E型沙眼衣原体MOMP基因重组腺病毒转染树突状细胞免疫保护作用的研究

发布时间：2018-01-13 23:18

本文关键词：E型沙眼衣原体MOMP基因重组腺病毒转染树突状细胞免疫保护作用的研究　出处：《山东大学》2008年硕士论文　论文类型：学位论文

【摘要】： 研究背景: 沙眼衣原体(Chlamvdia trachomatis,Ct)泌尿生殖道感染是当今世界上最常见的性传播疾病(STD),每年全球有近9亿的新发病例。Ct的持续感染可以导致女性慢性盆腔炎、不孕症和异位妊娠等严重并发症,同时也提高HIV和HPV传播的几率。虽然抗生素可用于治疗衣原体的生殖道感染,由于无症状Ct感染的普遍存在给预防Ct感染和并发症带来很大困难,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蛋白抗原为基础,Ct主要外膜蛋白(Major outermembrane protein,MOMP)是引起宿主免疫反应的主要靶抗原,多数疫苗的研究以MOMP为研究对象,包括亚单位疫苗、重组疫苗、DNA疫苗、树突状细胞疫苗等,目前,这些疫苗研究均处在动物实验阶段,且免疫保护效果差异较大。在人型Ct的19个血清型中,E型为国内外最流行的血清型,占全部Ct感染的40%。与鼠型沙眼衣原体相比,人E型Ct对小鼠的毒力较弱、小鼠感染模型的建立比较困难,因而目前有关E型Ct的疫苗研究较少。为探索新型的沙眼衣原体疫苗,我们以E型沙眼衣原体为研究对象,进行了以下研究: 研究目的: 1.获得高活性高滴度的E型Ct;建立BALB/c小鼠的E型Ct生殖道感染模型;筛选最佳感染剂量进行疫苗评价。 2.评价MOMP重组腺病毒转染DC疫苗的免疫保护作用,为预防E型Ct感染提供实验依据。 3.探讨Ct感染后对DC功能的影响,为Ct致病机制的研究提供新思路。研究内容: 1.E型Ct的培养扩增及小鼠生殖道感染模型的建立改进E型Ct的培养方法,并对Ct扩增及超声破碎细胞的最佳条件进行了摸索。利用细胞培养时高营养,Ct培养低营养,超声时保持低温的原则,按改进方法进行培养,可在175cm~2的细胞培养瓶中扩增得到10~8IFU的E型Ct,为此后的实验工作奠定了基础。以不同剂量(10~4、10~5、10~6、10~7 IFU)的E型Ct阴道内感染小鼠,感染后不同时间检测小鼠阴道排菌量及生殖道组织病理改变,探讨建立生殖道感染模型的合适感染剂量。实验结果表明:经阴道接种10~5-10~7IFU E型衣原体均能造成小鼠生殖道上行性感染(感染率达100%),而10~4IFU的感染率在60%左右。生殖道主要的病理改变包括:生殖道粘膜层水肿;粘膜层上皮细胞灶性坏死,糜烂;粘膜及粘膜下层中性粒细胞浸润;血管扩张充血。以上结果表明:我们成功建立了小鼠E型Ct生殖道感染模型,10~(6-7)IFU接种能造成小鼠严重的感染,10~5 IFU是使小鼠感染致病的最小感染剂量。 2、E型Ct MOMP重组腺病毒转染树突状细胞的免疫保护作用选用Ad-MOMP转染DC疫苗免疫小鼠来评价该疫苗的体内免疫保护效果。我们主要做了以下内容: 1)DC诱导培养及重组Ad-MOMP的转染常规方法诱导培养小鼠骨髓来源的DC,流式细胞术检测可知,诱导培养6d、LPS刺激2d时CD11c阳性的DC能够达到66%,MHC-Ⅱ阳性的DC达81.54%,双标的细胞占50%,说明成功诱导培养出DC。 LPS刺激48h后,收集DC,以不同滴度的Ad-EGFP转染DC,转染后48h倒置荧光显微镜下观察表达绿色荧光蛋白的阳性细胞数。发现当MOI为1000时,90%以上的DC表达绿色荧光蛋白,故选用MOI=1000为最佳感染滴度,进行重组Ad-MOMP的转染。 