【作者】 吴志洪；

【导师】 涂亚庭；

【作者基本信息】 华中科技大学 ， 皮肤病与性病学， 2009， 博士

【摘要】 第一部分淋球菌的基因分型、毒力岛分类及与女性无症状感染关系的研究目的评价Opa分型及NG-MAST对淋球菌分离株进行基因分型的能力及性伴侣分离淋球菌基因型别之间的关系，研究淋球菌不同基因型别的毒力岛类型及其所引起女性无症状感染关系。方法12对性接触者淋球菌分离株培养鉴定后Opa基因以HpaⅡ酶切及聚丙烯酰胺凝胶电泳银染显色分析；12株男性标本和81株女性标本PCR扩增por和tbpB后测序用NG-MAST方法从分子水平上进行基因分型，并用ClustalX软件做进化树分析；并对毒力岛分类与女性无症状感染关系进行了研究。结果12对性接触患者分离株中，NG-MAST区分出10种ST型，而使用Opa分型则呈现12种OT型；新发现的ST（217-679）是国内特有的流行株。武汉地区女性感染的81株野生株中，GGI阳性菌株占了64株（80．43％）；无症状组有GGI阳性株也有GGI阴性株，有症状组也是如此。结论患者-性伴组除了45／46之外每对菌株的ST型及OT型均相同，表明患者与性伴相互接触传染。Opa-typing及NG-MAST可区分性关系网中的淋球菌菌株，二种方法分型结果总的趋向一致。Opa分型能力高于NG-MAST，ST型别可进一步的分成opa-types（OT）亚型。GGI毒力岛与女性症状感染之间并无明显的相关性，即女性有脓性分泌物感染并不是感染了强毒力的菌株所致。第二部分衣原体感染、性激素水平等因素与女性无症状感染关系的研究目的探讨女性淋球菌感染症状与衣原体感染及体内性激素水平等因素之间的关系。方法选择136例无明显症状女性淋病患者为观察组，45例有症状者为对照组，检测宫颈分泌物衣原体及淋球菌DNA；放射免疫法测定E2和P浓度；用RT-PCR和ELISA的方法在mRNA水平和蛋白水平检测炎症因子TNF-α、IL-1β的表达。用SPSS12．0软件进行统计学分析。结果淋病的无明显症状感染与衣原体感染无显著相关性，但其炎症因子TNF-α、IL-1β减少与血清孕激素水平升高的相关性具有统计学意义。结论女性淋病患者体内孕激素水平的增高可能是导致无症状感染的重要原因。第三部分孕激素体外调节Ngo-PMNs炎症反应的作用机制研究目的选择了有症状组和无症状组的非独特NG-MAST亚型各3种菌株，进一步研究孕激素在这些淋球菌野生株引起的中性粒细胞炎症反应的调节作用，探讨其作用差异及信号转导的可能机制。方法分离人外周血中性粒细胞（PMNs），用免疫荧光和RT-PCR检测PMNs上PR的表达：细胞维持在含10％热灭活胎牛血清的RPMI培养液中，细胞浓度为5×10⁶，淋球菌培养过夜后重悬在PBS中调整为OD₆₀₀=0．15(～10⁸ gonococci ml^-1)，感染复数（MOI）为10 bacteria／cell，在孕激素作用的不同时段用荧光定量PCR的方法检测iNOS，eNOS，TNF-a和IL-1β的变化，用Western Blot测定iNOS，eNOS，Akt和p-Akt（Ser473）蛋白水平，比较分析其差异。结果我们通过RT-PCR和免疫荧光的方法发现人的外周中性粒细胞并不表达经典的PR受体；孕激素能抑制iNOS，TNF-α和IL-1β的mRNA表达，在有症状组NgoST2951、ST735、ST436和无症状组ST809、ST369、ST2948中并没有显著差别；孕激素显著下调了iNOS蛋白的表达水平，然而eNOS蛋白没有检测到显著的改变；在孕激素处理前后没有能观察到磷酸化的Akt蛋白，总的Akt也没有改变。结论孕激素的压力导致PMNs中加剧iNOS表达的抑制和PMNs前炎症因子的下调，而这种作用是不依赖于PR介导受体途径和Akt信号通路。第四部分孕激素体外调节Ngo-PMNs呼吸爆发、凋亡应答的机制研究目的研究孕激素体外调节淋球菌引起的中性粒细胞呼吸爆发和凋亡的作用。方法分离人外周血中性粒细胞，分别用孕激素和十字孢碱处理，十字孢碱作为阳性对照。在0，3，8，12小时等时段用荧光定量RT-PCR分别测定各组中性粒细胞中凋亡抑制蛋白cIAP2和XIAP的表达水平；同时用流式细胞仪检测PMNs凋亡，用鲁米诺化学发光法检测淋球菌ST2951株刺激PMNs后呼吸爆发。结果cIAP2在孕激素处理3h后显著上调而XIAP和ST2951+medium组相比上调轻微；cIAP2在十字孢碱组是下调的。此外，我们发现孕激素对淋球菌ST2951刺激后的中性粒细胞有明显的延迟凋亡作用，尤其是在12h的时候；10ng／mL的孕激素对中性粒细胞的呼吸爆发并没有显著的影响，而十字孢碱加强了淋球菌刺激后中性粒细胞超氧负离子的产生。结论孕激素对淋球菌引起的中性粒细胞炎症反应有很重要的调节作用，孕激素介导的延迟感染了淋球菌的中性粒细胞凋亡可能是其逃避天然免疫和建立女性生殖道长期的低水平感染的重要机制，可能在女性无症状感染中起重要作用。更多还原

【Abstract】 PartⅠStudy on Classification of the gonococcal genetic island ofdifferent genotypes and the relationship of GGI with femalegonococcal asymptomatic infectionsObjective Two molecular epidemiology methods which was Opa Genotyping andNG-MAST Genotyping were carried out to identify the genomic species of Neisseriagonorrhoeae.To evaluate effectivity of two molecular epidemiology methods and study therelationship between sex partner and different genotypes and female gonococcalasymptomatic infections.Methods The methods of Opa Genotyping and NG-MAST Genotyping was used todifferentiate 12 strains from male and 81 strains from female patients who attended theoutpatients of sexually transmitted disease clinics and the relationship between differentgenotypes and phenotypes was