本文關(guān)鍵詞：E型沙眼衣原體MOMP基因重組腺病毒轉(zhuǎn)染樹(shù)突狀細(xì)胞免疫保護(hù)作用的研究　出處：《山東大學(xué)》2008年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 沙眼衣原體 E型 基因 MOMP 疫苗 重組腺病毒 樹(shù)突狀細(xì)胞

【摘要】： 研究背景: 沙眼衣原體(Chlamvdia trachomatis,Ct)泌尿生殖道感染是當(dāng)今世界上最常見(jiàn)的性傳播疾病(STD),每年全球有近9億的新發(fā)病例。Ct的持續(xù)感染可以導(dǎo)致女性慢性盆腔炎、不孕癥和異位妊娠等嚴(yán)重并發(fā)癥,同時(shí)也提高HIV和HPV傳播的幾率。雖然抗生素可用于治療衣原體的生殖道感染,由于無(wú)癥狀Ct感染的普遍存在給預(yù)防Ct感染和并發(fā)癥帶來(lái)很大困難,因此研制安全有效的疫苗是控制Ct泌尿生殖道感染的最好方法。 目前Ct疫苗研究主要以Ct蛋白抗原為基礎(chǔ),Ct主要外膜蛋白(Major outermembrane protein,MOMP)是引起宿主免疫反應(yīng)的主要靶抗原,多數(shù)疫苗的研究以MOMP為研究對(duì)象,包括亞單位疫苗、重組疫苗、DNA疫苗、樹(shù)突狀細(xì)胞疫苗等,目前,這些疫苗研究均處在動(dòng)物實(shí)驗(yàn)階段,且免疫保護(hù)效果差異較大。在人型Ct的19個(gè)血清型中,E型為國(guó)內(nèi)外最流行的血清型,占全部Ct感染的40%。與鼠型沙眼衣原體相比,人E型Ct對(duì)小鼠的毒力較弱、小鼠感染模型的建立比較困難,因而目前有關(guān)E型Ct的疫苗研究較少。為探索新型的沙眼衣原體疫苗,我們以E型沙眼衣原體為研究對(duì)象,進(jìn)行了以下研究: 研究目的: 1.獲得高活性高滴度的E型Ct;建立BALB/c小鼠的E型Ct生殖道感染模型;篩選最佳感染劑量進(jìn)行疫苗評(píng)價(jià)。 2.評(píng)價(jià)MOMP重組腺病毒轉(zhuǎn)染DC疫苗的免疫保護(hù)作用,為預(yù)防E型Ct感染提供實(shí)驗(yàn)依據(jù)。 3.探討Ct感染后對(duì)DC功能的影響,為Ct致病機(jī)制的研究提供新思路。 研究?jī)?nèi)容: 1.E型Ct的培養(yǎng)擴(kuò)增及小鼠生殖道感染模型的建立 改進(jìn)E型Ct的培養(yǎng)方法,并對(duì)Ct擴(kuò)增及超聲破碎細(xì)胞的最佳條件進(jìn)行了摸索。利用細(xì)胞培養(yǎng)時(shí)高營(yíng)養(yǎng),Ct培養(yǎng)低營(yíng)養(yǎng),超聲時(shí)保持低溫的原則,按改進(jìn)方法進(jìn)行培養(yǎng),可在175cm~2的細(xì)胞培養(yǎng)瓶中擴(kuò)增得到10~8IFU的E型Ct,為此后的實(shí)驗(yàn)工作奠定了基礎(chǔ)。 以不同劑量(10~4、10~5、10~6、10~7 IFU)的E型Ct陰道內(nèi)感染小鼠,感染后不同時(shí)間檢測(cè)小鼠陰道排菌量及生殖道組織病理改變,探討建立生殖道感染模型的合適感染劑量。實(shí)驗(yàn)結(jié)果表明:經(jīng)陰道接種10~5-10~7IFU E型衣原體均能造成小鼠生殖道上行性感染(感染率達(dá)100%),而10~4IFU的感染率在60%左右。生殖道主要的病理改變包括:生殖道粘膜層水腫;粘膜層上皮細(xì)胞灶性壞死,糜爛;粘膜及粘膜下層中性粒細(xì)胞浸潤(rùn);血管擴(kuò)張充血。