Ⅲ型前列腺炎患者精液和前列腺按摩液菌群結(jié)構(gòu)多樣性研究
發(fā)布時(shí)間：2018-09-14 14:32
【摘要】：背景：慢性前列腺炎(Chronic Prostatitis Syndrome,CPS)是目前成年男性中一種常見疾病,是世界范圍內(nèi)僅次于糖尿病影響男性健康的重要疾病,并且發(fā)病率呈上升的趨勢。在我國泌尿外科門診病人中CP患者約占25%,超過一半的男性在一生中的某一些階段有過CPS的癥狀。CPS可引起尿頻、尿急、排尿困難、疼痛。CPS與性功能障礙、不育的相關(guān)性研究也日益引起重視。其中90%為Ⅲ型(Chronic Prostatitis/Chronic plvic pain syndrome,CP/CPPS)。IIIA(慢性前列腺炎)、ⅢB(慢性骨盆疼痛綜合癥)各近50%。常規(guī)細(xì)菌方法培養(yǎng)無細(xì)菌,臨床常用抗感染治療并且有效,提示該型前列腺組織可能同樣存在微生物。對Ⅲ型CPS患者前列腺按摩液(EPS)采用選擇性培養(yǎng)基培養(yǎng)細(xì)菌,68%發(fā)現(xiàn)凝固酶陰性球菌,19.1%發(fā)現(xiàn)L型細(xì)菌。但是,因?yàn)檫x擇性培養(yǎng)基培養(yǎng)費(fèi)時(shí)費(fèi)力,且條件限制,部分微生物難以培養(yǎng),導(dǎo)致假陰性結(jié)果,影響了對該病的認(rèn)識和臨床療效。 分子生物學(xué)方法,如變性梯度凝膠電泳(denaturing gradient gel electrophoresis, DGGE)、高通量454測序技術(shù),為研究健康男性以及Ⅲ型前列腺炎患者的前列腺微生態(tài)及菌群改變提供了嶄新的思路。有文獻(xiàn)報(bào)道在前列腺穿刺活檢組織中查尋前列腺中全部菌群,試圖找到關(guān)鍵引起CP/CPPS發(fā)生的關(guān)鍵病原菌。但作為有創(chuàng)性方法,前列腺穿刺活檢有適應(yīng)證及取材的限制。而EPS和精液因取材方便,可多次取樣,易評價(jià)療效。 因此本研究將聯(lián)合以上兩種分子生物學(xué)技術(shù),研究Ⅲ型前列腺炎患者EPS和精液的微生態(tài)及菌群改變,作為創(chuàng)新點(diǎn)。 目的：基于16S rDNA基因V3區(qū)PCR-DGGEi(變性梯度凝膠電泳)半定量檢測和高通量454二代測序技術(shù),調(diào)查CP患者的前列腺按摩液和精液微生物群落結(jié)構(gòu)的組成,探討CP與菌群變化的相關(guān)性,期望為Ⅲ型慢性前列腺炎進(jìn)一步明確病因,為診斷和治療提供新的線索,從而針對性選擇治療措施,以達(dá)到改善療效、縮短療程目的。 方法：研究的對象為Ⅲ型慢性前列腺炎患者和正常健康男性對照組各30例,用兩種方法檢測前列腺按摩液及精液標(biāo)本： 1.基于16S rRNA的PCR-DGGE指紋圖譜分析精液和前列腺按摩液的微生物菌群多樣性變化； 2.基于高通量454二代測序技術(shù)研究精液和前列腺按摩液的微生物菌群組成結(jié)構(gòu)及變化。 結(jié)果：1.DGGE譜和高通量454二代測序技術(shù)檢測結(jié)果一致。健康男性生殖道存在一個(gè)正常的微生物群落。每個(gè)個(gè)體的前列腺按摩液與精液菌群有明顯同源條帶。在群落結(jié)構(gòu)組成比例上,每個(gè)個(gè)體的精液和前列腺按摩液的菌群整體組成結(jié)構(gòu)存在顯著差異；前列腺炎患者的前列腺按摩液與精液菌群組成的結(jié)構(gòu)相似性均顯著低于正常健康對照組。 2.前列腺按摩液和精液的主要菌群的組成門類相似,主要由厚壁菌門(Firmicutes)、放線菌門(Actinobacteria),擬桿菌門(Bacteroidetes),變形桿菌門(Proteobacteria),梭桿菌門(Fusobacteria),無壁菌門(Tenericutes),藍(lán)細(xì)菌門(Cyanobacteria),綠屈撓菌門(Chloroflexi),和酸桿菌門(Acidobacteria)等構(gòu)成。其組成除放線菌門和擬桿菌門外,其他優(yōu)勢菌在門的水平上均存在顯著差異(P0.05或P0.01)。例如厚壁菌門在EPS中占整個(gè)菌群組成的59.9%,而厚壁菌門在精液中雖然仍是主要優(yōu)勢菌群,但是僅占整個(gè)菌群的39.5%。