studied. Furthermore, the genetic diversity and phylogeneticrelationship of the 12 paired strains was investigated using the Clustaix software, whichwas based on the gene sequences information.Results 12 paired strains of Neisseria gonorrhoeae were classified into 10 ST genotypes byusing NG-MAST genotyping,whereas the specimens were classified into 12 OT Opagenotyping genotypes by using Opa genotyping; a new-found ST genotype (217-679)was aspecific epidemic strain in domestic. Some 80.43% of the 81 strains contained the island,different isolates from female asymptomatic infections carried different gonococcal geneticislands(positive and negative),so were the isolates from symptomatic infections.Conclusion Genotypes of each strains from patient-sex partner besides 45/46 are the samewhich indicated that contagious infection exist between patient and sex partner.Strainswithin sexual networks could be differentiated by Opa genotyping and NG-MASTgenotyping analysis. General agreement was found between these two techniques.Opa genotyping was more effective than NG-MAST genotyping in identify the genomic speciesof Neisseria gonorrhoeae.ST genotype could be further classfied into different opa-types.Different isolates carried different gonococcal genetic islands(Pathogenicity island) andcertain phenotypes, and there were little relationship between GGI and symptomaticgonococcal infections. PartⅡStudy of the relationship between the symptom of femalegonococcal infections, Chlamydia trachomatis infections and sexhormoneObjective To study the relationship between the symptom of female gonococcalinfections, Chlamydia trachomatis infections and sex hormone.Methods We studied 136 gonorrhea patients with less symptomatic (asymptomatic group)and 45 with symptomatic(symptomatic group ). Their endocervical specimens were testedfor Chlamydia trachomatis, Neisseria gonorrhoeae by PCR, the levels of TNF-αandIL-1βby ELISA. Serum progesterone (P) and estradiol(E2) levels were measured by radioimmunoassay (RIA). The results were evaluated by SPSS for Windows (version 12.0) usingstatistical analysis.Results Chlamydia trachomatis have no relationgnship with female asymptomaticgonococcal infections. However, the relationship between the decreased levels of TNF-α、IL-1βand the increased levels of progesterone was considered statistical significant at P＜0.05.Conclusion Progesterone may play a key role in female less symptomatic gonococcalinfections by down-regulatd inflammatory response. PartⅢEffect and mechanism of progesterone on PMNinflammatory responses to wild-type gonococci in vitroObjective We used 3 types of strains associated with female symptomatic Infections and 3with asymptomatic Infections to investigate the modulation effects of progesterone on PMNinflammatory responses to gonoccocus.Methods PMNs were isolated from Heparinized peripheral blood from healthy volunteers.After