沙眼衣原体E型DNA疫苗免疫效应的研究

发布时间：2018-01-23 00:07

本文关键词： 沙眼衣原体 E血清型 DNA疫苗细胞免疫体液免疫　出处：《天津医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 沙眼衣原体(Chlamydia trachomatis, C.t)所致的非淋球菌性尿道炎(non-gonococcal urethritis, NGU)是最常见的性传播疾病(STD, sexually transmitted diseases)。其感染所引起的的前列腺炎、不孕不育、异位妊娠、慢性盆腔炎等严重危害着患者的身心健康。对C.t感染的研究已成为当今国内外研究的热点之一，C.t的预防和控制方法亟待改进。沙眼衣原体疫苗的研究便是预防措施中的重点之一。在众多研究的C.t疫苗中，DNA疫苗凭借其不可比拟的优势成为发展的方向。遗憾的是到目前为止仍然没有成功的疫苗问世。目的：研究感染泌尿生殖道的C.t优势型—E型的DNA疫苗在小鼠体内诱发的免疫应答和免疫保护作用。方法：本研究以沙眼衣原体(C.t)细胞培养技术为基础，将C.t E血清型在McCoy细胞中培养，并经过连续传代使C.t增量。以4～5周龄BALB／c小鼠为实验动物，将小鼠分为接种空质粒的阴性对照组、DNA疫苗单独免疫组和接种灭活C.t E型原体的阳性对照组。分别于第0、2、4周双侧股四头肌肌肉接种，实验组接种本实验室所构建的DNA疫苗免疫，阴性对照组小鼠接种空质粒，阳性对照组接种灭活C.t E型原体同时辅以完全弗氏佐剂与不完全弗氏佐剂。第6周采集小鼠血清和生殖道分泌物冲洗液以检测血清和局部C.t特异性抗体、细胞因子、抗体中和试验；同时对部分小鼠进行迟发型超敏反应实验并处死采集其脾脏以完成脾淋巴细胞增殖试验。剩余部分右股部肌肉注射黄体酮以增强小鼠对C.t感染的敏感性。第7周用C.t E型原体从生殖道感染小鼠。第8周采集小鼠生殖道脱落细胞并进行小鼠生殖道C.t的免疫荧光检测与培养和计数。第9周取小鼠静脉血、生殖道分泌物冲洗液、脾淋巴细胞和小鼠完整子宫标本，检测其抗体、细胞因子、脾淋巴细胞增殖情况及宫颈组织病理，从而研究所构建的C.t E型DNA疫苗的免疫效应。结果：(1) C.t E型株成功培养并定量，结果显示，所有的孔板90％以上细胞感染，，收集6孔板的C.t悬液，高速离心后得到的C.t EB悬液其浓度为2.1750×10~7IFU／ml IFU／mL，考马斯亮蓝法进行C.t EB蛋白定量显示蛋白浓度为3081.74μg／ml。(2)生殖道C.t EB的攻击前后，检测血清抗体、细胞因子IFN-γ、抗体中和试验、小鼠进行迟发型超敏反应及脾淋巴细胞增殖试验示所构建的C.t E型DNA疫苗单独免疫小鼠能够诱导产生一定的C.t特异性细胞免疫和体液免疫。(3)生殖道分泌物衣原体免疫荧光测定、小鼠生殖道C.t培养和计数、组织病理活检示阴性对照组小鼠感染C.t，而阳性对照组与实验组未感染。结论：(1)所构建的C.t E型DNA疫苗单独免疫小鼠能够诱导产生一定的C.t特异性细胞免疫和体液免疫。(2)实验动物针对C.t omp1基因所编码的主要外膜蛋白(major outer membrane protein, MOMP)产生的抗体是保护性抗体，具有中和能力。(3)构建的C.t E型DNA疫苗能够在实验小鼠体内发挥一定的免疫保护作用。
[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.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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