,精液中以下細(xì)菌較EPS顯著增加：變形桿菌門(19.23%vs.7.2%),梭桿菌門(4.04%vs.3.57%),無壁菌門(2.28%vs.1.38%),藍(lán)細(xì)菌門(3.28%vs.0.95%),綠屈撓菌門(1.56%vs.0.44%)。相反,與EPS相比,精液中放線菌門比例顯著降低(0.26%vs.0.66%)。 3.Ⅲ型前列腺炎患者的精液菌群組成與健康對照組存在顯著差異。在門的水平上,前列腺炎患者的精液中疣微菌門(Verrucomicro)和互養(yǎng)菌門(Synergistetes)顯著高于正常健康對照組。 1.4.Ⅲ慢性前列腺患者的前列腺按摩液與健康對照組比較,在門的水平上,與精液的結(jié)果一致,疣微菌門顯著高于正常健康對照組,并且僅在在慢性前列腺炎患者中可以檢出,益生菌乳酸桿菌(Lactobacillus)的相對豐度顯著降低。 結(jié)論：本研究首次對男性精液和前列腺按摩液的微生物菌落進(jìn)行了全面分析,發(fā)現(xiàn)其組成遠(yuǎn)比以往認(rèn)識的豐富； 1.每個(gè)個(gè)體的精液、前列腺按摩液存在共有菌,但整體上二者菌群組成存在明顯差異,提示在菌群組成結(jié)構(gòu)上,精囊液與前列腺液可能存在不同； 2.Ⅲ前列腺炎患者的精液菌群結(jié)構(gòu)較健康男性發(fā)生了顯著變化,疣微菌門(Verrucomicro)和互養(yǎng)菌門(Synergistetes)顯著高于正常健康對照組； 3.Ⅲ前列腺炎患者的前列腺按摩液菌群雖然變化不顯著,但同樣檢測出疣微菌門(Verrucomicro)菌,值得動(dòng)物實(shí)驗(yàn)進(jìn)一步研究其作用。 4.Ⅲ前列腺炎患者精液和前列腺按摩液中益生菌乳酸桿菌的相對豐度顯著降低,提示可能與慢性前列腺炎的發(fā)生相關(guān)。
[Abstract]:BACKGROUND: Chronic Prostatitis Syndrome (CPS) is a common disease among adult males, and it is the second most important disease affecting male health in the world after diabetes mellitus. CPS can cause frequent urination, urgent urination, dysuria, pain, and the relationship between CPS and sexual dysfunction, and infertility has attracted increasing attention. 90% of them are type III (Chronic Prostatitis/Chronic plvic pain syndrome, CP/CPPS). IIIA (chronic prostatitis) and III B (chronic pelvic pain syndrome) are nearly 50% respectively. Bacterial culture is bacteria-free, anti-infective therapy is commonly used in clinic and effective, suggesting that microorganisms may also exist in this type of prostate tissue. In the prostatic massage fluid (EPS) of patients with type III CPS, 68% of coagulase-negative cocci and 19.1% of L-type bacteria were found in selective medium. It is difficult to cultivate some microorganisms because of laborious and limited conditions, which leads to false negative results and affects the understanding and clinical efficacy of the disease.