incubation of PMNs With 50 ng/mL 17β-estradiol for 6 hours at 37℃with 5% CO2,PR mRNA expression was determined by RT-PCR and immunofluorescence. Additionallythe effect of progesterone on N. gonorrhoeae-challenged polymorphonuclear leukocytesinflammatory responses at various time points were assayed by real-time RT-PCR. Akt,p-Akt (Ser473), iNOS and eNOS were detected respectively by Western blot.Results Among these non-unique multiple isolates from asymptomatic women(ST2951,ST735 and ST436) and from symptomatic women(ST809,ST369 and ST2948) we found nosignificant difference in Ngo-PMNs inflammatory responses. In vitro PMNs infectionexperiments, progesterone suppressed expression of iNOS、TNF-α、IL-1βtranscript andiNOS protein significantly(P＜0.01) while eNOS expression revealed a slight change. Wefound that human peripheral blood PMNs did not express the classical progesteronereceptor and the role of progesterone was independent of Akt signaling pathway.Conclusions Gonococci are susceptible to progesterone or their downstream effects invitro. Progesterone induced down-regulated expression of pro-inflammatory cytokines suchas iNOS etc., which is independent of akt signaling pathway in Ngo challanged PMNs. PartⅣEffect of progesterone on gonococci-induced apoptosisand respiratory burst of human polymorphonuclear leucocytesin vitroObjective To investigate the regulatory effect of progesterone on apoptosis and oxidativeburst activity of polymorphonuclear leucocytes(PMNs) challenged by Neisseriagonorrhoeae (Ngo) in vitro.Methods Isolated PMNs were incubated with progesterone or staurosporine. Staurosporinewas used as a positive control for our vitro model. Expression levels of inhibitory apoptosisproteins (IAPs), cellular IAP2(cIAP2) and X-linked IAP (XIAP) were determined byreal-time PCR. Additionally, PMNs apoptosis at various time points (3, 8, 12 and 24h) wereassayed by flow cytometry. Luminol amplified chemiluminescence methods were used toquantify the oxidative burst function of PMNs challenged with Ngo ST2951.Results cIAP2 was up-regulated significantly in PMNs with progesterone treatment in 3hand XIAP was up-regulated slightly compared to the medium+ST2951 group, while cIAP2was down-regulated in staurosporine-challenged PMNs. Additionally we foundprogesterone delayed the onset of apoptosis activity in Ngo ST2951 challenged PMNs,notably at 12h. No statistically significant changes in PMNs oxidative burst activity wereobserved at 10 ng/mL level of progesterone. Staurosporine enhanced the production ofsuperoxide anion (respiratory burst) of human PMNs stimulated by Ngo.Conclusion Progesterone plays an important regulatory role in the interaction of PMNs andNgo. Delayed PMNs apoptosis induced by progesterone presumably acts as a mechanismfor Ngo to avoid the innate immune response and establish long-term, low-level infection inthe female reproductive tract.更多还原