【摘要】：目的：本研究主要研究法尼醇對(duì)白念珠菌生物膜藥物易感性、形態(tài)、麥角甾醇的作用,探討法尼醇作為一種抗微生物化學(xué)活性分子對(duì)白念珠菌生物膜耐藥性的作用。 方法：本研究分為白念珠菌標(biāo)準(zhǔn)株未處理組(標(biāo)準(zhǔn)株組、對(duì)照組)、標(biāo)準(zhǔn)株法尼醇處理組、耐藥株未處理組(耐藥株組)及耐藥株法尼醇處理組,針對(duì)白念珠菌不同時(shí)相(6h、12h、24h、36h)生物膜進(jìn)行研究。。研究采用下述方法探討法尼醇對(duì)白念珠菌生物膜耐藥相關(guān)生物學(xué)特點(diǎn)的作用：XTT分析檢測(cè)不同濃度法尼醇對(duì)白念珠菌生物膜的抑制作用；XTT分析用于不同生物膜時(shí)相法尼醇作用于白念珠菌生物膜的藥物易感性實(shí)驗(yàn)；掃描電鏡觀察比較法尼醇處理后白念珠菌菌體形態(tài)的改變,激光共聚焦比較各組生物膜厚度；液相色相質(zhì)譜聯(lián)用儀對(duì)各組細(xì)胞質(zhì)膜成分進(jìn)行檢測(cè)并比較。 結(jié)果：實(shí)驗(yàn)一：在生物膜形成6、12h,標(biāo)準(zhǔn)株和耐藥株高濃度法尼醇處理組抑菌率比低濃度法尼醇處理組高；而標(biāo)準(zhǔn)株法尼醇處理組和耐藥株法尼醇處理組的24h生物膜在法尼醇處理濃度為0-150μM時(shí),抑菌率沒有明顯升高。標(biāo)準(zhǔn)株法尼醇處理組在法尼醇處理濃度為50μM時(shí),四個(gè)時(shí)相生物膜的抑菌率無明顯改變；在法尼醇處理濃度為100、150、200、250、300μM時(shí),同一法尼醇濃度作用于標(biāo)準(zhǔn)株法尼醇處理組四個(gè)時(shí)相生物膜,抑制作用隨時(shí)間延長(zhǎng)而減弱。在同一生物膜時(shí)相,同一濃度法尼醇對(duì)耐藥株生物膜的抑制作用弱于標(biāo)準(zhǔn)株。 實(shí)驗(yàn)二：XTT法檢測(cè)白念珠菌(標(biāo)準(zhǔn)株組、標(biāo)準(zhǔn)株法尼醇處理組、耐藥株組及耐藥株法尼醇處理組)生物膜MIC,發(fā)現(xiàn)標(biāo)準(zhǔn)株法尼醇處理組生物膜SMICgo在6h、12h、24h、36h均低于標(biāo)準(zhǔn)株組,且差異在36h變��；耐藥株法尼醇處理組生物膜SMIC80在6h、12h、24h低于耐藥株組,在36h,四組生物膜SMIC80均無明顯差異。耐藥株生物膜SMIC80在6h、12h、24h及36h均高于標(biāo)準(zhǔn)株生物膜。 實(shí)驗(yàn)三：掃描電鏡觀察四組白念珠菌生物膜的附著狀態(tài)及菌體形態(tài),發(fā)現(xiàn)法尼醇處理使白念珠菌菌絲形成較晚且菌絲變短,這個(gè)變化在6h、12h和24h時(shí)較為明顯,在36h則變化不明顯。法尼醇使白念珠菌菌體表面皺縮和形成凹陷,在生物膜早期及中期尤為顯著,隨著生物膜的成熟,凹陷逐漸變少,至生物膜成熟期凹陷已經(jīng)較為少見。同一時(shí)段下耐藥株法尼醇處理組菌體表面的凹陷少于標(biāo)準(zhǔn)株法尼醇處理組。同一時(shí)段下耐藥株組細(xì)胞外基質(zhì)多于標(biāo)準(zhǔn)株法尼醇處理組。激光共聚焦觀察發(fā)現(xiàn),在生物膜形成6h、12h、24h,標(biāo)準(zhǔn)株組生物膜厚度高于標(biāo)準(zhǔn)株法尼醇處理組,耐藥株組生物膜厚度高于耐藥株法尼醇處理組。在36h,法尼醇處理后生物膜厚度未顯著降低。在6h及12h,耐藥株生物膜厚度高于標(biāo)準(zhǔn)株。 實(shí)驗(yàn)四：HPLC檢測(cè)分析麥角甾醇結(jié)果顯示,在生物膜形成6h、12h、24h,同一時(shí)段法尼醇處理組生物膜麥角甾醇含量低于未處理組,標(biāo)準(zhǔn)株法尼醇處理組生物膜麥角甾醇含量均低于標(biāo)準(zhǔn)株組,耐藥株法尼醇處理組生物膜麥角甾醇含量均低于耐藥株組,且耐藥株組麥角甾醇含量高于標(biāo)準(zhǔn)株組。 隨著生物膜的逐漸成熟,標(biāo)準(zhǔn)株組生物膜麥角甾醇含量逐漸升高,標(biāo)準(zhǔn)株法尼醇處理組24h生物膜麥角甾醇含量較6h生物膜升高,標(biāo)準(zhǔn)株法尼醇處理組36h生物膜麥角甾醇含量較24h生物膜顯著降低；耐藥株組24h及36h生物膜麥角甾醇含量較6h生物膜顯著降低。 結(jié)論： 實(shí)驗(yàn)一：法尼醇對(duì)白念珠菌生物膜的抑制作用與法尼醇濃度及白念珠菌的生物膜時(shí)相相關(guān)。在生物膜早期及中期,高濃度法尼醇對(duì)生物膜的抑制效果比低濃度法尼醇強(qiáng)；在生物膜成熟期,法尼醇對(duì)白念珠菌生物膜無明顯抑制作用。法尼醇對(duì)白念珠菌各時(shí)期生物膜的抑制作用隨生物膜成熟而減弱,對(duì)早期生物膜抑制作用最強(qiáng)。 實(shí)驗(yàn)二：法尼醇可以抑制白念珠菌生物膜耐藥性,且法尼醇對(duì)早期及中期生物膜耐藥性的抑制作用明顯,對(duì)晚期成熟生物膜耐藥性的抑制作用不如早期及中期明顯。 實(shí)驗(yàn)三：研究結(jié)果顯示法尼醇抑制白念珠菌胞外基質(zhì)的形成,造成菌體表面損傷,抑制菌絲的形成和伸長(zhǎng),影響生物膜的形成,降低生物膜厚度。法尼醇的這些作用對(duì)早期和中期生物膜較明顯,對(duì)成熟期生物膜無明顯作用。 實(shí)驗(yàn)四：麥角甾醇含量與耐藥性呈正相關(guān)。法尼醇能降低早期及中期白念珠菌生物膜中的麥角甾醇含量,推測(cè)法尼醇抑制白念珠菌生物膜耐藥性的機(jī)制是法尼醇降低生物膜中的麥角甾醇含量,使細(xì)胞質(zhì)膜穩(wěn)定性下降,對(duì)唑類藥物通透性改變,從而導(dǎo)致白念珠菌對(duì)唑類藥物的耐藥性下降。
[Abstract]:Objective: To study the effect of farnesol on the drug susceptibility, morphology and ergoline, and to explore the effect of farnesol as an anti-microbial chemical active molecule on the drug resistance of Candida albicans. Methods: The study was divided into two groups: non-treated group (standard plant group, control group), standard strain FENOL treatment group, resistant strain non-treatment group (drug resistant strain group) and drug-resistant strain method, and