本文選題：抗菌肽 + 17BIPHE2��； 參考：《寧夏醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的研究抗菌肽17BIPHE2單獨使用及聯(lián)合抗生素對金黃色葡萄球菌(Staphylococcus aureus,SA)和銅綠假單胞菌(Pseudomonas aeruginosa,PA)生物被膜的抑制作用,并初步探討抗菌肽17BIPHE2作用細菌后對生物被膜相關(guān)基因轉(zhuǎn)錄水平的影響,為治療金黃色葡萄球菌、銅綠假單胞菌生物被膜相關(guān)感染及藥物研發(fā)提供了一個新思路。方法1.采用剛果紅平板測試法(金黃色葡萄球菌)和結(jié)晶紫染色法評估受試菌形成生物被膜的能力;2.微量肉湯稀釋法和瓊脂平板測試法測定金黃色葡萄球菌、銅綠假單胞菌最小抑菌濃度(MIC)、最小殺菌濃度(MBC);3.利用生物被膜早期黏附實驗觀察17BIPHE2單獨使用及聯(lián)合抗生素對生物被膜黏附階段的影響;4.利用生物被膜形成抑制實驗觀察17BIPHE2單獨使用及聯(lián)合抗生素對生物被膜形成階段的影響;5.通過掃描電子顯微鏡(SEM)觀察17BIPHE2單獨使用及聯(lián)合抗生素是否具有清除成熟階段生物被膜的作用;6.采用激光共聚焦激光掃描顯微鏡(LSCM)觀察17BIPHE2是否具有殺傷成熟銅綠假單胞菌生物被膜內(nèi)細菌的作用;7.利用Real-Time PCR分析17BIPHE2作用細菌后對生物被膜相關(guān)基因轉(zhuǎn)錄水平的影響。結(jié)果1.經(jīng)剛果紅平板測試法和結(jié)晶紫染色法評估臨床分離株SA 05具有較強形成生物被膜能力,臨床分離株SA 07不具有形成生物被膜的能力;經(jīng)結(jié)晶紫染色法評估臨床分離株P(guān)A 03具有較強的形成生物被膜能力,臨床分離株P(guān)A 07不具有形成生物被膜的能力;2.17BIPHE2對金黃色葡萄球菌MIC值為8μmol/L,1/2×MIC就可以有效抑制浮游菌的生長。17BIPHE2對銅綠假單胞菌MIC值為48μmol/L,亞抑菌濃度也可以有效抑制浮游菌的生長。17BIPHE2聯(lián)合抗生素使用較單一使用MIC值均有所下降;3.單獨使用17BIPHE2在金黃色葡萄球菌黏附階段抑制率為40%,17BIPHE2聯(lián)合抗生素使用較單獨使用抗生素其抑制效果有所提升。單獨使用17BIPHE2在銅綠假單胞菌黏附階段抑制率為60%,17BIPHE2聯(lián)合抗生素使用與上述結(jié)果相似,較單獨使用抗生素抑制率有所提高;4.在金黃色葡萄球菌生物被膜形成階段17BIPHE2單獨使用抑制率達到35%,而在銅綠假單胞菌生物被膜形成階段17BIPHE2單獨使用抑制率達到80%。值得注意的是,17BIPHE2在1/4×MIC也可以顯著抑制兩種細菌產(chǎn)生生物被膜。17BIPHE2聯(lián)合抗生素結(jié)果顯示較單獨使用抗生素其抑制生物被膜產(chǎn)生能力均有所提高;5.經(jīng)掃描電子顯微鏡觀察,17BIPHE2在1/4×MIC濃度即可促進金黃色葡萄球菌成熟生物被膜消散,1×MIC生物被膜消散同時細菌黏附量有所下降,聯(lián)合萬古霉素促進生物被膜消散同時細菌胞質(zhì)大量外泄。17BIPHE2促進銅綠假單胞菌成熟生物被膜消散效果與上述結(jié)果相似,亞抑菌濃度即可促進銅綠假單胞菌生物被膜消散;隨著藥物濃度提高細菌黏附量急劇下降,并且呈現(xiàn)劑量依賴關(guān)系。當(dāng)17BIPHE2濃度達到1×MIC時細菌完整形態(tài)幾乎消失,生物被膜消散同時細胞質(zhì)外泄;抗菌肽聯(lián)合環(huán)丙沙星清除生物被膜作用尤為顯著;6.采用激光共聚焦掃描顯微鏡觀察單獨17BIPHE2處理銅綠假單胞菌生物被膜后發(fā)現(xiàn),隨著藥物濃度的提高,細菌黏附量逐漸下降,死菌數(shù)逐漸增多,并且呈現(xiàn)明顯的劑量依賴關(guān)系。當(dāng)抗菌肽濃度提升至1×MIC,活菌數(shù)和死菌數(shù)均明顯下降。17BIPHE2聯(lián)合抗生素處理組較單獨使用抗生素處理組細菌黏附量顯著下降,殺菌作用明顯提高;7.Real-Time PCR結(jié)果表明,隨著17BIPHE2濃度提高,金黃色葡萄球菌和銅綠假單胞菌生物被膜相關(guān)基因在轉(zhuǎn)錄水平表達量均出現(xiàn)不同程度的抑制,且高濃度處理組抑制作用更為明顯。結(jié)論抗菌肽17BIPHE2具有良好的抑制金黃色葡萄球菌及銅綠假單胞菌生物被膜黏附、聚集作用,并可促進成熟生物被膜消散。17BIPHE2輔助抗生素抗生物被膜作用進一步提高。17BIPHE2可以有效抑制生物被膜相關(guān)基因的轉(zhuǎn)錄,這將為治療由金黃色葡萄球菌及銅綠假單胞菌生物被膜引起的相關(guān)感染提供了一個新思路。
[Abstract]:Objective to study the antibacterial peptide 17BIPHE2 used alone and combined with antibiotics against Staphylococcus aureus (Staphylococcus, aureus, SA) and Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA) by inhibition of biological membranes, and to investigate the effect of antibacterial peptide 17BIPHE2 bacteria effect on biofilm associated gene transcription, for the treatment of Staphylococcus aureus, Pseudomonas Areuginosa provides a new idea of membrane associated infection and drug development. Methods 1. Congo red plate test method (Staphylococcus aureus) and crystal violet staining method to assess the bacteria the ability to form biofilm; Determination of 2. Staphylococcus aureus broth dilution method and agar the test method, the minimum inhibitory concentration of Pseudomonas aeruginosa (MIC), minimum bactericidal concentration (MBC); 3. with biofilm adhesion experimental observation of 17BIPHE2 used alone and combined with antibiotics on Students Is the 4. stage of membrane adhesion; the biofilm formation inhibition of experimental observation of 17BIPHE2 used alone and combined with antibiotics on biofilm formation stage; 5. by scanning electron microscopy (SEM) observation of 17BIPHE2 used alone and combined with antibiotics is clear the mature stage of biofilm; 6. by laser confocal laser scanning electron microscopy (LSCM) observed whether 17BIPHE2 could kill the mature biofilm of Pseudomonas aeruginosa in bacteria; 7. using Real-Time PCR 17BIPHE2 analysis of bacteria on biofilm related genes affecting transcriptional level. Results 1. by Congo red plate test method and crystal violet staining method to