以上結(jié)果表明:我們成功建立了小鼠E型Ct生殖道感染模型,10~(6-7)IFU接種能造成小鼠嚴(yán)重的感染,10~5 IFU是使小鼠感染致病的最小感染劑量。 2、E型Ct MOMP重組腺病毒轉(zhuǎn)染樹(shù)突狀細(xì)胞的免疫保護(hù)作用 選用Ad-MOMP轉(zhuǎn)染DC疫苗免疫小鼠來(lái)評(píng)價(jià)該疫苗的體內(nèi)免疫保護(hù)效果。我們主要做了以下內(nèi)容: 1)DC誘導(dǎo)培養(yǎng)及重組Ad-MOMP的轉(zhuǎn)染 常規(guī)方法誘導(dǎo)培養(yǎng)小鼠骨髓來(lái)源的DC,流式細(xì)胞術(shù)檢測(cè)可知,誘導(dǎo)培養(yǎng)6d、LPS刺激2d時(shí)CD11c陽(yáng)性的DC能夠達(dá)到66%,MHC-Ⅱ陽(yáng)性的DC達(dá)81.54%,雙標(biāo)的細(xì)胞占50%,說(shuō)明成功誘導(dǎo)培養(yǎng)出DC。 LPS刺激48h后,收集DC,以不同滴度的Ad-EGFP轉(zhuǎn)染DC,轉(zhuǎn)染后48h倒置熒光顯微鏡下觀察表達(dá)綠色熒光蛋白的陽(yáng)性細(xì)胞數(shù)。發(fā)現(xiàn)當(dāng)MOI為1000時(shí),90%以上的DC表達(dá)綠色熒光蛋白,故選用MOI=1000為最佳感染滴度,進(jìn)行重組Ad-MOMP的轉(zhuǎn)染。 2)小鼠的免疫接種 選用6～8周齡BALB/c雌性小鼠,實(shí)驗(yàn)分組:Ad-MOMP轉(zhuǎn)染DC組(Ad-MOMP-DC組)、對(duì)照空腺病毒Ad轉(zhuǎn)染DC組(Ad-DC組)、死EB沖擊DC組(死EB-DC組)、未轉(zhuǎn)染DC組(DC組)。每只小鼠尾靜脈注射1×10~6細(xì)胞/100μl,間隔兩周重復(fù)免疫一次。 3)E型Ct攻擊前小鼠免疫效果的測(cè)定 末次免疫后兩周,孕酮處理后一周,小鼠尾靜脈采血,ELISA方法檢測(cè)血清中Ct特異型抗體(IgG、SIgA、IgG2a/IgG1)及陰道SIgA;無(wú)菌制備小鼠脾細(xì)胞培養(yǎng)上清,ELISA檢測(cè)細(xì)胞因子(IFN-γ/和IL-10)。實(shí)驗(yàn)結(jié)果表明,與Ad-DC組相比,Ad-MOMP-DC組的特異性抗體(IgG、sIgA、IgG2a)水平明顯升高,陰道沖洗液中能夠檢測(cè)到少量的sIgA;與死EB-DC組相比,Ad-MOMP-DC組特異性抗體(IgG、sIgA、IgG2a)的水平與之相當(dāng)。提示Ad-MOMP-DC免疫能夠誘導(dǎo)小鼠產(chǎn)生體液免疫反應(yīng)和生殖道局部粘膜免疫反應(yīng)。Ad-MOMP-DC組的IgG2a水平明顯升高(P＜0.05),脾細(xì)胞培養(yǎng)上清中可檢測(cè)到高水平IFN-γ,提示免疫鼠體內(nèi)建立了Th1型免疫應(yīng)答。 4)E型Ct攻擊后小鼠免疫效果的測(cè)定 末次免疫后兩周,孕酮處理一周,小鼠陰道內(nèi)注射10~5IFU/10μl的E型沙眼衣原體。觀察衣原體攻擊后小鼠的一般狀態(tài)、體重變化、陰道排菌量、生殖道Ct的定植以及小鼠生殖道的病理改變。結(jié)果發(fā)現(xiàn):攻擊后的小鼠,精神狀態(tài)普遍不佳,進(jìn)食和活動(dòng)量都有減少;與Ad-DC組相比,Ad-MOMP-DC免疫組小鼠的體重下降較慢,恢復(fù)較快;陰道排菌量和生殖道Ct定植量檢測(cè)發(fā)現(xiàn),Ad-MOMP-DC組陰道排菌量和定植量要顯著低于Ad-DC組(P＜0.05);攻擊后10d的生殖道組織病理切片觀察發(fā)現(xiàn),未免疫組、DC組及Ad-DC組小鼠的輸卵管有彌漫炎細(xì)胞浸潤(rùn);與之相比,Ad-MOMP-DC組和死EB-DC組小鼠的輸卵管炎癥不明顯,僅有少數(shù)炎細(xì)胞浸潤(rùn)。