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  本文關(guān)鍵詞： 沙眼衣原體 E血清型 DNA疫苗 細(xì)胞免疫 體液免疫　出處：《天津醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 沙眼衣原體(Chlamydia trachomatis, C.t)所致的非淋球菌性尿道炎(non-gonococcal urethritis, NGU)是最常見的性傳播疾病(STD, sexually transmitted diseases)。其感染所引起的的前列腺炎、不孕不育、異位妊娠、慢性盆腔炎等嚴(yán)重危害著患者的身心健康。對C.t感染的研究已成為當(dāng)今國內(nèi)外研究的熱點之一，C.t的預(yù)防和控制方法亟待改進(jìn)。沙眼衣原體疫苗的研究便是預(yù)防措施中的重點之一。在眾多研究的C.t疫苗中，DNA疫苗憑借其不可比擬的優(yōu)勢成為發(fā)展的方向。遺憾的是到目前為止仍然沒有成功的疫苗問世。 目的：研究感染泌尿生殖道的C.t優(yōu)勢型—E型的DNA疫苗在小鼠體內(nèi)誘發(fā)的免疫應(yīng)答和免疫保護(hù)作用。 方法：本研究以沙眼衣原體(C.t)細(xì)胞培養(yǎng)技術(shù)為基礎(chǔ)，將C.t E血清型在McCoy細(xì)胞中培養(yǎng)，并經(jīng)過連續(xù)傳代使C.t增量。以4～5周齡BALB／c小鼠為實驗動物，將小鼠分為接種空質(zhì)粒的陰性對照組、DNA疫苗單獨(dú)免疫組和接種滅活C.t E型原體的陽性對照組。分別于第0、2、4周雙側(cè)股四頭肌肌肉接種，實驗組接種本實驗室所構(gòu)建的DNA疫苗免疫，陰性對照組小鼠接種空質(zhì)粒，陽性對照組接種滅活C.t E型原體同時輔以完全弗氏佐劑與不完全弗氏佐劑。第6周采集小鼠血清和生殖道分泌物沖洗液以檢測血清和局部C.t特異性抗體、細(xì)胞因子、抗體中和試驗；同時對部分小鼠進(jìn)行遲發(fā)型超敏反應(yīng)實驗并處死采集其脾臟以完成脾淋巴細(xì)胞增殖試驗。剩余部分右股部肌肉注射黃體酮以增強(qiáng)小鼠對C.t感染的敏感性。第7周用C.t E型原體從生殖道感染小鼠。第8周采集小鼠生殖道脫落細(xì)胞并進(jìn)行小鼠生殖道C.t的免疫熒光檢測與培養(yǎng)和計數(shù)。第9周取小鼠靜脈血、生殖道分泌物沖洗液、脾淋巴細(xì)胞和小鼠完整子宮標(biāo)本，檢測其抗體、細(xì)胞因子、脾淋巴細(xì)胞增殖情況及宮頸組織病理，從而研究所構(gòu)建的C.t E型DNA疫苗的免疫效應(yīng)。 結(jié)果：(1) C.t E型株成功培養(yǎng)并定量，結(jié)果顯示，所有的孔板90％以上細(xì)胞感染，，收集6孔板的C.t懸液，高速離心后得到的C.t EB懸液其濃度為2.1750×10~7IFU／ml IFU／mL，考馬斯亮藍(lán)法進(jìn)行C.t EB蛋白定量顯示蛋白濃度為3081.74μg／ml。(2)生殖道C.t EB的攻擊前后，檢測血清抗體、細(xì)胞因子IFN-γ、抗體中和試驗、小鼠進(jìn)行遲發(fā)型超敏反應(yīng)及脾淋巴細(xì)胞增殖試驗示所構(gòu)建的C.t E型DNA疫苗單獨(dú)免疫小鼠能夠誘導(dǎo)產(chǎn)生一定的C.t特異性細(xì)胞免疫和體液免疫。(3)生殖道分泌物衣原體免疫熒光測定、小鼠生殖道C.t培養(yǎng)和計數(shù)、組織病理活檢示陰性對照組小鼠感染C.t，而陽性對照組與實驗組未感染。 結(jié)論：(1)所構(gòu)建的C.t E型DNA疫苗單獨(dú)免疫小鼠能夠誘導(dǎo)產(chǎn)生一定的C.t特異性細(xì)胞免疫和體液免疫。(2)實驗動物針對C.t omp1基因所編碼的主要外膜蛋白(major outer membrane protein, MOMP)產(chǎn)生的抗體是保護(hù)性抗體，具有中和能力。(3)構(gòu)建的C.t E型DNA疫苗能夠在實驗小鼠體內(nèi)發(fā)揮一定的免疫保護(hù)作用。
[Abstract]:Chlamydia trachomatis (Chlamydia trachomatis, C.t) caused by non gonococcal urethritis (non-gonococcal urethritis NGU) is the most common sexually transmitted disease (STD sexually, transmitted diseases). The infection caused by prostatitis, infertility, ectopic pregnancy, chronic pelvic inflammatory disease and other serious harm to the physical and mental health of patients. The study of C.t the infection has become one of the hot research at home and abroad, the prevention and control methods of C.t need to be improved. Research on C.T vaccine is the key point of prevention measures. In a large number of C.t vaccine, DNA vaccine has become a development direction with its incomparable advantages. Unfortunately so far still not the advent of successful vaccines.
Objective: To study the immune response and immunological protection of C.t dominant E type DNA vaccine infected with genitourinary tract in mice.
