本文選題：白念珠菌 + 逆轉耐藥��； 參考：《廣州中醫(yī)藥大學》2016年博士論文

【摘要】：目的：1.調(diào)查本院近期外陰陰道念珠菌病致病菌種的分布及其耐藥情況。2.探討香蓮復方中抗真菌及抗耐藥的有效成分,及其逆轉耐藥的作用機制,為今后中醫(yī)藥抗真菌研究,尤其是針對耐藥真菌的研究提供新方法、新思路。方法：1.回顧性調(diào)查本院近期診斷為外陰陰道念珠菌病的患者的臨床表現(xiàn)、白帶常規(guī)、真菌培養(yǎng)及藥敏結果并進行統(tǒng)計分析,了解近期單純性外陰陰道念珠菌病(VVC)和復發(fā)性外陰陰道念珠菌病(RVVC)患者的致病菌種的分布及耐藥情況。2.采用香蓮外洗液、丁香酚、小檗堿和丁香酚、小檗堿的混合液分別連續(xù)誘導培養(yǎng)白念珠菌標準耐藥菌株CA17和臨床分離耐藥菌株,觀察上述誘導藥物對耐藥白念珠菌的逆轉耐藥作用,比較各組誘導藥物之間的作用差異。3.采用實時熒光定量PCR (qRT-PCR)檢測耐藥原代菌株和其成功逆轉耐藥的各敏感子代的外排泵基因CDR1、CDR2和MDR1表達水平的變化,探討香蓮外洗液、丁香酚、小檗堿對外排泵相關基因的作用。4.采用流式細胞術檢測耐藥原代菌株和其成功逆轉耐藥的各敏感子代外排熒光染色劑羅丹明6G(R6G)功能的變化,探討香蓮外洗液、丁香酚、小檗堿對外排泵蛋白功能的影響。結果：1.近期本院外陰陰道念珠菌病患者中,主要致病菌種仍為白念珠菌(69.8%),非白念珠菌中以光滑念珠菌(27.6%)最為常見；所檢測5種抗真菌藥物中,出現(xiàn)耐藥率最高的是伊曲康唑(14.7%),其次為氟康唑(6.9%)。VVC和RVVC患者的致病菌種分布和耐藥率統(tǒng)計學分析差異沒有顯著性。白念珠菌比非白念珠菌對氟康嘩的耐藥率更高,而二者對其他藥物的耐藥率統(tǒng)計學分析差異沒有顯著性。所檢測三種唑類藥物之間交叉耐藥情況明顯。2.香蓮外洗液、丁香酚、小檗堿以及丁香酚、小檗堿混合液可在對三株臨床分離耐藥菌株連續(xù)誘導培養(yǎng)6-8代,實現(xiàn)對氟康唑逆轉耐藥。逆轉耐藥后,連續(xù)培養(yǎng)三代,菌株對于氟康唑的最小抑菌濃度(MIC)穩(wěn)定于敏感范圍內(nèi)。而三株臨床耐藥株采用兩種培養(yǎng)基的空白轉種株,均未能在10代之內(nèi)實現(xiàn)逆轉。上述誘導藥物均沒有在30代以內(nèi)對標準耐藥菌株CA17實現(xiàn)逆轉耐藥。3.臨床耐藥菌株的CDR1、CDR2、MDRl的表達水平明顯高于臨床敏感株；4組經(jīng)誘導藥物作用后的敏感子代的上述外排泵基因的表達水平較其耐藥原代菌株明顯下降。其中由香蓮外洗液和兩中藥單體混合液誘導培養(yǎng)的敏感子代,上述三種基因表達下降較丁香酚、小檗堿單獨誘導培養(yǎng)的敏感子代更加明顯,而丁香酚組的敏感子代的上述基因表達比小檗堿組下降更為明顯。4.與臨床耐藥株相比,白念珠菌標準耐藥株CA17外排R6G的功能更強,但主要表現(xiàn)在有葡萄糖(即有能量來源)的情況下。臨床耐藥株外排R6G的功能在有或無能量來源的條件下均明顯高于臨床敏感株。臨床耐藥株經(jīng)香蓮外洗液、小檗堿以及兩單體混合液誘導培養(yǎng)后的敏感子代在無能量來源的條件下,其外排率均顯著下降。但丁香酚組與耐藥原代差異沒有統(tǒng)計學意義。但加入葡萄糖(即有能量來源)后,各組與耐藥組相比,外排率均有顯著下降。其中對外排泵功能抑制作用最強的是香蓮外洗液,其次為丁香酚和小檗堿的混合液,丁香酚、小檗堿各自單獨作用差異沒有統(tǒng)計學意義。結論：1.所調(diào)查VVC和RWC患者的致病菌仍以白念珠菌為優(yōu)勢菌種,其次為光滑念珠菌,對氟康唑耐藥的主要菌種仍為白念珠菌,較以往流行病學變化不大,但所有念珠菌中伊曲康唑的耐藥率首次超過了氟康唑。2.香蓮外洗液、丁香酚、小檗堿以及丁香酚和小檗堿的混合液對臨床分離耐藥菌株均具有逆轉耐藥作用,以香蓮外洗液出現(xiàn)逆轉的代數(shù)最早。3.經(jīng)香蓮外洗液、丁香酚、小檗堿以及丁香酚、小檗堿的混合液誘導培養(yǎng),可以顯著降低白念珠菌臨床耐藥菌株的CDR1、CDR2、MDR1三種外排泵相關耐藥基因的表達水平,并明顯抑制其編碼的外排泵蛋白的外排功能。推測香蓮外洗液中的包括丁香酚和小檗堿在內(nèi)的部分有效成分是通過降低外排泵基因表達從而抑制真菌細胞外排抗真菌藥物達到抗耐藥作用的。4.在抑制外排泵基因表達和外排泵蛋白功能方面,香蓮外洗液原方作用均強于兩個中藥單體的單獨作用,部分情況下接近兩個單體的混合液的作用,推測丁香酚、小檗堿在香蓮外洗液中有相互協(xié)同的抗耐藥作用,香蓮外洗液中其他成分的上述作用有待進一步驗證。
[Abstract]:Objective: 1. to investigate the distribution and drug resistance of the pathogenic bacteria of vulvovaginal candidiasis in our hospital in the near future.2. to explore the effective components of antifungal and anti drug resistance in the fragrant lotus compound, and the mechanism of reversing drug resistance, so as to provide a new method for the study of antifungal antifungal drugs in the future, especially for the study of drug-resistant fungi. The new method is 1. cycles. The recent diagnosis of vulvovaginal candidiasis in our hospital, the clinical manifestations of the patients with vulvovaginal candidiasis, the routine leucorrhea, the fungal culture and the results of drug sensitivity and statistical analysis, the distribution and drug resistance of the recent simple vulvovaginal candidiasis (VVC) and recurrent