Molecular biological methods, such as denaturing gradient gel electrophoresis (DGGE) and high throughput 454 sequencing, provide a new idea for the study of prostatic microecology and microflora in healthy men and patients with prostatitis type III. But as a invasive method, prostate biopsy has indications and limitations of sampling. EPS and semen can be sampled many times because of the convenience of sampling.
Therefore, this study will combine the above two molecular biology techniques to study the changes of Microecology and microflora of EPS and semen in patients with type III prostatitis.
Objective: To investigate the composition of microbial community in prostatic massage fluid and semen of CP patients by semi-quantitative PCR-DGGEi (denaturing gradient gel electrophoresis) in V3 region of 16S rDNA gene and high-throughput 454 second-generation sequencing technique, and to explore the correlation between CP and microbial community changes. The treatment provides new clues, so as to select targeted treatment measures to improve the curative effect and shorten the course of treatment.
Methods: 30 cases of chronic prostatitis type III and 30 cases of normal male control group were studied.
1. PCR-DGGE fingerprint based on 16S rRNA was used to analyze the microbial diversity of semen and prostatic massage fluid.
2. The microbial community composition and changes of semen and prostatic massage fluid were studied based on high-throughput 454 second-generation sequencing.
Results: 1. The results of DGGE spectrum and high-throughput 454 second-generation sequencing were consistent. There was a normal microbial community in the genital tract of healthy men. The structure similarity of prostatic massage fluid and semen flora in patients with prostatitis was significantly lower than that in healthy controls.
2. The major flora of prostatic massage fluid and semen are similar, mainly composed of Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, Fusobacteria, Tenericutes, Cyanobacteria, Chloroflexi. In addition to actinomycetes and bacteroides, there were significant differences in the composition of other dominant bacteria at phylum level (P 0.05 or P 0.01). The following bacteria in semen were significantly increased as compared with EPS: Proteus (19.23% vs. 7.2%), Fusobacterium (4.04% vs. 3.57%), Anechocystis (2.28% vs. 1.38%), Cyanobacteria (3.28% vs. 0.95%), Flexible Green (1.56% vs. 0.44%).
3. The semen microflora and Synergistetes in patients with prostatitis type III were significantly higher than those in healthy controls.
1.4.III Prostate massage fluid from patients with chronic prostatitis was significantly higher than that of healthy controls at the door level, consistent with the semen results. Verrucous microflora was significantly higher than that of healthy controls, and was detected only in patients with chronic prostatitis, and the relative abundance of probiotic Lactobacillus was significantly reduced.
Conclusion: For the first time, the microbial colonies of male semen and prostatic massage fluid were analyzed comprehensively in this study, and it was found that the composition of microbial colonies was much more abundant than previously known.
1. There are common bacteria in semen and prostatic massage fluid of each individual, but there are obvious differences in the composition of bacteria between them, suggesting that there may be differences in the composition of bacteria between seminal vesicle fluid and prostatic fluid.
2. The semen microflora structure of prostatitis patients was significantly different from that of healthy men. Verrucomicro and Synergistetes were significantly higher than those of healthy controls.
3. Although the microflora of prostatic massage fluid in patients with prostatitis did not change significantly, Verrucomicro was also detected, which is worthy of further study in animal experiments.
4. The relative abundance of probiotic Lactobacillus in semen and prostatic massage fluid of prostatitis patients decreased significantly, suggesting that it may be related to the occurrence of chronic prostatitis.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R697.33
本文編號：2242984
[Abstract]:BACKGROUND: Chronic Prostatitis Syndrome (CPS) is a common disease among adult males, and it is the second most important disease affecting male health in the world after diabetes mellitus. CPS can cause frequent urination, urgent urination, dysuria, pain, and the relationship between CPS and sexual dysfunction, and infertility has attracted increasing attention. 90% of them are type III (Chronic Prostatitis/Chronic plvic pain syndrome, CP/CPPS). IIIA (chronic prostatitis) and III B (chronic pelvic pain syndrome) are nearly 50% respectively. Bacterial culture is bacteria-free, anti-infective therapy is commonly used in clinic and effective, suggesting that microorganisms may also exist in this type of prostate tissue. In the prostatic massage fluid (EPS) of patients with type III CPS, 68% of coagulase-negative cocci and 19.1% of L-type bacteria were found in selective medium. It is difficult to cultivate some microorganisms because of laborious and limited conditions, which leads to false negative results and affects the understanding and clinical efficacy of the disease.
