宫颈沙眼衣原体感染基因型临床特点及与宫颈上皮内瘤变关系的研究

发布时间：2018-06-23 06:57

本文选题：限制性片段长度多态性 + 测序　；参考：《广西医科大学》2015年博士论文

【摘要】：沙眼衣原体(Chlamydia Trachomais, CT)是一类介于病毒与细菌之间的特殊微生物。由于基因组小(基因组大小约1,038,842-1,044,459bp),生物合成途径缺乏或功能不全,只能侵入并生长于活体宿主细胞内,在普通培养基上不能存活。其感染宿主广泛,包括人、鼠、猪、羊、鸟类等,是一类人畜共患病。国外的流行病学资料显示,CT是目前性传播性疾病(Sexually Transmitted Disease, STD)感染率最高的病原体,约40%的STD由CT引起,尚有上升的趋势。人体感染CT后,在男性患者出现非淋球菌尿道炎、淋病后尿道炎、附睾炎、前列腺炎等。在女性出现宫颈炎、尿道炎、盆腔炎。很多研究结果显示,大多数生殖道的CT感染是无症状的隐形感染,或者症状轻微的亚临床感染,如果没有得到及时的诊断和治疗,将会导致一系列严重的后果,如不孕不育、异位妊娠、新生儿肺炎、结膜炎：近年来发现其与宫颈疾病发生、发展相关。是一项主要的健康及经济负担。既往大量的临床研究显示CT不同的亚型,不同的亚型具有不同的组织嗜性和致病力,可能与CT感染临床表现多样性有关。A-C型主要引起眼结膜炎症,是致盲的原因之一。D-K型引起泌尿生殖道的感染,在女性引起宫颈炎,病原体可沿生殖道黏膜上行引发上生殖道感染,是不孕、流产、盆腔炎性疾病、异位妊娠的主要病因。性病淋巴肉芽肿则归因于L1-3型感染。在动物实验中观察到不同的型别致病性及毒力不同。CT主要外膜蛋白(MOMP)是其外膜蛋白的主要成分,维持细胞完整性,构成物质及信号交换通道,同时也是CT主要的抗原成分,血清学检测通过检测感染血清中抗MOMP单克隆或多克隆抗体的差异可以对CT进行分型;MOMP氨基酸序列变化由主外膜蛋白基因(ompl)决定,基于ompl基因多态性分析的基因分型方法与血清检测法有较好的对应关系,较血清学检测方法更具有检测即时性,且敏感性高,可以提供病原体分子结构信息,用于菌株间比对和更进一步的细菌进化,药敏及耐药机理和疫苗学研究等等,目前关于CT基因分型尚无商用试剂盒,关于CT基因分型的临床运用,CT感染与宫颈疾病发生风险以及不同基因型在临床病例,尤其下生殖道感染和宫颈病变中的分布的相关资料甚少。本研究以临床横断面调查分析结合CT感染与CIN及CC发生风险的Meta分析从不同方面阐述CT感染与宫颈病变,尤其是宫颈上皮内瘤变的关系；在临床观察中,收集CT核酸扩增实验阳性的标本,CIN患者为观察组,非CIN患者为对照组,利用CT ompl基因PCR-RFLP结合ompl基因测序法进行CT基因分型,探讨CT基因型临床特点,以及与CIN发生的关系。第一章宫颈沙眼衣原体与宫颈上皮内瘤变关系的临床观察目的了解门诊就诊妇女人群中CT感染与CIN发生风险,与HR-HPV感染的相关性。方法纳入了2010年1月至2014年5月在柳州市人民医院门诊就诊的进行宫颈癌筛查12644名女性,年龄20-72岁,询问病史并记录临床症状、既往病史、孕产史、避孕方式及服用激素或抗生素情况。进行妇科检查及阴道分泌物、清洁度、乳酸杆菌、滴虫、细菌性阴道病(BV)、念珠菌,宫颈拭子沙眼衣原体(CT)、高危型人乳头瘤病毒(HR-HPV)PCR检测,并进行宫颈液基细胞学检查,对于宫颈视诊异常,HR-HPV阳性者或液基细胞学阳性者进行阴道镜检查,醋酸白试验和碘染阳性者直视下活检术,记录结果。应用SPSS软件包分析CT感染与CIN发生风险。结果 12644例门诊进行宫颈癌筛查的妇女中发生CIN例数260例,其中HR-HPV(+),CT(+)组18.00%(36/200),HR-HPV(+),CT(-)组10.26%(148／1443), HR-HPV(-),CT(+)组1.97%(29/1470),HR-HPV(-),CT(-)组0.48%(47/9531)；经过Kruskal-Wallis检验,各组间CIN发生率差异有统计学意义。多因素logistic回归分析显示,HR-HPV,合并CT感染,年龄,妊娠次数为CIN发生危险因素,产次,放置工UD避孕未增加CIN发生风险。以HR-HPV不同的感染状态进行分层,HR-HPV阴性组中,CT阳性增加CIN发生风险(OR 3.60,95%CI 2.26-5.74);而在HR-HPV阳性组中,CT阳性者较CT阴性更易发生CIN (OR 1.636,95%CI 1.104-2.424)。结论宫颈CT感染与CIN发生率增高有阳性关系。第二章沙眼衣原体感染与CIN/CC发生风险的Meta分析目的结合全球范围内的相关文献,对CT感染与CIN/CC发生风险进行Meta分析,以评价CT感染与发生宫颈恶性病变及癌前病变的风险。方法计算机检索Medline在线数据库(Pubmed)等外文及中国生物医学文献数据库(CBM)中文数据库,纳入符合标准的研究进行质量评价,对同质性研究采用Review Manager5.2软件进行Meta分析。结果在219篇文献中,筛选出8个队列研究和4个横断面调查。经过异质性检验和发表偏倚检验后,进行Meta分析,在队列研究中,CT感染与CIN/ICC发生有关(OR 2.10,95%CI 1.68-2.63),在限定HR-HPV(+)妇女中的队列研究显示CT感染是HR-HPV的协同危险因素,OR 1.77,95%CI1.48-2.12；横断面研究中亦得出相似结论,CT感染增加CIN/ICC发生风险,OR 2.89,95%CI 2.15-3.90。结论CT感染增加CIN/CC发生的风险,可能是HR-HPV致CIN的协同因素或者危险因素之一。第三章沙眼衣原体基因分型方法的构建目的通过基于编码MOMP的ompl基因PCR-RFLP方法,结合ompl基因测序建立沙眼衣原体的基因分型的实验体系和参考酶切图谱。用于临床标本的CT分型以了解不同的基因型在宫颈感染的分布以及与宫颈上皮内瘤变关系。方法收集2010年1月至2014年5月在柳州市人民医院就诊的宫颈脱落细胞CT-PCR阳性女性宫颈拭子标本167例,成功扩增ompl基因128例,使用限制性片段长度多态性(restriction fragment length polymorphism, RFLP)结合ompl基因测序法进行分型,建立反应体系,通过ompl基因测序结果比对,确定本实验条件的参考酶切图谱,用于临床标本的CT分型检测。结果CT各型ompl基因长度略有差异,总长度均在1.1kbp左右,结合ompl基因测序法,构建出不同实验条件下ompl基因PCR-RFLP酶切图谱,便于临床判读,并且通过BLASTA及多序列比对发现ompl基因序列碱基变异。结论运用PCR-RFLP结合ompl基因测序对宫颈拭子标本CT基因分型,成功通过基因测序构建本实验条件下的PCR-RFLP酶切图谱。第四章沙眼衣原体基因型分布特点与宫颈上皮内瘤变关系的研究目的使用PCR-RFLP结合ompl基因测序法进一步检测CT不同基因型别在宫颈感染中的分布情况。方法标本来自于2010年1月至2014年5月在柳州市人民医院门诊妇女CT阳性167例(包括临床观察中CT阳性CIN65例,同期宫颈活检非CIN102例),其中128例标本ompl基因成功扩增(其中CIN41例,同期宫颈活检非CIN87例),使用ompl基因PCR-RFLP进行分型,分析CT不同基因型在临床病例中不同年龄、临床表现以及CIN中分布差异。结果在门诊就诊妇女中,CT-D型最为常见(n=38,29.69%),其次为E(n=28,21.88%),G(n=21,16.41%)和F(n=16,12.50%),J、H、K较为少见,分别只有11,7,7例,未发现I型。CT感染多见于性活跃期妇女,在年龄及有无临床症状上CT基因型别分布无差别,G型易导致粘液脓性宫颈炎,易合并其他细菌或原虫感染；F型易合并其他细菌或原虫感染。CT-G易导致宫颈病理异常,E型易合并HR-HPV感染。而D型感染在合并感染及导致宫颈病理改变相对“低危”。结论 CT基因型在宫颈感染中分布不尽相同,与基因序列差异存在一定的关系。
