Ⅲ型前列腺炎患者精液和前列腺按摩液菌群结构多样性研究
发布时间：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种常见疾病,是世界范围内仅次于糖尿病影响男性健康的重要疾病,并且发病率呈上升的趋势。在我国泌尿外科门诊病人中CP患者约占25%,超过一半的男性在一生中的某一些阶段有过CPS的症状。CPS可引起尿频、尿急、排尿困难、疼痛。CPS与性功能障碍、不育的相关性研究也日益引起重视。其中90%为Ⅲ型(Chronic Prostatitis/Chronic plvic pain syndrome,CP/CPPS)。IIIA(慢性前列腺炎)、ⅢB(慢性骨盆疼痛综合症)各近50%。常规细菌方法培养无细菌,临床常用抗感染治疗并且有效,提示该型前列腺组织可能同样存在微生物。对Ⅲ型CPS患者前列腺按摩液(EPS)采用选择性培养基培养细菌,68%发现凝固酶阴性球菌,19.1%发现L型细菌。但是,因为选择性培养基培养费时费力,且条件限制,部分微生物难以培养,导致假阴性结果,影响了对该病的认识和临床疗效。 分子生物学方法,如变性梯度凝胶电泳(denaturing gradient gel electrophoresis, DGGE)、高通量454测序技术,为研究健康男性以及Ⅲ型前列腺炎患者的前列腺微生态及菌群改变提供了崭新的思路。有文献报道在前列腺穿刺活检组织中查寻前列腺中全部菌群,试图找到关键引起CP/CPPS发生的关键病原菌。但作为有创性方法,前列腺穿刺活检有适应证及取材的限制。而EPS和精液因取材方便,可多次取样,易评价疗效。 因此本研究将联合以上两种分子生物学技术,研究Ⅲ型前列腺炎患者EPS和精液的微生态及菌群改变,作为创新点。 目的：基于16S rDNA基因V3区PCR-DGGEi(变性梯度凝胶电泳)半定量检测和高通量454二代测序技术,调查CP患者的前列腺按摩液和精液微生物群落结构的组成,探讨CP与菌群变化的相关性,期望为Ⅲ型慢性前列腺炎进一步明确病因,为诊断和治疗提供新的线索,从而针对性选择治疗措施,以达到改善疗效、缩短疗程目的。 方法：研究的对象为Ⅲ型慢性前列腺炎患者和正常健康男性对照组各30例,用两种方法检测前列腺按摩液及精液标本： 1.基于16S rRNA的PCR-DGGE指纹图谱分析精液和前列腺按摩液的微生物菌群多样性变化； 2.基于高通量454二代测序技术研究精液和前列腺按摩液的微生物菌群组成结构及变化。 结果：1.DGGE谱和高通量454二代测序技术检测结果一致。健康男性生殖道存在一个正常的微生物群落。每个个体的前列腺按摩液与精液菌群有明显同源条带。在群落结构组成比例上,每个个体的精液和前列腺按摩液的菌群整体组成结构存在显著差异；前列腺炎患者的前列腺按摩液与精液菌群组成的结构相似性均显著低于正常健康对照组。 2.前列腺按摩液和精液的主要菌群的组成门类相似,主要由厚壁菌门(Firmicutes)、放线菌门(Actinobacteria),拟杆菌门(Bacteroidetes),变形杆菌门(Proteobacteria),梭杆菌门(Fusobacteria),无壁菌门(Tenericutes),蓝细菌门(Cyanobacteria),绿屈挠菌门(Chloroflexi),和酸杆菌门(Acidobacteria)等构成。其组成除放线菌门和拟杆菌门外,其他优势菌在门的水平上均存在显著差异(P0.05或P0.01)。例如厚壁菌门在EPS中占整个菌群组成的59.9%,而厚壁菌门在精液中虽然仍是主要优势菌群,但是仅占整个菌群的39.5%。,精液中以下细菌较EPS显著增加：变形杆菌门(19.23%vs.7.2%),梭杆菌门(4.04%vs.3.57%),无壁菌门(2.28%vs.1.38%),蓝细菌门(3.28%vs.0.95%),绿屈挠菌门(1.56%vs.0.44%)。相反,与EPS相比,精液中放线菌门比例显著降低(0.26%vs.0.66%)。 3.Ⅲ型前列腺炎患者的精液菌群组成与健康对照组存在显著差异。在门的水平上,前列腺炎患者的精液中疣微菌门(Verrucomicro)和互养菌门(Synergistetes)显著高于正常健康对照组。 1.4.Ⅲ慢性前列腺患者的前列腺按摩液与健康对照组比较,在门的水平上,与精液的结果一致,疣微菌门显著高于正常健康对照组,并且仅在在慢性前列腺炎患者中可以检出,益生菌乳酸杆菌(Lactobacillus)的相对丰度显著降低。 结论：本研究首次对男性精液和前列腺按摩液的微生物菌落进行了全面分析,发现其组成远比以往认识的丰富； 1.每个个体的精液、前列腺按摩液存在共有菌,但整体上二者菌群组成存在明显差异,提示在菌群组成结构上,精囊液与前列腺液可能存在不同； 2.Ⅲ前列腺炎患者的精液菌群结构较健康男性发生了显著变化,疣微菌门(Verrucomicro)和互养菌门(Synergistetes)显著高于正常健康对照组； 3.Ⅲ前列腺炎患者的前列腺按摩液菌群虽然变化不显著,但同样检测出疣微菌门(Verrucomicro)菌,值得动物实验进一步研究其作用。 4.Ⅲ前列腺炎患者精液和前列腺按摩液中益生菌乳酸杆菌的相对丰度显著降低,提示可能与慢性前列腺炎的发生相关。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R697.33
本文编号：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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R697.33
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