本文關鍵詞： 鹽酸氨溴索 氟康唑 耐藥白色念珠菌 協(xié)同作用 協(xié)同機制　出處：《山東大學》2017年碩士論文　論文類型：學位論文

【摘要】：背景近年來,真菌,尤其是侵襲性真菌,已經(jīng)成為引起院內感染的重要病原菌。最常見的侵襲性真菌主要包括念珠菌和曲霉,其中念珠菌中的白色念珠菌是最為常見的人體機會致病菌之一,由白色念珠菌造成的感染致死率也很高,已超過30%,這主要是由于目前臨床上可用的抗真菌藥物異常匱乏以及該病原菌易產(chǎn)生高耐藥性所致。目前,臨床可用的抗真菌藥物種類不多,僅有唑類、棘白菌素類、多烯類、嘧啶類和烯胺類等為數(shù)不多的幾個品種。在這些抗真菌藥物中,作為唑類抗真菌藥物之一的氟康唑由于其具有生物利用度高和毒性低的特點,已成為臨床上治療白色念珠菌感染的首選藥物。然而,廣泛、高頻率、高劑量使用氟康唑卻導致了白色念珠菌耐藥性的出現(xiàn),使得臨床上對抗耐藥白色念珠菌所致的感染變得十分棘手。此外,白色念珠菌還容易在體內留置導管上形成難以清除的生物膜,而生物膜的形成也在一定程度上增加了其耐藥性。因此,白色念珠菌的耐藥現(xiàn)象以及耐藥白色念珠菌所致感染為臨床治療帶來的挑戰(zhàn)已經(jīng)不容忽視。目前尚未找到克服白色念珠菌耐藥現(xiàn)象的有效途徑和手段,所以感染耐藥白色念珠菌的醫(yī)院患者只能接受高毒性或昂貴的抗真菌藥物的治療。因此,開發(fā)能夠克服真菌耐藥的治療手段迫在眉睫。目前,尋找可以增加氟康唑敏感性的非抗真菌藥物,將其和氟康唑聯(lián)合用藥來對抗耐藥白色念珠,已成為較為成功的方法,備受關注。鹽酸氨溴索具有強大的祛痰作用,能改善呼吸作用,且毒性十分低,因而被廣泛用于呼吸系統(tǒng)疾病的治療。到目前為止,該藥在臨床上的療效、耐受性和安全性均已被證實和肯定。近來實驗研究表明,鹽酸氨溴索單用還具有抗微生物作用,可以對抗敏感白色念珠菌、非白色念珠菌和某些細菌。然而,目前尚無關于鹽酸氨溴索與氟康唑聯(lián)用抗真菌的相關實驗研究報道。目的本實驗的目的首先是測定氟康唑與鹽酸氨溴索的體內、外抗念珠菌活性,分析二者聯(lián)用是否有協(xié)同抗真菌作用,并進一步探索氟康唑與鹽酸氨溴索的藥物組合對耐藥白色念珠菌的協(xié)同抗真菌作用機制。方法按照CLSI M27-A3藥物敏感性測定方案,采用微量稀釋法測定氟康唑和鹽酸氨溴索單用或聯(lián)用分別對敏感白色念珠菌、耐藥白色念珠菌的體外抗真菌作用,并以部分抑菌濃度指數(shù)(fractional inhibitory concentration index,FICI)模型和生長差異百分率(percentage ofgrowth difference,ΔE)模型共同地評價這兩個藥物聯(lián)合抗真菌作用效果。使用大蠟螟作為白色念珠菌感染模型,通過測定大蠟螟的生存率、載菌量和組織學改變綜合評價氟康唑和鹽酸氨溴索單用與聯(lián)用在體內的聯(lián)合作用效果。在機制研究方面,使用羅丹明6G作為氟康唑的熒光替代物,借助流式細胞儀評價鹽酸氨溴索對耐藥白色念珠菌藥物外轉運泵活性的影響。此外,本研究還測定了藥物聯(lián)用前后對耐藥白色念珠菌的菌絲生長情況和胞外磷脂酶活性的變化,評價該藥物組合對耐藥白色念珠菌毒力因子表達情況的影響,并以實時定量PCR法測定藥物聯(lián)用對菌絲相關基因(HWP1)和磷脂酶編碼基因(PLB1和PLC1)的影響。結果1藥物聯(lián)用的體外抗真菌作用加入鹽酸氨溴索(濃度為128μg/mL)可以使氟康唑對耐藥白色念珠菌CA10和CA16的MIC值從512μg/mL以上降至2μg/mL,FICI模型和ΔE模型均表示兩藥在體外聯(lián)用具有很好的協(xié)同抗耐藥白色念珠菌作用。兩藥在體外聯(lián)用對抗敏感白色念珠菌作用的FICI值均大于1、ΔE絕對值均遠大于200%,表明該藥物組合對敏感白色念珠菌沒有協(xié)同作用。2藥物聯(lián)用的體內抗真菌作用氟康唑和鹽酸氨溴索單用時對提高感染耐藥白色念珠菌的大蠟螟的生存率沒有顯著的作用,但是聯(lián)用組可以明顯提高感染后大蠟螟的生存率,由此初步確定了該藥物組合的體內抗真菌療效。另外,與其他組三相比,兩藥聯(lián)用組可明顯降低感染大蠟螟體內的載菌量。通過觀察四組感染耐藥白色念珠菌后的大蠟螟病理組織切片之后我們發(fā)現(xiàn),與其他組三相比,藥物聯(lián)用組的大蠟螟體內黑色真菌斑塊明顯減少。我們的結果證實了氟康唑聯(lián)合鹽酸氨溴索在體內的抗耐藥白色念珠菌作用。3鹽酸氨溴索可顯著抑制藥物轉運泵的活性羅丹明6G和氟康唑,兩者都是真菌細胞膜上藥物轉運泵的作用底物。本實驗將羅丹明6G用作氟康唑的替代物。與對照組相比,鹽酸氨溴索干預后,白色念珠菌胞內羅丹明6G明顯吸收加快、外排明顯減慢。由此可見,兩藥聯(lián)用的協(xié)同抗真菌機制可能與它們抑制真菌細胞膜上的藥物轉運泵的活性進而增加氟康唑的胞內濃度有關。4鹽酸氨溴索聯(lián)合應用氟康唑可抑制菌白色念珠菌毒力因子的表達4.1藥物聯(lián)用可抑制菌絲生長借助熒光顯微鏡可以看到,對照組、氟康唑單用組和鹽酸氨溴索單用組均有出大量含有細長的菌絲,且菌絲有聚集成團現(xiàn)象,但是在藥物聯(lián)用組中的菌絲十分稀少,且菌絲很短,甚至有被破壞的現(xiàn)象。此外,與對照組相比,藥物聯(lián)用組中的菌絲生長調控基因(HWP1)明顯降低。由此我們可以確定,該藥物組合聯(lián)用的協(xié)同抗真菌機制可能與抑制耐藥白色念珠菌的菌絲的生長有關。4.2藥物聯(lián)用可抑制胞外磷脂酶活性白色念珠菌可分泌胞外磷脂酶,卵黃瓊脂培養(yǎng)基是一種能夠快速檢測該酶活性的培養(yǎng)基。結果發(fā)現(xiàn),藥物單用組的Pz值與對照組相比無顯著性差異,但是藥物聯(lián)用組的Pz值對照組相比卻明顯升高,表明鹽酸氨溴索與氟康唑聯(lián)合應用具有很強的抑制耐藥白色念珠菌胞外磷脂酶活性的作用。通過進一步的實時定量PCR的結果可以發(fā)現(xiàn),藥物聯(lián)用可明顯降低磷脂酶編碼基因PLB1和PLC1的表達,進一步揭示了該藥物組合對耐藥白色念珠菌胞外磷脂酶活性具有抑制作用,同時也表明PLB1和PLC1可能為該藥物組合協(xié)同抗耐藥白色念珠菌作用的潛在靶點。結論本研究揭示了鹽酸氨溴索與氟康唑聯(lián)用對抗耐藥白色念珠菌的體內、外作用效果。此外,我們發(fā)現(xiàn)該藥物組合協(xié)同抗耐藥白色念珠菌的作用機制與抑制抗真菌藥物轉運泵活性、抑制毒力因子(菌絲生長、胞外磷脂酶活性)的表達有關。我們的實驗第一次揭示了鹽酸氨溴索可與氟康唑聯(lián)用逆轉白色念珠菌的耐藥性。此外,我們的研究還將有助于新的抗真菌藥物靶點的發(fā)現(xiàn)。