[Abstract]:Chlamydia Trachomais (CT) is a special kind of microorganism between virus and bacteria. Because of the small genome (the genome size is about 1038842-1044459bp), the biosynthetic pathway is deficient or functional, it can only invade and grow in the living host cell, and can not survive on the ordinary medium. Including human, rat, pig, sheep, bird and so on, it is a type of zoonosis. Foreign epidemiological data show that CT is the highest infection rate of Sexually Transmitted Disease (STD), and about 40% of STD is caused by CT, and there is a rising trend. After human body infection is infected with CT, there is no gonococcal urethritis and gonorrhea in male patients. Urethritis, epididymitis, prostatitis, and so on. In women, cervicitis, urethritis, and pelvic inflammation are found in women. Many studies show that CT infection in most genital tract is asymptomatic, or mild subclinical infection, and if not diagnosed and treated in time, it will lead to a series of serious consequences, such as infertility, and difference. Pregnancy, pneumonia, conjunctivitis: it has been found in recent years that it is associated with the occurrence and development of cervix disease. It is a major health and economic burden. A large number of previous clinical studies have shown that different subtypes of CT, different subtypes have different tissue basophilia and pathogenicity, and may be related to the diversity of the clinical manifestations of CT infection related to the main.A-C type. Eye conjunctivitis is one of the causes of blindness..D-K causes the infection of the genitourinary tract. In women, it causes cervicitis. The pathogen can lead to the genital tract infection along the genital mucosa. It is the main cause of infertility, abortion, pelvic inflammatory disease, and ectopic pregnancy. The venereal granuloma of the gage is attributed to L1-3 infection. .CT main outer membrane protein (MOMP) is the main component of its outer membrane protein, which maintains cell integrity, components and signal exchange channels, and is also the main antigen component of CT. Serological detection can be applied to CT by detecting the difference of anti MOMP monoclonal or polyclonal antibody in infected serum. The variation of MOMP amino acid sequence is determined by the main epicardial protein gene (ompl). The gene typing method based on the ompl gene polymorphism analysis has a better correspondence with the serological detection method. It has more immediate detection and sensitivity than the serological detection method. It can be used to provide the molecular structure information of the pathogen, and is used for the comparison of strains and more. Further bacterial evolution, drug sensitivity and drug resistance mechanisms and vaccine studies, and so on, there are no commercial kits for the CT genotyping. The clinical use of CT genotyping, the risk of CT infection and cervical disease, and the distribution of different genotypes in clinical cases, especially in the lower genital tract infection and cervical lesions In this study, the relationship between the CT infection and cervical lesions, especially the cervical intraepithelial neoplasia, was explained in different aspects with the clinical cross-sectional survey and the Meta analysis of the CT infection and the risk of CIN and CC. In clinical observation, the specimens of CT nucleic acid amplification