本文關(guān)鍵詞： 乳酸桿菌 陰道感染 微生態(tài) 益生菌 拮抗 黏附　出處：《南昌大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 婦女陰道是一個(gè)復(fù)雜的微生態(tài)系統(tǒng)，乳酸桿菌是其中占主導(dǎo)地位的細(xì)菌，對維持陰道微生態(tài)環(huán)境的平衡起到了關(guān)鍵作用。乳酸桿菌發(fā)揮作用的機(jī)制推測為：1)粘附上皮細(xì)胞，阻止病原菌初始粘附；2)分泌有機(jī)酸，起到陰道自凈作用；3)產(chǎn)生細(xì)菌素；4)產(chǎn)生H_2O_2；5)產(chǎn)生生物表面活性蛋白物質(zhì)；6)刺激宿主局部免疫。研究表明乳酸桿菌制劑不僅可以阻止病原菌對陰道的感染，而且對治療陰道感染有顯著的效果。目前國內(nèi)外都有相關(guān)的商品化乳酸桿菌制劑，在預(yù)防治療陰道感染中起到了重要的作用。 本文采用改良MRS培養(yǎng)基對162份健康孕婦陰道樣本中的嗜酸性細(xì)菌進(jìn)行了定向篩選。通過革蘭氏染色、細(xì)胞形態(tài)觀察以及生理生化實(shí)驗(yàn)初步將分離的細(xì)菌確定為乳酸桿菌，共獲得326株乳酸桿菌。通過TMB-HRP-MRS培養(yǎng)基對其產(chǎn)H_2O_2能力進(jìn)行了半定量分析。獲得H_2O_2陽性菌株244株。其中H_2O_2弱陽性菌株為89株，陽性116株，強(qiáng)陽性39株。統(tǒng)計(jì)分析結(jié)果表明：產(chǎn)H_2O_2菌株在孕婦中的檢出率為87.59％，其分布與孕周以及孕婦年齡相關(guān)；產(chǎn)H_2O_2乳酸桿菌的檢出率隨著孕周的增加而升高，隨著孕婦年齡的增長而降低。 乳酸桿菌屬涵蓋了102種，德氏乳桿菌、植物乳桿菌、嗜酸乳桿菌、唾液乳桿菌等菌種在食品藥品領(lǐng)域顯示出良好的應(yīng)用前景。傳統(tǒng)的乳酸桿菌鑒定方法主要是依據(jù)形態(tài)學(xué)觀察和簡單生理生化反應(yīng)。本研究除了運(yùn)用API細(xì)菌學(xué)鑒定金標(biāo)準(zhǔn)進(jìn)行生理生化鑒定之外，還運(yùn)用現(xiàn)代分子生物學(xué)的技術(shù)對乳酸桿菌的鑒定進(jìn)行了探討，嘗試采用兩次PCR，即組間和組內(nèi)PCR的方法將分離菌株鑒定到種。結(jié)果表明，在孕婦陰道中檢出率較高的是嗜酸乳桿菌和卷曲乳桿菌，H_2O_2產(chǎn)量較高的也是嗜酸乳桿菌和卷曲乳桿菌。 篩選具有較強(qiáng)拮抗病原菌能力的陰道乳酸桿菌，可以作為益生菌制劑用于治療陰道細(xì)菌感染。本研究采用Hela細(xì)胞黏附實(shí)驗(yàn)?zāi)Ｐ涂疾炝巳樗釛U菌分離株的黏附能力，采用雙層平板法考察各分離株對致病菌的拮抗能力，結(jié)果發(fā)現(xiàn)乳酸桿菌的黏附能力具有菌種的差異，同一種不同株的差異也很大。乳酸桿菌分離株中有53.24％的菌株對白色假絲酵母有抑制作用，13.65％的菌株有較強(qiáng)的拮抗作用。黏附拮抗實(shí)驗(yàn)表明乳酸桿菌可以阻止白色假絲酵母對于Hela細(xì)胞的黏附。 以拮抗白色假絲酵母能力為主要指標(biāo)，綜合對其它三種致病菌(大腸桿菌、金黃色葡萄球菌、銅綠假單胞菌)的拮抗作用以及對Hela細(xì)胞的黏附能力，從326株陰道乳酸桿菌分離株中篩選得到11株有益生效果的乳酸桿菌，對其分類學(xué)地位、益生的生物學(xué)特性進(jìn)行了較全面的分析。首先測定了11株陰道乳酸桿菌的16S rRNA基因(即16S rDNA)全序列，在基因數(shù)據(jù)庫(GenBank、EMBL、DDBJ、PDB)中進(jìn)行了同源性搜索。取相似度大于99％的序列為參考，，對其進(jìn)行了鑒定，并構(gòu)建了系統(tǒng)發(fā)育進(jìn)化樹，確定了其在系統(tǒng)發(fā)育中的分支地位。通過拮抗致病菌實(shí)驗(yàn)發(fā)現(xiàn)，11株乳酸桿菌對致病菌有較強(qiáng)的拮抗作用，提示可以用于治療致病菌感染，恢復(fù)乳酸桿菌的主導(dǎo)地位。通過對其生物學(xué)特性進(jìn)行研究，初步掌握了其生長特性，并測定了其抗菌譜，分析了個(gè)別菌株的抑菌機(jī)理，為進(jìn)一步研究和應(yīng)用奠定了基礎(chǔ)。 本研究篩選的11株乳酸桿菌是正常婦女陰道的原籍菌，具有較強(qiáng)的粘附和拮抗病原菌能力，可以作為微生態(tài)制劑的候選菌株進(jìn)行深入研究。
[Abstract]:The vagina is a complex micro ecosystem, lactobacillus is one of the dominant bacteria, to maintain vaginal micro ecological environment balance plays a key role in the mechanism of the role of lactic acid bacteria. That is: 1) adhesion to epithelial cells, prevent pathogen initial adhesion; 2) the secretion of organic acids, play vaginal self purification; 3) produce bacteriocin; 4) H_2O_2; 5) to produce bio surfactant protein; 6) stimulate the host local immunity. The research showed that lactobacillus can not only prevent the infection of the vagina, but also has a significant effect on the treatment of vaginal infection. At home and abroad have commercial lactic acid preparation of relevant, in the prevention and treatment of vaginal infection plays an important role.
This paper uses the modified MRS medium for directional screening of 162 vaginal samples of healthy pregnant women in the acidophilic bacteria. By Gram staining, observe cell morphology and physiological and biochemical experiments will be the separation of bacteria identified as Lactobacillus, a total of 326 strains of Lactobacillus. Medium was studied by semi quantitative analysis of the H_2O_2 production by TMB-HRP-MRS training. H_2O_2 244 positive isolates. The H_2O_2 weakly positive 89 strains, 116 strains were positive, 39 strong positive strains. The results showed that the H_2O_2 producing strains in pregnant women in the detection rate was 87.59%, and the distribution of gestational age and maternal age; H_2O_2 producing lactic acid bacteria detection rate increased with the increase of weeks of pregnancy, pregnant women decreased with age.
