cultured with Herba Epimedii extract. There was no significant change in the expression of IL-10 IDO gene in CD _ 4 ~ + CTLA-4 ~ (- 4) FoxP _ (3) and FoxP _ (3). The expression of IL-10 in M 1 macrophages stimulated by Herba Epimedii extract was increased by 11.83 times and FoxP3 by 4.55 times, while CD4 CTLA-4 did not change, but TGF- 尾 was not detected. However, the expression of CTLA-4 gene in M1 macrophages stimulated by Morinda officinalis extract increased 3.22 times and FoxP3 increased 3.69 times. The CCR5 gene of M 2c macrophages stimulated by Herba Epimedii extract increased 5.94 times and the TGF- 尾 gene increased 3.53 times. The increase of CCR5 gene in M 2c macrophages stimulated by Morinda officinalis extract was 6.43 times. CTLA-4 gene increased 3.28 times and IL-10 gene increased 3.76 times. TGF- 尾 gene increased 11.05 times and CD4 gene increased 3.77 times. There was no significant change in two genes: FoxP3 and IDO. Conclusion: the MO type macrophages cultured with Herba Epimedii extract can up-regulate the gene expression of CCR5 and TGF- 尾, which can promote inflammation and fibrosis. After culture of Herba Epimedii and Morinda officinalis extracts, M 1 macrophages could up-regulate the expression of anti-inflammatory genes, such as IL-10, FoxP3, CTLA-4, reduce inflammatory reaction and tissue fibrosis, and help to maintain and protect the tissue structure. After cultured with Herba Epimedii and Morinda officinalis extract, the expression of CCR5, CD4 and TGF- 尾 genes in M2c macrophages was significantly increased. It showed immunomodulatory effect of promoting inflammation and fibrosis. The results showed that the extracts of Herba Epimedii and Morinda officinalis had different effects on the gene expression of macrophages under different polarization conditions, and played a bidirectional role in promoting inflammation and inhibiting inflammation, promoting fibrosis and inhibiting fibrosis.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R285

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