綜合分析說(shuō)明,Ad-MOMP-DC疫苗免疫對(duì)E型Ct生殖道攻擊具有良好的免疫保護(hù)作用。各組小鼠的卵巢基本正常無(wú)明顯炎癥,說(shuō)明E型Ct的感染比較緩慢,感染10天時(shí)還沒(méi)有上行至卵巢。 3.鼠型沙眼衣原體感染DC后有利于DC向DC1方向分化 為探討Ct感染對(duì)DC功能的影響,我們做了兩部分實(shí)驗(yàn):1)選用鼠型沙眼衣原體(Chlamydia muridarum,C.muridarum)感染小鼠骨髓來(lái)源的DC,不同的時(shí)間進(jìn)行包涵體的染色,發(fā)現(xiàn)C.muridarum在DC中可以存活并緩慢生長(zhǎng);與Hep-2細(xì)胞中形成的包涵體相比,在鏡下觀察DC內(nèi)沒(méi)有感染性的原體,其衣原體處于非感染狀態(tài)。2)對(duì)感染后DC分泌細(xì)胞因子及促進(jìn)T細(xì)胞增殖的功能進(jìn)行了檢測(cè),發(fā)現(xiàn)感染后DC能夠分泌較高水平的IL-12;并能促進(jìn)同種異體T細(xì)胞增殖和分泌較高水平的IFN-γ,說(shuō)明其抗原遞呈功能沒(méi)有受到明顯的影響,C.muridarum感染可誘導(dǎo)DC向DC1分化。 研究結(jié)論: 1.成功培養(yǎng)出高活性高滴度的E型Ct;建立了E型Ct小鼠生殖道感染模型;篩選出疫苗評(píng)價(jià)時(shí)所用最佳的感染劑量為10~5IFU。 2.Ad-MOMP-DC疫苗免疫可誘導(dǎo)小鼠產(chǎn)生以Th1為主的免疫反應(yīng),對(duì)E型Ct生殖道攻擊具有良好的免疫保護(hù)作用。 3.成功建立沙眼衣原體感染DC的體外細(xì)胞模型,初步的功能分析發(fā)現(xiàn)沙眼衣原體感染可誘導(dǎo)DC向DC1分化。
[Abstract]:Research background:
Chlamydia trachomatis (Chlamvdia trachomatis, Ct) of genitourinary tract infection is the most common sexually transmitted disease in the world today (STD), every year nearly 900 million new cases of.Ct infection can cause the female chronic pelvic inflammatory disease, infertility and ectopic pregnancy and other serious complications, but also improve the probability of transmission of HIV and HPV although antibiotics can be used for the treatment of chlamydial genital tract infection, due to widespread asymptomatic Ct infection has caused great difficulties to the prevention of Ct infection and complications, so the development of safe and effective vaccine is the best way to control Ct infection in urogenital tract.