test positive, the CIN patients as the observation group, the non CIN patients as the control group, and the CT ompl. Gene PCR-RFLP combined with ompl gene sequencing for CT genotyping, to explore the clinical characteristics of CT genotypes, and the relationship with CIN. Chapter 1 the clinical observation of the relationship between Chlamydia trachomatis and cervical intraepithelial neoplasia in order to understand the risk of CT infection and CIN birth in the outpatient women and the correlation with HR-HPV infection. From January 2010 to May 2014, 12644 women were screened at the outpatient clinic of the Liuzhou people's Hospital for screening cervical cancer. They were aged 20-72 years old. They asked for medical history and recorded clinical symptoms, history, history of pregnancy, contraception and use of hormone or antibiotics. Gynecologic examination and vaginal secretions, cleanliness, Lactobacillus, trichomonas, bacteria, and bacteria Sexual vaginosis (BV), Candida, cervix swab, Chlamydia trachomatis (CT), high risk human papillomavirus (HR-HPV) PCR detection, and cervical cytological examination of the cervix, for abnormal cervical diagnosis, HR-HPV positive or liquid based cytology positive examination by colposcopy, white acetic acid test and iodine staining positive eyes biopsy, record the results. The SPSS software package was used to analyze the risk of CT infection and CIN. Results there were 260 cases of CIN in 12644 women screening for cervical cancer, of which HR-HPV (+), CT (+) group 18% (36/200), HR-HPV (+), CT (-) group 10.26% (148 / 1443), HR-HPV (-), CT (+) group 1.97% (29/1470), 1.97% (-), 0.48%). There was significant difference in the incidence of CIN in each group. Multiple factor Logistic regression analysis showed that HR-HPV, CT infection, age, and pregnancy times were CIN, and UD contraception did not increase the risk of CIN. The risk of HR-HPV in different infection States was stratified, and CT positive increased CIN risk in HR-HPV negative group (OR) %CI 2.26-5.74), and in HR-HPV positive group, CIN (OR 1.636,95%CI 1.104-2.424) is more likely to occur in CT positive than CT negative. Conclusion there is a positive relationship between CT infection and higher incidence of CIN in cervical cervix. The second chapter of the analysis of the risk of Chlamydia trachomatis and the risk of CIN/CC The risk was analyzed by Meta to evaluate the risk of CT infection and cervical malignant lesions and precancerous lesions. Methods computer retrieved foreign languages such as Medline online database (Pubmed) and Chinese biomedical literature database (CBM) Chinese database, and included the standard study for quality evaluation. Review Manager5.2 soft was used for the homogeneity study. Meta analysis. Results in the 219 literature, 8 cohort studies and 4 cross-sectional studies were screened. After heterogeneity test and publication bias test, Meta analysis was performed. In the cohort study, CT infection was associated with CIN/ICC (OR 2.10,95%CI 1.68-2.63). The cohort study in limited HR-HPV (+) women showed that CT infection was HR-HPV. Synergetic risk factors, OR 1.77,95%CI1.48-2.12, and a similar conclusion in cross-sectional study, CT infection increases the risk of CIN/ICC, OR 2.89,95%CI 2.15-3.90. conclusion CT infection increases the risk of CIN/CC, may be one of CIN synergistic factors or risk factors for HR-HPV. The third chapter of Chlamydia trachomatis