【作者】 郑冰洁；

【导师】 尹跃平；

【作者基本信息】 北京协和医学院 ， 皮肤病与性病学， 2014， 博士

【摘要】 支原体是一类缺乏细胞壁、基因组较小的原核微生物。目前已被证实对人类有致病性的6种支原体中,3种为生殖道支原体：分别为生殖支原体(Mycoplasma genitalium, Mg)、解脲脲原体(Ureaplasma urealyticum, Uu)和人型支原体(Mycoplasma hominis, Mh)。生殖道支原体主要吸附于泌尿生殖道上皮及精子等细胞表面,可导致非淋菌性尿道炎(Non-gonococcal urethritis, NGU)、持续性或复发性尿道炎、附睾炎、子宫内膜炎、盆腔炎(Pelvic inflammatory disease, PID)、及早产、胎膜早破等不良妊娠结局等。Mg近年来被证实为一种性传播疾病(Sexual transmitted diseases, STD)病原体。流行病学研究表明,患有急性NGU的男性患者中Mg感染率为5%-42%,而在非淋菌性非沙眼衣原体性尿道炎(Non-chlamydial non-gonococcal urethritis, NCNGU)患¨者中,感染率为18%-46%。此外,越来越多的研究发现Mg感染后可促进HIV的感染和传播。然而不同于其他支原体或STD病原体,国内外在Mg感染现状的流行病学报道较少。仅有的一些流行病学研究也集中于女性性工作者(Female sex workers,FSWs)及STD门诊患者等群体,不能反映其他人群中的Mg感染情况。男男性行为者(Men who have sex with men, MSM)作为HIV感染及传播的高危人群,因其HIV感染率逐年增加而受到了性病预防控制中心的高度重视,但目前仅有数篇文献报道了该人群中Mg的感染情况。因此,了解该人群中Mg的流行情况并分析Mg与HIV之间的关系,对HIV的控制和预防有着重要意义。流行病学研究是基于临床或人群的研究,可提供横断面数据。考虑到人体生殖道局部环境较为复杂,可并存其他多种微生物,在临床症状方面也有一定的协同作用。动物模型则是一种比较直观的研究方法,可尽最大可能统一宿主条件,避免众多临床因素引起干扰,可进一步探讨致病机制及致病因素。Mg生长缓慢、对培养要求条件苛刻、培养极其困难,自1981年首次被报道以来,仅1例成功建立动物模型的研究。而Uu的体外培养较为容易,较多研究使用Uu建立动物模型。Uu被分为生物群1(Parvo)及生物群2(T960)两个亚群：生物群1包括血清型1、3、6、14型,生物群2包括2、4、5、7-13型等10个血清型。Uu感染后可导致多种泌尿生殖道疾病甚至垂直传播导致新生儿支原体感染等,但目前关于Uu的致病性是否和特定血清型相关、Uu致病性与宿主之间的关系等仍不清楚。多数流行病学研究仅从生物群水平探讨两个生物群间感染率的差异,而不足以解释Uu各血清型之间的致病性差异。相关的动物研究也多关注于单个型别或一些常见型别,而对少见型别如血清型2、9、5、7等的致病性情况并无较多了解。此外,目前多使用Uu常见型别建立药效评价模型,而忽视了其致病特征与特定血清型之间的关系。因此,建立Uu的14个型别的动物模型可深入了解Uu各型别间致病性差异,并为筛选最适血清型建立药效评价模型提供基础。全文分为两部分,各部分主要内容分述如下：第一部分：深圳男男性行为者生殖支原体流行病学调查及危险因素分析于2010年1月至5月,对深圳的MSM人群进行抽样调查。在深圳MSM人群聚集的娱乐场所或公共场所纳入合适的研究对象并采集人口社会学资料、收集受试者首段尿(First void urine,FVU)及直肠拭子标本,采用Taqman MGB荧光定量PCR法检测Mg DNA,并分析Mg感染的危险因素。研究期间共有409名MSM被纳入,以汉族、外来人口为主。其中95.5%(374/390)自我认同为同性恋,89.4%(349/390)仅与男性或较少女性发生性行为,22.9%(93/406)曾参加过性交易活动。95.1%(386/406)最近半年内曾与男性发生过肛交性行为,其中71.4%(275/385)使用了安全套。26.1%(106/405)最近半年内曾与女性发生性关系。55.7%(228/405)借助互联网寻找性伴。共收集811份直肠拭子和尿液标本,Mg总阳性率为8.1%(33/406,95%CI5.7%-10.6%)。406份FVU中Mg阳性率为3.4%(14/406,95%CI1.7%-5.4%),405份直肠拭子标本中阳性率为5.4%(22/405,95%CI3.5%-7.7%)。单因素分析及多因素分析均显示：较多与女性发生性关系者、在过去6个月内发生过无保护肛交者较仅与男性发生性关系者及未发生无保护肛交者Mg感染风险升高。提示MSM人群中Mg感染的主要部位可能是直肠,且双性恋人群是Mg感染及传播的高危人群,应引起进一步的重视。第二部分：解脲脲原体14血清型BALB/c小鼠感染模型中致病性初探采用雌性7周龄BALB/c小鼠及ATCC保藏的Uu14个血清型标准菌株建立动物模型。设立空白对照组、雌激素对照组及14个血清型感染组,每组随机分配15只小鼠。除空白组外,雌激素对照组与各型别感染组小鼠分别经阴道接种无菌Uu液体培养基及颜色改变单位(Color change unit,CCU)浓度为1×107CCU/mL的各型别标准菌液,接种后定期观察小鼠外阴形态变化。接种后第3、7、14、21天,对各组小鼠采集阴道冲洗液并进行培养、CCU法定量并检测肿瘤坏死因子-α(Tumornecrosis factor,TNF)-α、白介素(Interleukin,IL)-1β水平。接种后第3、21天,每组随机处死5只小鼠,取阴道、子宫标本,甲醛溶液固定后进行组织病理检查。研究发现接种后空白对照组和雌激素对照组两组小鼠阴道冲洗液中均无Uu生长、各时间点阴道冲洗液中TNF-αα及IL-1p均维持在较低水平。UUR各感染组各时间点小鼠阴道冲洗液培养阳性率较UPA高,且随着时间延长CCU逐渐下降。各感染组TNF-α及IL-1β水平变化呈现不同趋势：血清型1/4型接种后第3天炎性因子水平达到峰值,而至接种后21天则下降至对照组水平；血清型8/13组小鼠阴道冲洗液中两炎性因子水平至感染后第14天达到最高值,此后逐渐降低至对照组水平；血清型2/7/9/11/12/14型接种后两炎性因子水平逐渐升高,至接种后第21天炎性因子达到峰值。组织病理结果显示,各型别接种后小鼠阴道组织几乎不被累及,子宫则出现不同程度的急慢性炎症反应,其中4型主要导致急性炎症反应,1/3/11/8型主要导致慢性炎症反应。提示BALB/c小鼠下生殖道感染Uu后,主要导致宫颈炎、子宫内膜炎。血清型1、3、14、4、8、11型可能是14个型别中的主要致病型别,而其他型别可能是条件致病菌或非下生殖道常驻菌群。血清型13型可能导致隐性感染。血清型3、14、8、11型感染后在BALB/c小鼠下生殖道中定植时间较长,可导致子宫持慢性炎症反应,可能更适合作为药效评价模型的感染型别。更多还原