[Abstract]:Background: in recent years, fungi, especially invasive fungi, has become an important pathogen causing nosocomial infection. The most common invasive fungi including Candida and Aspergillus, Candida in Candida albicans is one of the most common opportunistic human pathogen, caused by Candida albicans infection mortality rate is very high and has more than 30%, which is mainly due to the current clinical available antifungal drugs and the lack of abnormal pathogenic bacteria to produce high resistance caused. At present, the anti fungal drug clinical types are not many, only azole, echinocandins, polyenes, pyrimidine and enamine for several the number of varieties. In these antifungal agents, as one of the azole antifungal drugs fluconazole because of its high bioavailability and low toxicity characteristics, has become the first choice drugs for treatment of Candida albicans infection in clinic. However, extensive, high frequency, high dose of fluconazole has led to the emergence of drug resistance of Candida albicans, the clinical resistant Candida albicans infection becomes very difficult. In addition, Candida albicans also easy in vivo biofilm formation and removal of indwelling catheter, and biofilm formation also has increased to a certain extent the drug resistance. Therefore, drug resistance of Candida albicans and Candida albicans infection caused by drug resistance for clinical treatment of the challenge has been ignored. To overcome the drug resistance of Candida albicans has yet to find effective ways and means of the phenomenon, so the resistance of Candida albicans infection in hospital patients can receive treatment of highly toxic or expensive antifungal drugs. Therefore, to overcome the development of fungal resistance treatment is imminent. At present, looking for non really can increase the sensitivity of fluconazole resistance The bacteria drug, and fluconazole combination against drug-resistant Candida albicans, has become a successful method of concern. Ambroxol hydrochloride has a strong expectorant effect, can improve the respiratory function, and the toxicity is very low, so it is widely used for the treatment of respiratory diseases. So far, the effect of the drug in the clinic the tolerance and safety have been confirmed and affirmed. Recent experimental research shows that ambroxol hydrochloride alone has antimicrobial effects against sensitive Candida albicans, Candida albicans and some bacteria. However, there is no about ambroxol hydrochloride and fluconazole combined with related research reports. The purpose of the anti fungi the first is the determination of fluconazole and ambroxol hydrochloride in vivo, and anti Candida activity, combined with analysis of two have synergistic antifungal effects, and further explore and fluconazole Synergistic antifungal mechanism of ambroxol hydrochloride combination on drug resistance of Candida albicans. Methods according to the CLSI M27-A3 determination method of drug sensitivity determination of fluconazole and ambroxol hydrochloride alone or in combination were sensitive to Candida albicans by microdilution method, the antifungal activity of resistant Candida albicans in vitro, and the fractional inhibitory concentration index (fractional inhibitory concentration index, FICI) model and the growth difference percentage (percentage ofgrowth