本文關(guān)鍵詞：天然植物抗菌液（PAMs）對皮膚炎癥的抑制作用及初步機制研究　出處：《西南交通大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 天然植物抗菌液 ITS2 皮膚炎癥 銀屑病 黑色素瘤

【摘要】：皮膚類的疾病是影響人身體健康的一大問題,其中以皮膚炎癥疾病和腫瘤疾病最具有代表性。患者往往很難通過常規(guī)的手段被治愈且傳統(tǒng)的治療藥物副作用大也損傷機體,然而中藥毒副作用小,有利于改善病人生存質(zhì)量,因而越來越受到重視。天然植物抗菌液源自我國民間藥用植物,具備多種功效且在民間已經(jīng)被廣泛使用,具有良好的開發(fā)前景和研究意義。本論文利用現(xiàn)代分子生物學(xué)的方法,為抗菌液配方中的原料藥材提供了分子鑒定技術(shù),使其質(zhì)量安全能夠從源頭上得以保證,并且進一步解釋其抗炎作用的分子機制和對皮膚黑色素瘤中的基因調(diào)控情況,從而使抗菌液的藥物安全和分子作用機制更加完善。本文首先對其中的紅花、刺天茄、艾納香和紫草四種關(guān)鍵藥材進行基因組DNA的提取,通過PCR技術(shù)擴增其ITS2序列,分析各種藥材的ITS2序列與其易混物種之間的ITS2序列差異,研究ITS2序列鑒定的能力。結(jié)果表明紅花藥材的ITS2序列種內(nèi)出現(xiàn)了第57位堿基中的C-T突變,艾納香藥材的ITS2序列種內(nèi)出現(xiàn)了第213位堿基中的C-T突變和227位堿基的T-C突變,刺天茄和新疆軟紫草藥材的ITS2序列種內(nèi)未發(fā)現(xiàn)堿基突變;通過對各種藥材的ITS2序列與對應(yīng)的近緣物種的序列差異性分析,發(fā)現(xiàn)與近緣物種的序列均有較大的堿基差異,且差異明顯大于種內(nèi)堿基差異;構(gòu)建進化樹也發(fā)現(xiàn)上述四種藥材的ITS2序列能夠各自聚類為一支,表明ITS2序列的獨特性,表明其可能用來作為鑒定藥材的條形碼序列。其次,開展了體內(nèi)和體外實驗探究PAMs對皮膚炎癥的作用機制。在體外,以TNF-α和IFN-y誘導(dǎo)HaCaT細胞產(chǎn)生炎癥反應(yīng)為模型,PAMs能夠在基因水平和蛋白水平降低MDC、IL-8和IL-6的表達且與模型組有顯著的差異。在體內(nèi),PAMs也能夠顯著性改善咪喹莫特誘導(dǎo)的小鼠銀屑病皮膚損傷,通過每日的觀察和評分發(fā)現(xiàn),PAMs組和陽性組地塞米松均能夠有效的抑制鱗屑、紅斑和棘層肥厚的形成;通過HE病理切片分析,也表明PAMs組有對損傷的恢復(fù)有顯著效果;對背部皮損組織的基因分析表明PAMs能夠顯著性下調(diào)IL-8、TNF-α、IC4M-1和IL-23的表達,小鼠血清中TNF-α的含量也被顯著性的降低;免疫組化分析表明,與細胞增值相關(guān)的蛋白Ki67和與銀屑病相關(guān)的蛋白ICAM-1的表達也被顯著性抑制。最后又通過細胞和動物組織的免疫熒光染色發(fā)現(xiàn)PAMs能夠顯著性抑制NF-κB信號通路的激活。這些結(jié)果從形態(tài)學(xué)和分子水平共同分析了 PAMs對皮膚炎癥的作用機制,使其有望成為一個潛在的治療銀屑病類皮膚炎癥的藥物,同時也為臨床藥物研究提供參考價值。隨后,我們又用PAMs處理小鼠黑色瘤B16細胞24 h,通過轉(zhuǎn)錄組測序分析空白組和藥物組基因差異表達情況,統(tǒng)計結(jié)果顯示PAMs處理后有919個基因上調(diào),1412個基因下調(diào),并對部分上調(diào)和下調(diào)基因通過qRT-PCR進行驗證和功能分析,進一步從基因水平揭示PAMs對黑色素瘤的作用機制。
[Abstract]:The disease of the skin is a major problem affecting the health of the human body, among which skin inflammation and tumor disease are the most representative. Patients are often difficult to be cured by conventional means, and the traditional side effects of treatment are also harmful to the body. However, the side effects of traditional Chinese medicine are small, which is conducive to improving the quality of life of patients. Natural plant antibacterial liquid is the source of Chinese folk medicinal plants. It has many functions and has been widely used in the folk. It has a good development prospect and research significance. In this paper, using modern molecular biology method, provides molecular identification of antibacterial liquid as raw material in the formulation, the quality and safety can be guaranteed from the source, and further explain the molecular mechanism of the anti-inflammatory effect of cutaneous melanoma in gene regulation, so that the function of drug safety and molecular mechanism the antibacterial liquid is more perfect. Firstly, the extraction of safflower, Solanum khasianum, Blumea and Lithospermum four key medicinal materials of genomic DNA, the ITS2 sequence was amplified by PCR technology, ITS2 sequence analysis of a variety of herbs and mixed