evaluate clinical isolates with SA 05 strong ability of biofilm formation, clinical isolates of SA 07 does not have the ability to form biofilm by crystal violet staining; evaluation of clinical isolates of PA 03 has Strong ability of biofilm formation, clinical isolates of PA 07 does not have the ability to form biofilm; 2.17BIPHE2 of Staphylococcus aureus MIC value is 8 mol/L, the growth of.17BIPHE2 1/2 * MIC can effectively inhibit bacteria of Pseudomonas aeruginosa MIC was 48 mol/L, compared with single use MIC value decreased use of subinhibitory concentration can effectively inhibit the growth of planktonic bacteria.17BIPHE2 combined with antibiotics; 3. 17BIPHE2 alone in the adhesion stage of Staphylococcus aureus inhibitory rate was 40%, the use of 17BIPHE2 combined with antibiotics than single use of antibiotics which inhibit effect is improved. The single use of 17BIPHE2 in Pseudomonas aeruginosa adhesion stage inhibition rate was 60% 17BIPHE2, combined with the use of antibiotics and the results were similar, compared with antibiotic inhibition rate increased; 4. in Staphylococcus aureus biofilm formation stage using 17BIPHE2 alone The inhibition rate reached 35%, while in the Pseudomonas Areuginosa stage 17BIPHE2 membrane formation alone inhibition rate was 80%. it is worth noting that 17BIPHE2 can also inhibit two kinds of bacteria biofilm.17BIPHE2 combined with antibiotics showed than single antibiotics which inhibit biofilm production capacity were improved in 1/4 * MIC; 5. by scanning electron microscopy, 17BIPHE2 in 1/4 * MIC concentration can promote Staphylococcus aureus biofilms dissipated 1 * MIC biofilm and dissipation of bacterial adhesion decreased, combined with vancomycin promote biofilm and bacterial cytoplasm dissipate a lot of leakage of.17BIPHE2 promoting Pseudomonas aeruginosa biofilm maturation effect the film dissipation and similar results, subinhibitory concentration could promote Pseudomonas aeruginosa biofilm dissipation; with the increasing of drug concentration of bacterial adhesion sharply under Fall, and in a dose-dependent manner. When the 17BIPHE2 concentration reached 1 * MIC intact bacterial biofilm morphology almost disappeared, while the cytoplasm leakage dissipation of antibacterial peptide; ciprofloxacin combined biofilm removal effect is particularly significant; 6. using confocal laser scanning microscopy 17BIPHE2 alone treatment of Pseudomonas aeruginosa biofilm found with the increase of drug concentration, bacterial adhesion decreased, the number of dead bacteria increased gradually, and showed a dose-dependent manner. When the concentration of antibiotic peptide increased to 1 x MIC, down.17BIPHE2 combined with antibiotic group compared with antibiotics alone treatment group of bacterial adhesion was significantly decreased the number of viable cells and dead cells were significantly lower, sterilization the role of 7.Real-Time increased significantly; PCR results showed that with the increase of 17BIPHE2 concentration, Staphylococcus aureus and Pseudomonas aeruginosa biofilm related genes at the transcriptional level The expression was inhibited, and the higher concentration was inhibited obviously. Conclusion the antibacterial peptide 17BIPHE2 has good inhibition of Staphylococcus aureus and Pseudomonas aeruginosa biofilm adhesion, aggregation, and can promote the mature biofilm dissipation.17BIPHE2 assisted.17BIPHE2 was improved further biological antimicrobial film can effectively inhibit the biological membrane by transcription related genes, which will provide a new way for treatment of infection by Staphylococcus aureus and Pseudomonas aeruginosa biofilm caused.

【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R446.5

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