difference, E) model to evaluate the two common drugs combined with anti fungal effect. The use of wax moth as Candida albicans infection model, the survival rate of determination of wax moth, changes of the comprehensive evaluation of fluconazole and ambroxol hydrochloride alone and their combination with the combined effects of the in vivo effects of microbial load and organization. In terms of mechanism research, using Luo Danming 6G as fluorescence of fluconazole Substitute, with effect evaluation of ambroxol hydrochloride pump on the activity of drug resistant Candida albicans. Flow cytometry. In addition, this study also measured the changes of drug resistance of Candida albicans after mycelial growth and extracellular phospholipase activity, the evaluation of effect of the drug combination on the expression of drug resistance of Candida albicans virulence factor, and real time quantitative PCR method for the determination of drugs on the mycelial associated gene (HWP1) encoding gene and phospholipase (PLB1 and PLC1). The influence of the antifungal effect of the 1 drugs in vitro with ambroxol hydrochloride (concentration 128 g/mL) can make the fluconazole resistant Candida albicans CA10 and CA16 the MIC value from 512 Mu g/mL to 2 mu g/mL, FICI model and E model are delta two drugs have synergistic anti drug resistant Candida albicans in vitro with good medicine against white. Two sensitive in vitro with The role of Candida albicans FICI values are greater than 1, the absolute value of E was greater than 200%, indicating that the drug combination has no synergistic effect of.2 in combination with in vivo antifungal effects of fluconazole sensitive Candida albicans and ambroxol alone had no significant effect on improving the survival rate of Galleria mellonella infection resistant Candida albicans. But the combination group can significantly improve the survival rate of infection after the wax moth, to determine the initial antifungal efficacy of the combination of drugs in vivo. In addition, with other groups of three phase ratio, the two drug combination group can significantly reduce the microbial load of infection of Galleria mellonella in vivo. After infection were observed in four groups of drug resistance of Candida albicans after the wax moth pathological sections we found that with the other groups compared to three drug group the wax moth in black fungus plaque decreased. Our results confirmed that fluconazole combined with ambroxol hydrochloride in vitro Resistance of Candida albicans.3 in Ambroxol Hydrochloride can significantly inhibit drug transporter activity of Luo Danming 6G and fluconazole, both substrates on fungal cell membrane drug transport pump. This experiment will be used as a substitute for Luo Danming 6G of fluconazole. Compared with the control group, the ambroxol hydrochloride intervention, Candida albicans intracellular Luo Danming 6G significantly accelerates absorption, efflux was reduced. Thus, can inhibit the mycelial growth by fluorescence microscope can see that the combination of the two drug combination synergistic antifungal mechanism may be related