The present study mainly by Ct protein antigen Ct vaccine based on Ct, the major outer membrane protein (Major outermembrane protein, MOMP) is a major target antigen causing the host immune response, most vaccine research with MOMP as the research object, including subunit vaccines, recombinant vaccines, DNA vaccines, dendritic cell vaccine, at present, these vaccines study on the stage of animal experiment, and the immune protective effect of differences. In 19 serum type Ct, serotype E is the most popular at home and abroad, the total Ct infection with Chlamydia trachomatis 40%. rats compared to E Ct on mice was weak, difficult to establish infection in mice the model, therefore the current vaccine research on E type Ct is less. In order to explore the new Chlamydia trachomatis vaccines, we with Chlamydia trachomatis E as the research object, has carried on the following research:
The purpose of the study is:
1. E type Ct with high activity and high titer was obtained; E type Ct reproductive tract infection model of BALB/c mice was established, and the best infection dose was screened for vaccine evaluation.
2. the immuno protective effect of MOMP recombinant adenovirus transfected to DC vaccine was evaluated to provide experimental basis for preventing E type Ct infection.
3. to explore the effect of Ct infection on the function of DC, and to provide a new idea for the study of the pathogenesis of Ct.
Research content:
The culture and amplification of 1.E type Ct and the establishment of a mouse reproductive tract infection model
Cultivation method of improved E type Ct, and the best conditions for Ct amplification and ultrasonic cell disruption were studied by cell culture. When high nutrition, Ct culture and low nutrition, low temperature ultrasound according to the principle of maintaining and improving methods of training, training can be amplified to E Ct in 10~8IFU in 175cm~2 cells. The experimental work has laid the foundation for this post.
At different doses (10~4,10~5,10~6,10~7 IFU) in mice infected with E type Ct vagina, different time after infection detection of mouse vaginal discharge of bacteria and reproductive tract tissue pathological changes, to explore the establishment of appropriate infection dose model of reproductive tract infection. The experimental results show that the 10~ 5-10~7IFU E type vaginal inoculation of chlamydia can cause genital tract of mice ascending the infection (infection rate 100%), while the 10~4IFU infection rate was about 60%. The main pathological changes of the reproductive tract including: genital tract mucosa edema; mucosa epithelial cell necrosis, mucosa and mucosal erosion; neutrophil infiltration layer; vascular dilatation and congestion. The above results show that we have successfully established a model the reproductive tract infection in mice E type Ct, 10~ (6-7) IFU inoculation can cause serious infection in mice, 10~5 IFU is the minimum infective dose of the mice were infected with the disease.
2, the immuno protective effect of recombinant adenovirus E Ct MOMP recombinant adenovirus transfected on dendritic cells
Ad-MOMP transfected DC vaccine was used to immunize mice to evaluate the protective effect of the vaccine in vivo.
1) DC induced culture and transfection of recombinant Ad-MOMP
Mice bone marrow derived DC were induced by routine method. Flow cytometry showed that CD11c positive DC reached 66%, 6D positive DC reached 81.54% and double labeled cells accounted for 50% when induced LPS 2D stimulation, indicating successful induction of DC..
LPS after 48h stimulation, DC collected with different titer of Ad-EGFP transfected DC, 48h after transfection under inverted fluorescence microscope to observe the expression of positive cells of green fluorescent protein. It is found that when MOI was 1000, the expression of green fluorescent protein more than 90% DC, so MOI=1000 was selected as the best infection titer, transfection of recombinant Ad-MOMP.
2) immunization of mice
Select 6 ~ 8 week old BALB/c female mice, experimental groups: Ad-MOMP transfection of DC group (Ad-MOMP-DC group) and control adenovirus transfected Ad DC group (group Ad-DC), group DC (EB shock death death EB-DC group) and non transfected DC group (DC group). Each mouse tail vein injection of 1 * 10~6 /100 cell l, two week interval repeated immunization at a time.