ITS2 sequence differences between species, identification of ITS2 sequence can force. The results show that the ITS2 sequence of Flos Carthami appeared in fifty-seventh bases in the C-T mutation, ITS2 sequence for Blumea herbs appeared in the 213rd base of the 227 base C-T mutation and T-C mutation, Solanum khasianum and Xinjiang soft puccoon herbs ITS2 sequences have been found by sequence mutation; different species of ITS2 sequence and corresponding to the analysis of a variety of herbs, and found the species sequence has great base difference, and the difference is significantly greater than the nucleotide differences within species; the phylogenetic tree also showed that ITS2 sequence of the four kinds of Chinese herbal medicines can respectively cluster as a show, unique ITS2 sequence, suggesting that it may be used as a bar code sequence identification of medicinal materials. Secondly, in vivo and in vitro experiments have been carried out to explore the mechanism of PAMs on skin inflammation. In vitro, TNF- and IFN-y induce HaCaT cells to produce inflammatory response. PAMs can reduce the expression of MDC, IL-8 and IL-6 at the gene level and protein level, and has significant difference with the model group. In vivo, PAMs can significantly improve the mice psoriasis skin damage induced by imiquimod, daily observation and scoring, PAMs group and positive group dexamethasone can effectively inhibit scaling, erythema and acanthosis formed by HE; pathological analysis also shows that the PAMs group has significant effect on the injury recovery; analysis of the lesion on the back of the gene showed that PAMs could significantly downregulate IL-8 expression, TNF-, IC4M-1 and IL-23 alpha, alpha TNF- content in mouse serum was significantly decreased; immunohistochemical analysis showed that expression and cell proliferation related protein Ki67 and protein ICAM-1 is associated with psoriasis was significantly inhibited. Finally, it was found that PAMs could significantly inhibit the activation of NF- kappa B signaling pathway through immunofluorescence staining in cell and animal tissues. These results jointly analyzed the mechanism of PAMs on skin inflammation from morphological and molecular level, making it possible to become a potential drug for psoriatic skin inflammation, and also provide a reference for clinical drug research. Then, we used PAMs treated mice melanoma B16 cells for 24 h, the expression of the difference between the blank group and drug group genes by transcriptome sequencing, statistics showed that after the treatment of PAMs with 919 genes up-regulated and 1412 genes were down regulated, and some up-regulated and down regulated genes by qRT-PCR analysis and verification function, further reveal the mechanism of PAMs melanoma from gene level.
【學(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2;R285.5

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