vulvovaginal candidiasis (RVVC) patients were found and the drug resistance of.2. was used outside the fragrant lotus. The mixture of berberine, berberine and eugenol, berberine and berberine were continuously induced by the mixture of berberine and berberine, and the effect of the induced drug on the reversal of drug resistance of Candida albicans was observed, and the difference of the effect between the induced drugs was compared in each group.3. by real-time fluorescence quantitative PCR (qRT-PCR). The changes in the expression level of CDR1, CDR2 and MDR1 in the drug resistant primary strain and the susceptible progeny of its successful reversal of drug resistance, the effects of the external efflux genes of the external lotion, eugenol and berberine on the related genes of the pump.4. were examined by flow cytometry to detect the resistance of the original strain and the sensitive subgeneration of the sensitive subgeneration of the drug. The influence of the function of Luo Danming 6G (R6G) on the function of the external efflux pump protein of Xiang lotus lotion, eugenol and berberine. Results: 1. the main pathogenic bacteria in the patients with vulvovaginal candidiasis in our hospital are Candida albicans (69.8%) and Candida albicans (27.6%) in non Candida albicans, and 5 kinds of antifungal agents were detected. Among the drugs, the highest rate of drug resistance was itraconazole (14.7%). There was no significant difference in the distribution of pathogenic bacteria and the rate of drug resistance in the patients with fluconazole (6.9%).VVC and RVVC. The drug resistance rate of Candida albicans was higher than that of non Candida albicans, while the difference in the statistical analysis of the two of the other drugs was not significant. Cross resistance between three kinds of azoles was obvious. The mixture of eugenol, berberine and eugenol, berberine, and berberine, can be cultured for 6-8 generations continuously in three strains of clinical isolates, to achieve the reversal of fluconazole resistance. After reversing drug resistance, the.2. was continuously cultured for three generations, and the minimum bacteriostasis for fluconazole was found. The concentration (MIC) was stable in the sensitive range, but three strains of clinically resistant strains were failed to reverse in the 10 generation by using the two culture medium. All of the induced drugs did not reverse the CDR1, CDR2, MDRl expression level of the standard drug resistant strain CA17, and the expression level of CDR2 and MDRl was significantly higher than the clinical sensitivity. The expression level of the above efflux pump gene in the 4 groups was significantly lower than that of the drug resistant original strains. Among them, the sensitive subcultures induced by the mixture of fragrant lotus lotion and two Chinese medicine monomers decreased