Molecular biological methods, such as denaturing gradient gel electrophoresis (DGGE) and high throughput 454 sequencing, provide a new idea for the study of prostatic microecology and microflora in healthy men and patients with prostatitis type III. But as a invasive method, prostate biopsy has indications and limitations of sampling. EPS and semen can be sampled many times because of the convenience of sampling.
Therefore, this study will combine the above two molecular biology techniques to study the changes of Microecology and microflora of EPS and semen in patients with type III prostatitis.
Objective: To investigate the composition of microbial community in prostatic massage fluid and semen of CP patients by semi-quantitative PCR-DGGEi (denaturing gradient gel electrophoresis) in V3 region of 16S rDNA gene and high-throughput 454 second-generation sequencing technique, and to explore the correlation between CP and microbial community changes. The treatment provides new clues, so as to select targeted treatment measures to improve the curative effect and shorten the course of treatment.
Methods: 30 cases of chronic prostatitis type III and 30 cases of normal male control group were studied.
1. PCR-DGGE fingerprint based on 16S rRNA was used to analyze the microbial diversity of semen and prostatic massage fluid.
2. The microbial community composition and changes of semen and prostatic massage fluid were studied based on high-throughput 454 second-generation sequencing.
Results: 1. The results of DGGE spectrum and high-throughput 454 second-generation sequencing were consistent. There was a normal microbial community in the genital tract of healthy men. The structure similarity of prostatic massage fluid and semen flora in patients with prostatitis was significantly lower than that in healthy controls.
2. The major flora of prostatic massage fluid and semen are similar, mainly composed of Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, Fusobacteria, Tenericutes, Cyanobacteria, Chloroflexi. In addition to actinomycetes and bacteroides, there were significant differences in the composition of other dominant bacteria at phylum level (P 0.05 or P 0.01). The following bacteria in semen were significantly increased as compared with EPS: Proteus (19.23% vs. 7.2%), Fusobacterium (4.04% vs. 3.57%), Anechocystis (2.28% vs. 1.38%), Cyanobacteria (3.28% vs. 0.95%), Flexible Green (1.56% vs. 0.44%).
3. The semen microflora and Synergistetes in patients with prostatitis type III were significantly higher than those in healthy controls.
1.4.III Prostate massage fluid from patients with chronic prostatitis was significantly higher than that of healthy controls at the door level, consistent with the semen results. Verrucous microflora was significantly higher than that of healthy controls, and was detected only in patients with chronic prostatitis, and the relative abundance of probiotic Lactobacillus was significantly reduced.
Conclusion: For the first time, the microbial colonies of male semen and prostatic massage fluid were analyzed comprehensively in this study, and it was found that the composition of microbial colonies was much more abundant than previously known.
1. There are common bacteria in semen and prostatic massage fluid of each individual, but there are obvious differences in the composition of bacteria between them, suggesting that there may be differences in the composition of bacteria between seminal vesicle fluid and prostatic fluid.
2. The semen microflora structure of prostatitis patients was significantly different from that of healthy men. Verrucomicro and Synergistetes were significantly higher than those of healthy controls.
3. Although the microflora of prostatic massage fluid in patients with prostatitis did not change significantly, Verrucomicro was also detected, which is worthy of further study in animal experiments.
4. The relative abundance of probiotic Lactobacillus in semen and prostatic massage fluid of prostatitis patients decreased significantly, suggesting that it may be related to the occurrence of chronic prostatitis.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R697.33
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