Lactobacillus covers 102 kinds of Deshi, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus salivarius and other strains showed good application prospect in the field of food and drug. The traditional methods of identification of lactic acid bacteria is mainly based on the morphological observation and simple physiological and biochemical reactions. In this study, in addition to the gold standard for bacteriological identification by API physiological and biochemical identification, identification of the application of modern molecular biology technology of lactic acid bacteria were discussed, using two PCR, namely groups and PCR methods isolates were identified to species. The results show that the higher detection rate of maternal vaginal is Lactobacillus acidophilus and Lactobacillus casei, H_2O_2 high yield is Lactobacillus acidophilus and Lactobacillus plantarum.
Screening of vaginal Lactobacillus bacteria with strong antagonistic pathogens, can be used as probiotics in treating vaginal bacterial infection. This study investigated the adhesion ability of Lactobacillus isolates by Hela cell adhesion test model, effects of Antagonistic Ability of the isolates of the pathogen by double-layer plate method, has found differences in adhesion of lactic acid bacteria coli, with a different strain is also a great difference. Lactobacillus isolates and 53.24% strains had inhibitory effect on Candida albicans, 13.65% strains with strong antagonistic effect. Antagonism experiments show that lactic acid bacteria can prevent adhesion of Candida albicans to Hela cells.
The antagonism of Candida albicans is the main index of comprehensive ability, other three kinds of pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa) and the antagonistic effect on Hela cell adhesion from 326 strains of vaginal lactobacilli isolates screened 11 strains of Lactobacillus probiotic effects on. The taxonomic status and biological characteristics of probiotic were comprehensively analyzed. Firstly, the 16S rRNA gene of 11 strains of lactobacilli were determined (16S rDNA) sequence in the gene database (GenBank, EMBL, DDBJ, PDB) by homology search. The sequence similarity is greater than 99% for reference to they were identified, and construct phylogenetic tree, determine its phylogenetic position. Through antagonistic bacteria branch experiments showed that 11 Lactobacillus strains had strong antagonistic effect on pathogenic bacteria, that can be used for the treatment of The infection of pathogenic bacteria restored the dominant position of Lactobacillus. By studying its biological characteristics, its growth characteristics were preliminarily mastered, its antibacterial spectrum was determined, and the bacteriostatic mechanism of some strains was analyzed, which laid a foundation for further research and application.
The 11 lactobacilli screened from this study are the original vaginal bacteria of normal women, and have strong adhesion and antagonistic ability to pathogenic bacteria. They can be used as candidate strains for further research.

【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R371

【引證文獻(xiàn)】

相關(guān)期刊論文 前3條

1 陳勇輝;付桂明;萬茵;韓蓓;羅陽帆;王建濤;李紅歌;陳建芳;柴建新;;三唑磷降解菌C-Y106的分離·鑒定及其降解特性研究[J];安徽農(nóng)業(yè)科學(xué);2011年01期

2 楊欣;吳雅琨;陳麗仙;龍芳羽;王安如;;德氏乳桿菌特性及應(yīng)用研究進(jìn)展[J];安徽農(nóng)業(yè)科學(xué);2011年08期

3 陳勇輝;付桂明;萬茵;韓蓓;羅陽帆;王建濤;李紅歌;陳建芳;柴建新;;三唑磷降解菌Bacillus subtilis str. C-Y106的分離、鑒定及其降解特性研究(摘要)(英文)[J];Agricultural Science & Technology;2010年08期

相關(guān)碩士學(xué)位論文 前1條

1 王江;健康婦女陰道乳桿菌多樣性分析及具有益生菌特征菌株的篩選[D];重慶醫(yī)科大學(xué);2012年

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