3) determination of immune effect in mice before E Ct attack
Two weeks after the last immunization, a week after progesterone treatment, mice tail vein blood, Ct type specific ELISA method for detection of serum antibodies (IgG, SIgA, IgG2a/IgG1) and vaginal SIgA; aseptic preparation of mouse spleen cell culture supernatant, ELISA detection of cytokines (IFN- and gamma / IL-10). The experimental results show that compared to with the Ad-DC group, Ad-MOMP-DC group specific antibodies (IgG, sIgA, IgG2a) significantly increased, vaginal washing fluid can be detected in a small amount of sIgA; compared with the death of EB-DC group, Ad-MOMP-DC group specific antibody (IgG, sIgA, IgG2a) level of Ad-MOMP-DC. When prompted to produce humoral immune the reproductive tract reaction and local mucosal immune responses in.Ad-MOMP-DC group had significantly higher levels of IgG2a induced mice (P < 0.05), spleen cells were cultured to detect high levels of IFN- gamma supernatant, suggesting that the immune mice establish Th1 type immune response.
4) determination of immune effect in mice after E Ct attack
Two weeks after the last immunization, progesterone treatment week, mouse vaginal injection of 10~5IFU/10 l of Chlamydia trachomatis E. The changes of body weight in mice to observe the general state, Chlamydia attacks, vaginal discharge amount of bacteria, the change of genital Ct colonization and mouse reproductive tract pathology. Results: after the attack in spirit the state is generally poor, eating and activity had decreased; compared with Ad-DC group, Ad-MOMP-DC immunized mice weight decreased slowly, and fast recovery; vaginal discharge amount of bacteria and genital Ct colonization rate detected in Ad-MOMP-DC group, vaginal discharge amount of bacteria and colonization rate was significantly lower than in Ad-DC group (P < 0.05); after the attack the reproductive tract histopathology observation of 10d, non immune group, DC group and Ad-DC group of mice oviduct diffuse infiltration of inflammatory cells; compared with Ad-MOMP-DC group and EB-DC group of mice died of salpingitis is not obvious, only a few inflammatory cell infiltration Comprehensive analysis showed that Ad-MOMP-DC vaccine immunization had good immunological protection against E Ct reproductive tract attacks. Mice in each group were basically normal without obvious inflammation, indicating that the infection of E Ct was relatively slow, and the infection did not go up to the ovary on the 10 day of infection.
3. mice infected with Chlamydia trachomatis after DC are beneficial to the differentiation of DC into DC1 direction
In order to investigate the effect of Ct infection on the function of DC, we do the two part of the experiment: 1 mice) of Chlamydia trachomatis (Chlamydia muridarum, C.muridarum) infection of murine bone marrow derived DC staining, different time of inclusion body, found that C.muridarum could survive and slow growth in DC; compared with the inclusion formation in Hep-2 cells the observation of no infectious pathogens of DC in under the microscope, the Chlamydia noninfectious.2) on cytokine secretion of DC after infection and promote the proliferation of T cell function were detected, the infection was found DC can secrete higher levels of IL-12; and can promote the proliferation of allogenic T cells and the secretion of high level IFN- gamma, the antigen presenting function was not significantly affected, C.muridarum infection can induce DC1 to differentiate into DC.
The conclusions are as follows:
1., E type Ct with high activity and high titer was successfully cultivated. A E Ct mouse reproductive tract infection model was established, and the best dose of 10~5IFU. was selected for vaccine evaluation.
The immunization of 2.Ad-MOMP-DC vaccine can induce Th1 - based immune response in mice, which has a good protective effect on the E type Ct reproductive tract attack.
3. a cell model in vitro was successfully established for Chlamydia trachomatis infection in DC. Preliminary functional analysis showed that Chlamydia trachomatis infection could induce DC to differentiate into DC1.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392;R759

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