2)小鼠的免疫接种选用6～8周龄BALB/c雌性小鼠,实验分组:Ad-MOMP转染DC组(Ad-MOMP-DC组)、对照空腺病毒Ad转染DC组(Ad-DC组)、死EB冲击DC组(死EB-DC组)、未转染DC组(DC组)。每只小鼠尾静脉注射1×10~6细胞/100μl,间隔两周重复免疫一次。 3)E型Ct攻击前小鼠免疫效果的测定末次免疫后两周,孕酮处理后一周,小鼠尾静脉采血,ELISA方法检测血清中Ct特异型抗体(IgG、SIgA、IgG2a/IgG1)及阴道SIgA;无菌制备小鼠脾细胞培养上清,ELISA检测细胞因子(IFN-γ/和IL-10)。实验结果表明,与Ad-DC组相比,Ad-MOMP-DC组的特异性抗体(IgG、sIgA、IgG2a)水平明显升高,阴道冲洗液中能够检测到少量的sIgA;与死EB-DC组相比,Ad-MOMP-DC组特异性抗体(IgG、sIgA、IgG2a)的水平与之相当。提示Ad-MOMP-DC免疫能够诱导小鼠产生体液免疫反应和生殖道局部粘膜免疫反应。Ad-MOMP-DC组的IgG2a水平明显升高(P＜0.05),脾细胞培养上清中可检测到高水平IFN-γ,提示免疫鼠体内建立了Th1型免疫应答。 4)E型Ct攻击后小鼠免疫效果的测定末次免疫后两周,孕酮处理一周,小鼠阴道内注射10~5IFU/10μl的E型沙眼衣原体。观察衣原体攻击后小鼠的一般状态、体重变化、阴道排菌量、生殖道Ct的定植以及小鼠生殖道的病理改变。结果发现:攻击后的小鼠,精神状态普遍不佳,进食和活动量都有减少;与Ad-DC组相比,Ad-MOMP-DC免疫组小鼠的体重下降较慢,恢复较快;阴道排菌量和生殖道Ct定植量检测发现,Ad-MOMP-DC组阴道排菌量和定植量要显著低于Ad-DC组(P＜0.05);攻击后10d的生殖道组织病理切片观察发现,未免疫组、DC组及Ad-DC组小鼠的输卵管有弥漫炎细胞浸润;与之相比,Ad-MOMP-DC组和死EB-DC组小鼠的输卵管炎症不明显,仅有少数炎细胞浸润。综合分析说明,Ad-MOMP-DC疫苗免疫对E型Ct生殖道攻击具有良好的免疫保护作用。各组小鼠的卵巢基本正常无明显炎症,说明E型Ct的感染比较缓慢,感染10天时还没有上行至卵巢。 3.鼠型沙眼衣原体感染DC后有利于DC向DC1方向分化为探讨Ct感染对DC功能的影响,我们做了两部分实验:1)选用鼠型沙眼衣原体(Chlamydia muridarum,C.muridarum)感染小鼠骨髓来源的DC,不同的时间进行包涵体的染色,发现C.muridarum在DC中可以存活并缓慢生长;与Hep-2细胞中形成的包涵体相比,在镜下观察DC内没有感染性的原体,其衣原体处于非感染状态。2)对感染后DC分泌细胞因子及促进T细胞增殖的功能进行了检测,发现感染后DC能够分泌较高水平的IL-12;并能促进同种异体T细胞增殖和分泌较高水平的IFN-γ,说明其抗原递呈功能没有受到明显的影响,C.muridarum感染可诱导DC向DC1分化。研究结论: 1.成功培养出高活性高滴度的E型Ct;建立了E型Ct小鼠生殖道感染模型;筛选出疫苗评价时所用最佳的感染剂量为10~5IFU。 2.Ad-MOMP-DC疫苗免疫可诱导小鼠产生以Th1为主的免疫反应,对E型Ct生殖道攻击具有良好的免疫保护作用。 3.成功建立沙眼衣原体感染DC的体外细胞模型,初步的功能分析发现沙眼衣原体感染可诱导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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R759

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