香莲方对耐药白念珠菌外排泵基因表达影响的研究

发布时间：2018-06-10 15:10

本文选题：白念珠菌 + 逆转耐药　；参考：《广州中医药大学》2016年博士论文

【摘要】：目的：1.调查本院近期外阴阴道念珠菌病致病菌种的分布及其耐药情况。2.探讨香莲复方中抗真菌及抗耐药的有效成分,及其逆转耐药的作用机制,为今后中医药抗真菌研究,尤其是针对耐药真菌的研究提供新方法、新思路。方法：1.回顾性调查本院近期诊断为外阴阴道念珠菌病的患者的临床表现、白带常规、真菌培养及药敏结果并进行统计分析,了解近期单纯性外阴阴道念珠菌病(VVC)和复发性外阴阴道念珠菌病(RVVC)患者的致病菌种的分布及耐药情况。2.采用香莲外洗液、丁香酚、小檗碱和丁香酚、小檗碱的混合液分别连续诱导培养白念珠菌标准耐药菌株CA17和临床分离耐药菌株,观察上述诱导药物对耐药白念珠菌的逆转耐药作用,比较各组诱导药物之间的作用差异。3.采用实时荧光定量PCR (qRT-PCR)检测耐药原代菌株和其成功逆转耐药的各敏感子代的外排泵基因CDR1、CDR2和MDR1表达水平的变化,探讨香莲外洗液、丁香酚、小檗碱对外排泵相关基因的作用。4.采用流式细胞术检测耐药原代菌株和其成功逆转耐药的各敏感子代外排荧光染色剂罗丹明6G(R6G)功能的变化,探讨香莲外洗液、丁香酚、小檗碱对外排泵蛋白功能的影响。结果：1.近期本院外阴阴道念珠菌病患者中,主要致病菌种仍为白念珠菌(69.8%),非白念珠菌中以光滑念珠菌(27.6%)最为常见；所检测5种抗真菌药物中,出现耐药率最高的是伊曲康唑(14.7%),其次为氟康唑(6.9%)。VVC和RVVC患者的致病菌种分布和耐药率统计学分析差异没有显著性。白念珠菌比非白念珠菌对氟康哗的耐药率更高,而二者对其他药物的耐药率统计学分析差异没有显著性。所检测三种唑类药物之间交叉耐药情况明显。2.香莲外洗液、丁香酚、小檗碱以及丁香酚、小檗碱混合液可在对三株临床分离耐药菌株连续诱导培养6-8代,实现对氟康唑逆转耐药。逆转耐药后,连续培养三代,菌株对于氟康唑的最小抑菌浓度(MIC)稳定于敏感范围内。而三株临床耐药株采用两种培养基的空白转种株,均未能在10代之内实现逆转。上述诱导药物均没有在30代以内对标准耐药菌株CA17实现逆转耐药。3.临床耐药菌株的CDR1、CDR2、MDRl的表达水平明显高于临床敏感株；4组经诱导药物作用后的敏感子代的上述外排泵基因的表达水平较其耐药原代菌株明显下降。其中由香莲外洗液和两中药单体混合液诱导培养的敏感子代,上述三种基因表达下降较丁香酚、小檗碱单独诱导培养的敏感子代更加明显,而丁香酚组的敏感子代的上述基因表达比小檗碱组下降更为明显。4.与临床耐药株相比,白念珠菌标准耐药株CA17外排R6G的功能更强,但主要表现在有葡萄糖(即有能量来源)的情况下。临床耐药株外排R6G的功能在有或无能量来源的条件下均明显高于临床敏感株。临床耐药株经香莲外洗液、小檗碱以及两单体混合液诱导培养后的敏感子代在无能量来源的条件下,其外排率均显著下降。但丁香酚组与耐药原代差异没有统计学意义。但加入葡萄糖(即有能量来源)后,各组与耐药组相比,外排率均有显著下降。其中对外排泵功能抑制作用最强的是香莲外洗液,其次为丁香酚和小檗碱的混合液,丁香酚、小檗碱各自单独作用差异没有统计学意义。结论：1.所调查VVC和RWC患者的致病菌仍以白念珠菌为优势菌种,其次为光滑念珠菌,对氟康唑耐药的主要菌种仍为白念珠菌,较以往流行病学变化不大,但所有念珠菌中伊曲康唑的耐药率首次超过了氟康唑。2.香莲外洗液、丁香酚、小檗碱以及丁香酚和小檗碱的混合液对临床分离耐药菌株均具有逆转耐药作用,以香莲外洗液出现逆转的代数最早。3.经香莲外洗液、丁香酚、小檗碱以及丁香酚、小檗碱的混合液诱导培养,可以显著降低白念珠菌临床耐药菌株的CDR1、CDR2、MDR1三种外排泵相关耐药基因的表达水平,并明显抑制其编码的外排泵蛋白的外排功能。推测香莲外洗液中的包括丁香酚和小檗碱在内的部分有效成分是通过降低外排泵基因表达从而抑制真菌细胞外排抗真菌药物达到抗耐药作用的。4.在抑制外排泵基因表达和外排泵蛋白功能方面,香莲外洗液原方作用均强于两个中药单体的单独作用,部分情况下接近两个单体的混合液的作用,推测丁香酚、小檗碱在香莲外洗液中有相互协同的抗耐药作用,香莲外洗液中其他成分的上述作用有待进一步验证。
[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.
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R285

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