鞣花酸体外抗红色毛癣菌作用及机制研究

发布时间：2018-08-08 21:52

【摘要】：目的:以红色毛癣菌(Trichophyton rubrum)为研究对象,紫草素和特比奈酚为阳性对照药,探讨地锦草有效成分鞣花酸抗红色毛癣菌活性,研究其对真菌细胞超微结构、细胞凋亡及其细胞生物合成相关基因表达量的影响,阐明其抗真菌作用的机制,为深入研究鞣花酸抗红色毛癣菌靶位和其开发利用奠定基础,为其临床应用治疗皮肤癣菌病提供科学依据。方法:1)采用美国临床实验室标准化委员会(NCCLS)推荐的《产孢丝状真菌的液基稀释法抗真菌药物敏感性试验方案(M38-A),测定地锦草有效成分鞣花酸及其不同提取部位对4株红色毛癣菌(Trichophyton rubrum)、石膏样毛癣菌(Trichophyton mentagrophytes)、白色念珠菌(Candida albicans)、近平滑念珠菌(Candida prapsilosis)临床常见真菌的MIC值,确定其抗真菌谱,筛选出对其敏感的真菌菌株。参考MIC结果,进一步测定鞣花酸对红色毛癣菌细胞生长抑制率。2)鞣花酸作用于红色毛癣菌(T.rubrum)后,按常规方法进行样品处理,利用扫描电子显微镜(Scanning Electron Microscope,SEM),观察其对红色毛癣菌细胞表面形态结构的影响。3)鞣花酸作用于红色毛癣菌后,分离菌丝样品,经过洗涤、染色等样品前处理后,设低(64μg·mL-1)、中(128μg·mL-1)、高(256μg·mL-1)剂量组,采用AinexinV-FITC/PI双染法考察其对红色毛癣菌细胞凋亡的影响。4)鞣花酸作用于红色毛癣菌后,采用RT-PCR方法检测其对真菌细胞膜麦角甾醇生物合成通路有关ERG11、SUB1、MEP4、ERG6等基因表达的影响。结果:1)鞣花酸对念珠菌的平均MIC值为256μg·mL-1、对石膏样毛癣菌的平均MIC值为128μg·mL-1,其中对红色毛癣菌最为敏感,平均MIC值为64μg·mL-1。地锦草有效部位对红色毛癣菌(T.rubrum)、石膏样毛癣菌(T.mentagrophytes)的平均MIC值大于1024μg·mL-1,对近平滑念珠菌(C.parapsilosis)、白色念珠菌(C.albicans)的平均MIC均大于2048μg·mL-1;2)扫描电子显微镜(SEM)下观察,鞣花酸(64μg·mL-1)作用于红色毛癣菌后真菌细胞表面皱缩不平,明显变形膨胀,且可见表层剥离,菌丝破裂,呈粘糊状,细胞内容物溢出;3)鞣花酸作用于红色毛癣菌后,鞣花酸低、中、高浓度的细胞坏死率依次为:16.6%、20.6%、27.3%。4)鞣花酸低、中、高剂量的诱导下MEP4、SUB1和ERG11基因的表达量下调分别为0.1526、0.0726、0.0585,0.2093、0.1331、0.0857,0.0791、0.0423、0.0400等不同浓度的鞣花酸对ERG6的上调表达量分别为2.6950、4.1398、4.8764。差异有显著性(P0.05)。结论:鞣花酸具有显著的抗真菌活性,对浅部致病真菌有较强的抑制作用,其中对红色毛癣菌最敏感。可抑制红色毛癣菌生长,通过破坏真菌细胞膜的结构,诱导其坏死,其机制可能是麦角甾醇生物合成相关的MEP4、SUB1、ERG11基因相对表达量的下调,ERG6基因相对表达量的上调有关。
[Abstract]:Objective: To study the effect of tannin acid on the activity of tannic acid against Trichophyton ringworm (tannic Trichophyton rubrum), the active component of Herba euphorbium and terbiol as the positive control drug, and to study its effect on the cell ultrastructure, cell apoptosis and the expression of cell biosynthesis related genes, and to elucidate the antifungal effect of Trichophyton rubrum. The mechanism provides a scientific basis for the in-depth study of tannic acid against Trichophyton rubrum target and its development and utilization, and provides a scientific basis for its clinical application in the treatment of dermatitis. Method: 1) the test scheme of antifungal susceptibility test (M38-A) of the liquid based dilution method of filamentous fungi (NCCLS) recommended by the American clinical laboratory standardization committee (NCCLS) was used. Tannic acid and its different extraction sites on 4 strains of Trichophyton rubrum (Trichophyton rubrum), Trichophyton mentagrophytes, Candida albicans, and near Candida albicans (Candida prapsilosis) in clinical common fungi were determined and their antifungal spectrum was determined. Bacteria strain. Reference MIC results to further determine the growth inhibition rate of tannic acid to Trichophyton rubrum cell growth rate.2) after tannic acid was treated with Trichophyton rubrum (T.rubrum), the sample was treated by conventional method, and the effect of Scanning Electron Microscope (SEM) on the surface morphology and structure of Trichophyton rubrum cell was observed by scanning electron microscope (.3). After the effect of tannic acid on Trichophyton rubre, the hypha samples were separated, after washing, dyeing and other samples, low (64 G. ML-1), medium (128 G. ML-1), high (256 mu g. ML-1) dose group, AinexinV-FITC/PI double staining method was used to investigate the effect of its effect on cell withering of Trichophyton rubrum.4) tannic acid acting on Trichophyton ringworm (Trichophyton ringworm), using RT-PCR method The effects on the expression of ERG11, SUB1, MEP4 and ERG6 in the ergosterol biosynthesis pathway of fungal cell membrane were detected. Results: 1) the average MIC value of tannic acid to Candida albicans was 256 G. ML-1, and the average MIC value to Trichophyton plaster fungus was 128 u g. ML-1, which was the most sensitive to Trichophyton rubrum, and the average MIC value was 64 mu. The average MIC value of the effective sites for T.rubrum and T.mentagrophytes was greater than 1024 u g. ML-1. The average MIC of Candida albicans (C.parapsilosis) and Candida albicans (C.albicans) was greater than 2048 UU mL-1; 2) scanning electron microscopy (SEM). Tannin acid (64 mu g.) acted upon Trichophyton rubrum. The surface of bacteria cells crinkled uneven, obviously deformed and inflated, and visible surface peeling, mycelium rupture, sticky, cell content spillover; 3) tannic acid was low after tannic acid acted on Trichophyton rubrum, and the high concentration of cell necrosis rate was 16.6%, 20.6%, 27.3%.4) low tannic acid, medium and high dose induced MEP4, SUB1 and ERG11 gene table The up regulation of tannic acid with different concentrations of 0.1526,0.0726,0.0585,0.2093,0.1331,0.0857,0.0791,0.0423,0.0400 and other tannic acids, respectively, was 2.6950,4.1398,4.8764., respectively (P0.05). Conclusion: tannic acid has significant antifungal activity and has a strong inhibitory effect on the superficial pathogenic fungi, in which red is red. Trichophyton Trichophyton is most sensitive. It can inhibit the growth of Trichophyton rubrum and induce necrosis by destroying the structure of fungal cell membrane. The mechanism may be the down regulation of the relative expression of MEP4, SUB1, ERG11 gene related to ergosterol biosynthesis, and up regulation of the relative expression of ERG6 gene.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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