天然植物抗菌液（PAMs）对皮肤炎症的抑制作用及初步机制研究

发布时间：2017-12-27 17:18

本文关键词：天然植物抗菌液（PAMs）对皮肤炎症的抑制作用及初步机制研究　出处：《西南交通大学》2017年硕士论文　论文类型：学位论文

【摘要】：皮肤类的疾病是影响人身体健康的一大问题,其中以皮肤炎症疾病和肿瘤疾病最具有代表性。患者往往很难通过常规的手段被治愈且传统的治疗药物副作用大也损伤机体,然而中药毒副作用小,有利于改善病人生存质量,因而越来越受到重视。天然植物抗菌液源自我国民间药用植物,具备多种功效且在民间已经被广泛使用,具有良好的开发前景和研究意义。本论文利用现代分子生物学的方法,为抗菌液配方中的原料药材提供了分子鉴定技术,使其质量安全能够从源头上得以保证,并且进一步解释其抗炎作用的分子机制和对皮肤黑色素瘤中的基因调控情况,从而使抗菌液的药物安全和分子作用机制更加完善。本文首先对其中的红花、刺天茄、艾纳香和紫草四种关键药材进行基因组DNA的提取,通过PCR技术扩增其ITS2序列,分析各种药材的ITS2序列与其易混物种之间的ITS2序列差异,研究ITS2序列鉴定的能力。结果表明红花药材的ITS2序列种内出现了第57位碱基中的C-T突变,艾纳香药材的ITS2序列种内出现了第213位碱基中的C-T突变和227位碱基的T-C突变,刺天茄和新疆软紫草药材的ITS2序列种内未发现碱基突变;通过对各种药材的ITS2序列与对应的近缘物种的序列差异性分析,发现与近缘物种的序列均有较大的碱基差异,且差异明显大于种内碱基差异;构建进化树也发现上述四种药材的ITS2序列能够各自聚类为一支,表明ITS2序列的独特性,表明其可能用来作为鉴定药材的条形码序列。其次,开展了体内和体外实验探究PAMs对皮肤炎症的作用机制。在体外,以TNF-α和IFN-y诱导HaCaT细胞产生炎症反应为模型,PAMs能够在基因水平和蛋白水平降低MDC、IL-8和IL-6的表达且与模型组有显著的差异。在体内,PAMs也能够显著性改善咪喹莫特诱导的小鼠银屑病皮肤损伤,通过每日的观察和评分发现,PAMs组和阳性组地塞米松均能够有效的抑制鳞屑、红斑和棘层肥厚的形成;通过HE病理切片分析,也表明PAMs组有对损伤的恢复有显著效果;对背部皮损组织的基因分析表明PAMs能够显著性下调IL-8、TNF-α、IC4M-1和IL-23的表达,小鼠血清中TNF-α的含量也被显著性的降低;免疫组化分析表明,与细胞增值相关的蛋白Ki67和与银屑病相关的蛋白ICAM-1的表达也被显著性抑制。最后又通过细胞和动物组织的免疫荧光染色发现PAMs能够显著性抑制NF-κB信号通路的激活。这些结果从形态学和分子水平共同分析了 PAMs对皮肤炎症的作用机制,使其有望成为一个潜在的治疗银屑病类皮肤炎症的药物,同时也为临床药物研究提供参考价值。随后,我们又用PAMs处理小鼠黑色瘤B16细胞24 h,通过转录组测序分析空白组和药物组基因差异表达情况,统计结果显示PAMs处理后有919个基因上调,1412个基因下调,并对部分上调和下调基因通过qRT-PCR进行验证和功能分析,进一步从基因水平揭示PAMs对黑色素瘤的作用机制。
[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.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;R285.5

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