Methods: in this study, Chlamydia trachomatis (C.t) cell culture technique, C.t serotype E cultured in McCoy cells, and the increment of C.t. After continuous passages in 4 ~ 5 week old BALB / c mice as experimental animal, mice were divided into negative control group inoculated with empty plasmid DNA vaccine. Individual immunity group and positive control group. C.t vaccination with inactivated type E phytoplasma. Were in the 0,2,4 week four bilateral femoral biceps muscle inoculation, the experimental group were constructed by our laboratory DNA immunization, mice were inoculated with empty plasmid negative control group, positive control group were inoculated with inactivated C.t E primary body with complete Freund Freund's adjuvant and incomplete Freund's adjuvant. Sixth weeks of collected serum and genital tract secretions washing fluid of mice to detect serum and local C.t specific antibodies, cytokines, antibody neutralization test; the mice delayed hypersensitivity experiment and death acquisition The spleen lymphocyte proliferation test. In order to complete the remaining portion of the right thigh muscle injection of progesterone to enhance mouse susceptibility to C.t infection. Seventh weeks with C.t E phytoplasma from mouse reproductive tract infection. Eighth weeks were collected vaginal exfoliated cells and mouse reproductive tract C.t immunofluorescence detection and culture and count for ninth weeks. The mice of venous blood, genital tract secretions washing liquid, spleen cells and mice uterine specimens, detection of antibodies, cytokines, spleen lymphocyte proliferation and cervical pathology, and immune effect of C.t E DNA vaccine.
Results: (1) the results of C.t E strains were successfully cultured and quantitative, showed that all of the plate more than 90% cells infected, collected 6 hole plate C.t suspension obtained after high speed centrifugation C.t EB suspension concentration of 2.1750 * 10~7IFU / ml IFU / mL, Kaumas C.t EB protein brilliantblue method the amount that the protein concentration was 3081.74 g / ml. (2) before and after genital C.t EB attacks, serum antibodies, cytokines IFN-, antibody neutralization test, mice were immunized with C.t E vaccine DNA mice delayed hypersensitivity and lymphocyte proliferation test showed the able to induce a certain C.t specific cellular immunity and humoral immunity. (3) the determination of genital tract Chlamydia immunofluorescence, mouse reproductive tract C.t culture and counting, biopsy showed negative control group of mice infected with C.t, and the positive control group and experimental group were not infected.
Conclusion: (1) C.t E DNA vaccine immunized mice alone constructed can induce C.t specific cellular immunity and humoral immunity. (2) the major outer membrane protein of experimental animal for C.t gene encoding the omp1 (major outer membrane protein, MOMP) produced by the anti body is a protective antibody, neutralizing. (3) the C.t E DNA vaccine can play a certain protective immunity in mice.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392

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