本文選題：限制性片段長度多態(tài)性 + 測序�。� 參考：《廣西醫(yī)科大學(xué)》2015年博士論文

【摘要】：沙眼衣原體(Chlamydia Trachomais, CT)是一類介于病毒與細(xì)菌之間的特殊微生物。由于基因組小(基因組大小約1,038,842-1,044,459bp),生物合成途徑缺乏或功能不全,只能侵入并生長于活體宿主細(xì)胞內(nèi),在普通培養(yǎng)基上不能存活。其感染宿主廣泛,包括人、鼠、豬、羊、鳥類等,是一類人畜共患病。國外的流行病學(xué)資料顯示,CT是目前性傳播性疾病(Sexually Transmitted Disease, STD)感染率最高的病原體,約40%的STD由CT引起,尚有上升的趨勢。人體感染CT后,在男性患者出現(xiàn)非淋球菌尿道炎、淋病后尿道炎、附睪炎、前列腺炎等。在女性出現(xiàn)宮頸炎、尿道炎、盆腔炎。很多研究結(jié)果顯示,大多數(shù)生殖道的CT感染是無癥狀的隱形感染,或者癥狀輕微的亞臨床感染,如果沒有得到及時的診斷和治療,將會導(dǎo)致一系列嚴(yán)重的后果,如不孕不育、異位妊娠、新生兒肺炎、結(jié)膜炎：近年來發(fā)現(xiàn)其與宮頸疾病發(fā)生、發(fā)展相關(guān)。是一項主要的健康及經(jīng)濟(jì)負(fù)擔(dān)。既往大量的臨床研究顯示CT不同的亞型,不同的亞型具有不同的組織嗜性和致病力,可能與CT感染臨床表現(xiàn)多樣性有關(guān)。A-C型主要引起眼結(jié)膜炎癥,是致盲的原因之一。D-K型引起泌尿生殖道的感染,在女性引起宮頸炎,病原體可沿生殖道黏膜上行引發(fā)上生殖道感染,是不孕、流產(chǎn)、盆腔炎性疾病、異位妊娠的主要病因。性病淋巴肉芽腫則歸因于L1-3型感染。在動物實驗中觀察到不同的型別致病性及毒力不同。CT主要外膜蛋白(MOMP)是其外膜蛋白的主要成分,維持細(xì)胞完整性,構(gòu)成物質(zhì)及信號交換通道,同時也是CT主要的抗原成分,血清學(xué)檢測通過檢測感染血清中抗MOMP單克隆或多克隆抗體的差異可以對CT進(jìn)行分型;MOMP氨基酸序列變化由主外膜蛋白基因(ompl)決定,基于ompl基因多態(tài)性分析的基因分型方法與血清檢測法有較好的對應(yīng)關(guān)系,較血清學(xué)檢測方法更具有檢測即時性,且敏感性高,可以提供病原體分子結(jié)構(gòu)信息,用于菌株間比對和更進(jìn)一步的細(xì)菌進(jìn)化,藥敏及耐藥機(jī)理和疫苗學(xué)研究等等,目前關(guān)于CT基因分型尚無商用試劑盒,關(guān)于CT基因分型的臨床運用,CT感染與宮頸疾病發(fā)生風(fēng)險以及不同基因型在臨床病例,尤其下生殖道感染和宮頸病變中的分布的相關(guān)資料甚少。本研究以臨床橫斷面調(diào)查分析結(jié)合CT感染與CIN及CC發(fā)生風(fēng)險的Meta分析從不同方面闡述CT感染與宮頸病變,尤其是宮頸上皮內(nèi)瘤變的關(guān)系；在臨床觀察中,收集CT核酸擴(kuò)增實驗陽性的標(biāo)本,CIN患者為觀察組,非CIN患者為對照組,利用CT ompl基因PCR-RFLP結(jié)合ompl基因測序法進(jìn)行CT基因分型,探討CT基因型臨床特點,以及與CIN發(fā)生的關(guān)系。第一章 宮頸沙眼衣原體與宮頸上皮內(nèi)瘤變關(guān)系的臨床觀察目的了解門診就診婦女人群中CT感染與CIN發(fā)生風(fēng)險,與HR-HPV感染的相關(guān)性。方法 納入了2010年1月至2014年5月在柳州市人民醫(yī)院門診就診的進(jìn)行宮頸癌篩查12644名女性,年齡20-72歲,詢問病史并記錄臨床癥狀、既往病史、孕產(chǎn)史、避孕方式及服用激素或抗生素情況。進(jìn)行婦科檢查及陰道分泌物、清潔度、乳酸桿菌、滴蟲、細(xì)菌性陰道病(BV)、念珠菌,宮頸拭子沙眼衣原體(CT)、高危型人乳頭瘤病毒(HR-HPV)PCR檢測,并進(jìn)行宮頸液基細(xì)胞學(xué)檢查,對于宮頸視診異常,HR-HPV陽性者或液基細(xì)胞學(xué)陽性者進(jìn)行陰道鏡檢查,醋酸白試驗和碘染陽性者直視下活檢術(shù),記錄結(jié)果。應(yīng)用SPSS軟件包分析CT感染與CIN發(fā)生風(fēng)險。結(jié)果 12644例門診進(jìn)行宮頸癌篩查的婦女中發(fā)生CIN例數(shù)260例,其中HR-HPV(+),CT(+)組18.00%(36/200),HR-HPV(+),CT(-)組10.26%(148／1443), HR-HPV(-),CT(+)組1.97%(29/1470),HR-HPV(-),CT(-)組0.48%(47/9531)；經(jīng)過Kruskal-Wallis檢驗,各組間CIN發(fā)生率差異有統(tǒng)計學(xué)意義。多因素logistic回歸分析顯示,HR-HPV,合并CT感染,年齡,妊娠次數(shù)為CIN發(fā)生危險因素,產(chǎn)次,放置工UD避孕未增加CIN發(fā)生風(fēng)險。