more than eugenol, and the sensitivities of berberine alone induced by berberine were more obvious. The expression of the above gene in the sensitive subgroup of the eugenol group was more obvious than that of the berberine group. Compared with the clinical drug resistant strain, the function of the CA17 outer row R6G of the Candida albicans was stronger, but it was mainly manifested in the presence of glucose (that is, the source of energy). The function of the exosplatin R6G in the clinical drug resistant strain was in or without energy sources. The clinical sensitive strains were significantly higher than the clinical sensitive strains. The sensitive progenies of the clinical drug-resistant strains were significantly decreased under the conditions of no energy source, but there was no significant difference between the eugenol group and the drug resistant original generation. Compared with the drug resistant group, the outer row rate decreased significantly. The strongest inhibitory effect of the external pump function was the fragrant lotus lotion, followed by the mixture of eugenol and berberine, and there was no significant difference in the separate effects of eugenol and berberine. Conclusion: 1. of the pathogenic bacteria in the patients with VVC and RWC were still the dominant strains of Candida albicans. The second is Candida smooth and the main strains resistant to fluconazole are Candida albicans, but the drug resistance rate of itraconazole in all Candida is higher than that of fluconazole.2. lotus lotion for the first time, and the mixture of eugenol, berberine, berberine and berberine has adverse effects on the clinical isolates. The earliest.3. of the algebra of Xiang Lian lotion was induced by the mixture of fragrant lotus lotion, eugenol, berberine, berberine and berberine, which could significantly reduce the expression level of three kinds of resistance genes related to CDR1, CDR2, MDR1 of Candida albicans and inhibit the encoding of the outer row. Some effective components, including eugenol and berberine, are used to reduce the expression of the outer row pump gene and inhibit the anti fungal drug resistance of the fungal cells to inhibit the expression of the efflux pump gene and the function of the efflux pump protein by reducing the expression of the efflux pump gene in the lotion of the fragrant Lotus lotion. The original recipe of the fragrant lotus lotion was made by.4.. The action of two Chinese herbs was stronger than that of the mixture of two monomers in some cases. It was suggested that eugenol and berberine have synergistic anti drug resistance in the fragrant lotus lotion. The effects of other ingredients in the fragrant lotus lotion need to be further verified.
【學位授予單位】：廣州中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R285

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