the non-simultaneous phase of Candida albicans (6h, 12h,24). h,36 h) Biofilm ............................................................................................................ Application of XTT in the study of drug susceptibility to the membrane of Candida albicans in different biological membrane phases; the changes of the morphology of Candida albicans after the treatment with the method of scanning electron microscopy; and the thickness of the biofilms in each group were compared by laser confocal. The plasma membrane components of each group were detected and compared by a liquid phase-phase-phase-mass-mass spectrometer. The results were as follows:1. The inhibition rate of the high-concentration farnesol treatment group was higher than that of the low-concentration farnesol treatment group in the biofilm formation of 6,12 h, the standard strain and the drug-resistant strain. The inhibition rate is not clear at the time of the. m The results showed that the inhibition rate of the four-phase biofilms was not significantly changed when the treatment concentration of the normal plant was 50. m u.M. The concentration of the same method was 100,150,200,250,300. m u.M, and the concentration of the same method in the treatment group of the normal strain was 100,150,200,250 and 300. m Biofilm, inhibition over time In the same biological membrane phase, the inhibition of the same concentration of farnesol on the biofilms of drug-resistant strains was weaker than that of the same biofilms. Standard strain was used to test the MIC of the biofilms of Candida albicans (standard plant group, standard strain Fanyol treatment group, drug-resistant strain group and drug-resistant strain Fanyol treatment group) by means of XTT method, and it was found that the biofilms SMICgo of the standard strain method were lower than that of the standard plant group at 6 h,12 h,24 h and 36 h, and the difference was The treatment group biofilm SMIC80 was lower than that of the resistant strain group at 6 h,12 h and 24 h. There was no significant difference. The biological membrane of the resistant strain SMIC80 was higher than the standard in 6 h,12 h,24 h and 36 h. The attachment status and the cell morphology of the four groups of Candida albicans biofilm were observed by scanning electron microscope. The results showed that the hyphae of Candida albicans were formed later and the hyphae became shorter at 6 h,12 h and 24 h, and at 36 h,12 h and 24 h, In that early and mid-term of the biofilms, with the mature of the biofilms, the depression became less, and the maturation of the biofilms was concave. The depression of the cell surface of the drug-resistant strain in the same period is less than that of the standard. The extracellular matrix of drug-resistant strains in the same period was more than that of the standard. It was found that the membrane thickness of the standard plant group was higher than that of the standard method, the thickness of the biofilm was higher than that of the standard strain, the thickness of the biofilm was higher than