【Abstract】 Mycoplasma genitalium (Mg) is a member of the Mollicutes class that colonizes human mucosal surfaces of urogenital tracts. Accumulating evidence supports that Mg is a common sexually transmitted infection (STI) pathogen that can cause non-gonococcal urethritis (NGU) in men and pelvic inflammatory disease (PID) in women. In addition, several studies have shown that Mg infection can increase the risk of human immunodeficiency virus (HIV) transmission and/or acquisition. However, there have been limited number of epidemiological studies on Mg infection worldwide and data on the prevalence of this pathogen among men who have sex with men (MSM) are especially scarce. MSM plays a significant role in the spread of HIV and other sexually transmitted diseases (STD). Recent studies revealed an increasing trend of HIV/STD prevalence in Chinese MSM. However, to date, limited reports on the prevalence of Mg infection in this population have been reported. As part of a large cross-sectional study aimed at investigating the epidemiology of Mg infection among high risk populations in China, this study aims:(i) to determine the prevalence of urethral and rectal Mg infection in MSM in Shenzhen, Guangdong Province, China, and (ii) to identify the potential risk factors related to Mg infection.Ureaplasma spp. is another member of the Mollicutes class. It has14known serovars and is separated into the following2new species:Ureaplasma urealyticum (serovars2/4/5and7-13) and Ureapalsma parvum (serovars1/3/6/14). Although common in healthy people, ureaplasmas are also frequently reported to be associated with non-gonococcal urethritis (NGU), pelvic inflammatory disease (PID), infertility, adverse pregnancy outcomes (APO), chorioamnionitis and bronchopulmonary dysplasia (BPD) in neonates. However, there is conflicting evidence concerning whether there are differences in virulence among Ureaplasma serovars. And limited epidemiological studies have observed the prevalence of each serovars among neither high risk nor general population. Moreover, there has been no published study to date that has investigated the differences in the pathogenicity of the14serovars of Ureaplasma spp. Thus, the objectives of this part are to establish a murine model of genital tract by14serovars of Ureaplamsa spp, and to evaluate differences in the pathogenicity of these14serotypes.Section1:The prevalence of urethral and rectal Mycoplasma genitalium among men who have sex with men in Shenzhen, China.Between January and May2010, a total of409MSM were recruited in Shenzhen, Guangdong Province, China. An anonymous questionnaire was used to collect information regarding their