to their inhibition of drug transport pump on fungal cell membrane and increase the activity of the intracellular concentration of fluconazole combined with ambroxol hydrochloride on.4 fluconazole can inhibit bacteria the expression of virulence factors of Candida albicans 4.1 drug control group, fluconazole monotherapy group and ambroxol alone group had a slender hyphae containing a large number of, And the hyphae are gathered into a group phenomenon, but in combination with hyphae in the group is very scarce, and the mycelium is very short, even the destruction of the phenomenon. In addition, compared with the control group, drug group in the mycelial growth controlling gene (HWP1) was significantly reduced. In this way we can determine, can inhibit the extracellular the phospholipase activity of Candida albicans secreted extracellular phospholipase associated with the drug combination for hypha synergistic antifungal mechanism may be related to the inhibition of drug resistance of Candida albicans growth.4.2 drugs, egg yolk agar is a culture medium for rapid detection of the enzyme activity. The results showed that with the single drug group compared with the Pz value the control group had no significant difference, but the drug group Pz values compared to the control group, but significantly higher, showed a combination of ambroxol hydrochloride and fluconazole has strong inhibition of drug resistance of Candida albicans extracellular phospholipase activity The role of the real-time PCR further results can be found, can reduce the expression of phospholipase PLB1 gene encoding and PLC1 drug combination, further reveals the combination of drug resistant Candida albicans extracellular phospholipase activity has inhibitory effect, also showed the potential targets of PLB1 and PLC1 may have synergistic anti resistant Candida albicans for the drug combinations. Conclusions ambroxol hydrochloride combined with fluconazole resistant Candida albicans in vivo, external effect. In addition, we found that the mechanism of the drug combination synergistic anti Candida albicans resistance and inhibition of antifungal drug transporter activity, inhibition of virulence factors (mycelial growth, extracellular phospholipase activity) the expression. Our study reveals for the first time with fluconazole combined with ambroxol can reverse the drug resistance of Candida albicans. In addition, I have The study will also contribute to the discovery of new antifungal targets.

【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96

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3 孫人杰邋韓暉;37項專利托起“制藥大王”[N];常州日報;2008年

4 許關煜 李敏華 編譯;歐盟審查氨溴索溴己新安全性[N];醫(yī)藥經(jīng)濟報;2014年

5 安力;氨溴索片致腹瀉[N];醫(yī)藥養(yǎng)生保健報;2006年

6 翔實;2006年呼吸系統(tǒng)疾病治療藥物　市場規(guī)模增長二成　氨溴索表現(xiàn)不俗[N];中國醫(yī)藥報;2007年

7 本報記者 陳雅瓊;多家上市藥企應對“不良反應”風波 國藥股份稱過敏為臨床使用問題[N];證券日報;2012年

8 本報記者 胡磊;多家上市藥企身陷“過敏門”[N];濟南日報;2012年

9 上海華東醫(yī)院呼吸科 張月莉 等;鹽酸氨溴索治療老年慢阻肺合并肺部感染有效[N];醫(yī)藥經(jīng)濟報;2001年

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10 譚杰;鹽酸氨溴索對小兒心臟手術體外循環(huán)肺損傷作用的臨床觀察[D];新疆醫(yī)科大學;2011年

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