that of the standard strain, the thickness of the biofilm was higher than that of the standard strain. Plant farnesol treatment group. After 36 h, the biofilms were treated with farnesol. The thickness was not significantly reduced. In 6 h and 12 h, the biofilms of the resistant strains The results showed that the content of ergoline in the treatment group was lower than that of the untreated group in the same period of time, and the results showed that the content of the ergoline in the treatment group was lower than that of the untreated group. The content of ergoline was lower than that of the standard plant, and the content of ergoline in the treatment group of the drug-resistant strain was lower than that of the resistant strains. The alcohol content was higher than that of the standard plant group. With the gradual maturation of the biofilms, the content of ergoline in the standard plant group was gradually increased, and the content of the ergoline in the standard strain method of the treatment group was higher than that of the 6-h biofilms. The content of ergoline in the treatment group of the standard strain in the treatment group for 36h was higher than that of the standard plant group. The 24-hour and 36-hour biofilms of the drug-resistant strains were significantly lower than that of the 24-hour biofilms. The amount is 6 Conclusion: The inhibitory effect of farnesol on the biological membrane of Candida albicans and the inhibition of farnesol In the early and mid-term of the biofilms, the inhibition effect of the high-concentration farnesol on the biofilms is stronger than that of the low-concentration farnesol; in the mature period of the biofilms, the farnesol The inhibitory effect of farnesol on the biofilms of Candida albicans in various stages of Candida albicans was observed with the maturation of the biofilms. The results showed that the inhibition of the drug resistance of Candida albicans biofilm was inhibited, and the inhibition of the drug resistance of farnesol on the early and medium-term biofilm was evident. The results showed that the formation of extracellular matrix of Candida albicans was inhibited, the surface of the cells was damaged, and the formation of hyphae was inhibited. elongation, the formation of biofilm, and the reduction of biofilm thickness. These effects of farnesol on early and mid-term The biofilm is more obvious and has no obvious effect on the mature membrane of the mature period. The experimental results showed that the content of ergoline was positively correlated with the drug resistance. The results showed that the content of ergoline in the early and medium-term Candida albicans biofilm was reduced, and the mechanism of the inhibition of the drug resistance of the farnesol on the biofilms of Candida albicans was the decrease of the content of ergoline in the biofilm. the stability of the cytoplasmic membrane is reduced, the permeability of the plasma membrane is modified,
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R780.2

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