socio-demographic characteristics and sexual behaviors. In addition, first-void urine (FVU) samples and rectal swabs were collected for PCR-based Mg testing. Among the409participants,406valid FVU and405rectal swab samples collected. The overall Mg prevalence was8.1%(33/406,95%CI5.7%-10.6%), with a FVU positivity of3.4%(95%CI1.7%-5.4%) and a rectal positivity of5.4%(95%CI3.5%-7.7%). Multivariable logistic regression analyses indicating that urethral Mg infection was significantly associated with having more heterosexual behaviors (AOR7.16,95%CI1.89-27.13,) and having unprotected anal intercourse in the past six months (AOR4.80,95%CI1.40-16.47). Rectal Mg infection was significantly associated with HIV infection based on univariate logistic regression analysis (OR=4.49,95%CI1.18-17.12). In this study, we showed that Mg was more prevalent in MSM who had bisexual behaviors compared to those who only engaged in homosexual behaviors. Further work is needed to establish the mode of Mg transmission and to identify its relationship with HIV transmission. Meanwhile, more attention should be paid to Mg infection among MSMs, and especially bisexual MSMs, which might have critical implications for effective HIV/STD control in China.Section2:The pathogenicity of14serovars of Ureaplasma spps in the lower genital tract of female BALB/c miceThe objectives of this section are to establish a murine model of lower genital tract infection by14serotypes of Ureaplasma spp, and to evaluate the differences in pathogenicity of these14serovars. Female BALB/c mice of7-weeks old were randomly and equally divided into14infectious groups and two control groups. The presence of infection was determined on solid and liquid culture media. Tumor necrosis factor (TNF-α) and interleukin (IL)-1β expression in lower genital tract secretions was determined using ELSIA kit. And histological examination was used to evaluate the difference in pathogenicity in each serovar. The blank control group and estradiol group were negative for Uu after inoculation with a lower level of TNF-a around2.07-7.69pg/mL.The positive rates of mice among UUR groups were generally higher than that among UPA groups. Both of positive rate and color change unit (CCU) value in two groups reduced with time. Meanwhile, each group presented different changes in the expression of TNF-a and IL-1β. The expression of these factors were peaked at3days after infection among serotypes1and4group, but were peaked at14days and21days after infection among8/13groups and2/7/9/11/12/14groups. The main histopathological feature was interstitial infiltration of inflammatory cells in uterus. In addition, Uu13might be an infectious serotype without causing symptoms. Therefore, serotype1/3/14/4/8/11might be primary pathogenic types and the rest might be opportunistic pathogen.更多还原