genotyping method Gou Jianmu The experimental system of Chlamydia trachomatis genotyping and the reference enzyme cutting map were established by the ompl gene PCR-RFLP method based on the encoded MOMP gene and the ompl gene sequencing. Used in the CT typing of clinical specimens to understand the distribution of different genotypes in the cervical infection and the relationship with the cervical intraepithelial neoplasia. Methods collected from January 2010 to 2014 5 167 cervical swab specimens of cervical exfoliative CT-PCR positive women in Liuzhou people's hospital were successfully amplified by restriction fragment length polymorphism, RFLP combined with ompl gene sequencing, and the reaction system was established by sequencing the result of ompl gene, and the result was compared with the result of ompl gene sequencing. The reference enzyme cutting map of the experimental conditions was used to determine the CT typing of clinical specimens. The results showed that the length of the CT ompl genes was slightly different, the total length was around 1.1kbp, and the PCR-RFLP enzyme cutting map of the ompl gene under the different experimental conditions was constructed with the ompl gene sequencing method, which was convenient for clinical interpretation, and was transmitted through BLASTA and multiple sequence alignment. The present ompl gene sequences base variation. Conclusion using PCR-RFLP combined with ompl gene to sequence the CT genotyping of cervical swab specimens, the PCR-RFLP enzyme cutting map under the experimental conditions was successfully constructed by gene sequencing. The fourth chapter of the study on the relationship between the distribution characteristics of Chlamydia trachomatis and the intraepithelial neoplasia of cervix was combined with PCR-RFLP ompl The distribution of CT different genotypes in cervical infection was further detected by gene sequencing. Method specimens were derived from 167 cases of CT positive in outpatient hospital of Liuzhou people's Hospital from January 2010 to May 2014 (including CT positive CIN65 cases in clinical observation and non CIN102 cases of cervical biopsy in the same period), of which 128 cases were successfully amplified by ompl gene (C IN41 cases, non CIN87 cases of cervical biopsy in the same period, using ompl gene PCR-RFLP for typing, and analyzing the different ages, clinical manifestations and CIN distribution differences of different CT genotypes in clinical cases. The results are the most common in the women in the outpatient clinic (n=38,29.69%), followed by E (n=28,21.88%), G (n=21,16.41%) and PCR-RFLP. Rare, only 11,7,7 cases, no I type.CT infection was found in sexually active women, and there was no difference in the distribution of CT genotypes in age and without clinical symptoms. G was easy to cause mucous pyogenic cervicitis, easy to merge with other bacteria or protozoa infection; F type easily associated with other bacteria or protozoa infected.CT-G could lead to cervical pathological abnormalities, E type susceptibility HR-HPV infection and D type infection in combined infection and cervical pathological changes are relatively "low risk". Conclusion the distribution of CT genotypes in cervical infection is not the same, and there is a certain relationship with the difference of gene sequence.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.33

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