【摘要】：目的:以紅色毛癬菌(Trichophyton rubrum)為研究對(duì)象,紫草素和特比奈酚為陽(yáng)性對(duì)照藥,探討地錦草有效成分鞣花酸抗紅色毛癬菌活性,研究其對(duì)真菌細(xì)胞超微結(jié)構(gòu)、細(xì)胞凋亡及其細(xì)胞生物合成相關(guān)基因表達(dá)量的影響,闡明其抗真菌作用的機(jī)制,為深入研究鞣花酸抗紅色毛癬菌靶位和其開(kāi)發(fā)利用奠定基礎(chǔ),為其臨床應(yīng)用治療皮膚癬菌病提供科學(xué)依據(jù)。方法:1)采用美國(guó)臨床實(shí)驗(yàn)室標(biāo)準(zhǔn)化委員會(huì)(NCCLS)推薦的《產(chǎn)孢絲狀真菌的液基稀釋法抗真菌藥物敏感性試驗(yàn)方案(M38-A),測(cè)定地錦草有效成分鞣花酸及其不同提取部位對(duì)4株紅色毛癬菌(Trichophyton rubrum)、石膏樣毛癬菌(Trichophyton mentagrophytes)、白色念珠菌(Candida albicans)、近平滑念珠菌(Candida prapsilosis)臨床常見(jiàn)真菌的MIC值,確定其抗真菌譜,篩選出對(duì)其敏感的真菌菌株。參考MIC結(jié)果,進(jìn)一步測(cè)定鞣花酸對(duì)紅色毛癬菌細(xì)胞生長(zhǎng)抑制率。2)鞣花酸作用于紅色毛癬菌(T.rubrum)后,按常規(guī)方法進(jìn)行樣品處理,利用掃描電子顯微鏡(Scanning Electron Microscope,SEM),觀察其對(duì)紅色毛癬菌細(xì)胞表面形態(tài)結(jié)構(gòu)的影響。3)鞣花酸作用于紅色毛癬菌后,分離菌絲樣品,經(jīng)過(guò)洗滌、染色等樣品前處理后,設(shè)低(64μg·mL-1)、中(128μg·mL-1)、高(256μg·mL-1)劑量組,采用AinexinV-FITC/PI雙染法考察其對(duì)紅色毛癬菌細(xì)胞凋亡的影響。4)鞣花酸作用于紅色毛癬菌后,采用RT-PCR方法檢測(cè)其對(duì)真菌細(xì)胞膜麥角甾醇生物合成通路有關(guān)ERG11、SUB1、MEP4、ERG6等基因表達(dá)的影響。結(jié)果:1)鞣花酸對(duì)念珠菌的平均MIC值為256μg·mL-1、對(duì)石膏樣毛癬菌的平均MIC值為128μg·mL-1,其中對(duì)紅色毛癬菌最為敏感,平均MIC值為64μg·mL-1。地錦草有效部位對(duì)紅色毛癬菌(T.rubrum)、石膏樣毛癬菌(T.mentagrophytes)的平均MIC值大于1024μg·mL-1,對(duì)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)掃描電子顯微鏡(SEM)下觀察,鞣花酸(64μg·mL-1)作用于紅色毛癬菌后真菌細(xì)胞表面皺縮不平,明顯變形膨脹,且可見(jiàn)表層剝離,菌絲破裂,呈粘糊狀,細(xì)胞內(nèi)容物溢出;3)鞣花酸作用于紅色毛癬菌后,鞣花酸低、中、高濃度的細(xì)胞壞死率依次為:16.6%、20.6%、27.3%。4)鞣花酸低、中、高劑量的誘導(dǎo)下MEP4、SUB1和ERG11基因的表達(dá)量下調(diào)分別為0.1526、0.0726、0.0585,0.2093、0.1331、0.0857,0.0791、0.0423、0.0400等不同濃度的鞣花酸對(duì)ERG6的上調(diào)表達(dá)量分別為2.6950、4.1398、4.8764。差異有顯著性(P0.05)。結(jié)論:鞣花酸具有顯著的抗真菌活性,對(duì)淺部致病真菌有較強(qiáng)的抑制作用,其中對(duì)紅色毛癬菌最敏感。可抑制紅色毛癬菌生長(zhǎng),通過(guò)破壞真菌細(xì)胞膜的結(jié)構(gòu),誘導(dǎo)其壞死,其機(jī)制可能是麥角甾醇生物合成相關(guān)的MEP4、SUB1、ERG11基因相對(duì)表達(dá)量的下調(diào),ERG6基因相對(duì)表達(dá)量的上調(diào)有關(guān)。
[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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285

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