以HR-HPV不同的感染狀態(tài)進(jìn)行分層,HR-HPV陰性組中,CT陽性增加CIN發(fā)生風(fēng)險(OR 3.60,95%CI 2.26-5.74);而在HR-HPV陽性組中,CT陽性者較CT陰性更易發(fā)生CIN (OR 1.636,95%CI 1.104-2.424)。結(jié)論 宮頸CT感染與CIN發(fā)生率增高有陽性關(guān)系。第二章沙眼衣原體感染與CIN/CC發(fā)生風(fēng)險的Meta分析目的 結(jié)合全球范圍內(nèi)的相關(guān)文獻(xiàn),對CT感染與CIN/CC發(fā)生風(fēng)險進(jìn)行Meta分析,以評價CT感染與發(fā)生宮頸惡性病變及癌前病變的風(fēng)險。方法計算機(jī)檢索Medline在線數(shù)據(jù)庫(Pubmed)等外文及中國生物醫(yī)學(xué)文獻(xiàn)數(shù)據(jù)庫(CBM)中文數(shù)據(jù)庫,納入符合標(biāo)準(zhǔn)的研究進(jìn)行質(zhì)量評價,對同質(zhì)性研究采用Review Manager5.2軟件進(jìn)行Meta分析。結(jié)果在219篇文獻(xiàn)中,篩選出8個隊列研究和4個橫斷面調(diào)查。經(jīng)過異質(zhì)性檢驗和發(fā)表偏倚檢驗后,進(jìn)行Meta分析,在隊列研究中,CT感染與CIN/ICC發(fā)生有關(guān)(OR 2.10,95%CI 1.68-2.63),在限定HR-HPV(+)婦女中的隊列研究顯示CT感染是HR-HPV的協(xié)同危險因素,OR 1.77,95%CI1.48-2.12；橫斷面研究中亦得出相似結(jié)論,CT感染增加CIN/ICC發(fā)生風(fēng)險,OR 2.89,95%CI 2.15-3.90。結(jié)論CT感染增加CIN/CC發(fā)生的風(fēng)險,可能是HR-HPV致CIN的協(xié)同因素或者危險因素之一。第三章沙眼衣原體基因分型方法的構(gòu)建目的通過基于編碼MOMP的ompl基因PCR-RFLP方法,結(jié)合ompl基因測序建立沙眼衣原體的基因分型的實驗體系和參考酶切圖譜。用于臨床標(biāo)本的CT分型以了解不同的基因型在宮頸感染的分布以及與宮頸上皮內(nèi)瘤變關(guān)系。方法收集2010年1月至2014年5月在柳州市人民醫(yī)院就診的宮頸脫落細(xì)胞CT-PCR陽性女性宮頸拭子標(biāo)本167例,成功擴(kuò)增ompl基因128例,使用限制性片段長度多態(tài)性(restriction fragment length polymorphism, RFLP)結(jié)合ompl基因測序法進(jìn)行分型,建立反應(yīng)體系,通過ompl基因測序結(jié)果比對,確定本實驗條件的參考酶切圖譜,用于臨床標(biāo)本的CT分型檢測。結(jié)果CT各型ompl基因長度略有差異,總長度均在1.1kbp左右,結(jié)合ompl基因測序法,構(gòu)建出不同實驗條件下ompl基因PCR-RFLP酶切圖譜,便于臨床判讀,并且通過BLASTA及多序列比對發(fā)現(xiàn)ompl基因序列堿基變異。結(jié)論運用PCR-RFLP結(jié)合ompl基因測序?qū)m頸拭子標(biāo)本CT基因分型,成功通過基因測序構(gòu)建本實驗條件下的PCR-RFLP酶切圖譜。第四章沙眼衣原體基因型分布特點與宮頸上皮內(nèi)瘤變關(guān)系的研究目的 使用PCR-RFLP結(jié)合ompl基因測序法進(jìn)一步檢測CT不同基因型別在宮頸感染中的分布情況。方法標(biāo)本來自于2010年1月至2014年5月在柳州市人民醫(yī)院門診婦女CT陽性167例(包括臨床觀察中CT陽性CIN65例,同期宮頸活檢非CIN102例),其中128例標(biāo)本ompl基因成功擴(kuò)增(其中CIN41例,同期宮頸活檢非CIN87例),使用ompl基因PCR-RFLP進(jìn)行分型,分析CT不同基因型在臨床病例中不同年齡、臨床表現(xiàn)以及CIN中分布差異。結(jié)果 在門診就診婦女中,CT-D型最為常見(n=38,29.69%),其次為E(n=28,21.88%),G(n=21,16.41%)和F(n=16,12.50%),J、H、K較為少見,分別只有11,7,7例,未發(fā)現(xiàn)I型。CT感染多見于性活躍期婦女,在年齡及有無臨床癥狀上CT基因型別分布無差別,G型易導(dǎo)致粘液膿性宮頸炎,易合并其他細(xì)菌或原蟲感染；F型易合并其他細(xì)菌或原蟲感染。CT-G易導(dǎo)致宮頸病理異常,E型易合并HR-HPV感染。而D型感染在合并感染及導(dǎo)致宮頸病理改變相對“低�！薄＝Y(jié)論 CT基因型在宮頸感染中分布不盡相同,與基因序列差異存在一定的關(guān)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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R737.33

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