【作者】 董志炜；

【导师】 潘善培；

【作者基本信息】 暨南大学 ， 发育生物学， 2005， 硕士

【摘要】 目的:评价卵透明带DNA避孕疫苗pVAX1-pZP3α壳聚糖纳米微粒经滴鼻和阴道免疫两种途径诱发的粘膜免疫反应、抗生育作用和对卵巢结构的影响,提高卵透明带DNA避孕疫苗的避孕率,并进一步探讨用粘膜免疫避免卵透明带抗原免疫引起卵巢功能紊乱的可行性。方法:制备pVAX1-pZP3α壳聚糖纳米微粒,分别滴鼻和阴道免疫10只BALB/c小鼠,对照组免疫不含DNA的壳聚糖,第3周和第6周分别加强免疫一次,ELISA法检测小鼠血清、阴道冲洗液中抗pZP3 IgA和IgG抗体;首次免疫后第10周与有生育力的雄鼠合笼交配,统计怀孕雌鼠动物数;首次免疫后第13周解剖动物取出双侧卵巢,制作石蜡切片,作卵巢组织病理学观察。为检测卵透明带DNA避孕疫苗pZP3α抗原在体内的表达,构建重组质粒pEGFP-N1-pZP3α,与壳聚糖制备成纳米微粒,经鼻粘膜和阴道粘膜免疫小鼠,免疫后第2、4、6、8、10天解剖动物,取滴鼻免疫小鼠的气管、肺部组织和阴道免疫小鼠的阴道组织制作冰冻切片,直接在荧光显微镜下观察。结果:在滴鼻和阴道免疫小鼠血清和阴道冲洗液中可以检测到抗pZP3αIgA抗体,抗pZP3 IgA抗体反应强度与持续时间有明显的个体差异;滴鼻免疫组8只小鼠(8/10)未怀孕,阴道免疫组5只小鼠(5/7)未怀孕,对照组全部怀孕,抗生育效果与合笼时血清抗pZP3 IgA反应强度有关;免疫组未怀孕小鼠与免疫组怀孕小鼠及对照组小鼠的卵巢组织均未观察到病理学变化。小鼠免疫pEGFP-N1-pZP3α壳聚糖纳米微粒后制作的冰冻切片在荧光显微镜下观察显示,与对照组小鼠相比,从免疫后第4天开始在免疫组小鼠气管、肺部、阴道的上皮细胞中可以观察到比较微弱的绿色荧光,提示以壳聚糖为释放系统经滴鼻和阴道免疫后在这些部位有pZP3α抗原的表达。结论:pVAX1-pZP3α壳聚糖纳米微粒滴鼻和阴道免疫小鼠能诱发生殖道粘膜免疫反应,产生较好的避孕效果,并对卵巢组织结构没有影响。粘膜免疫是克服卵透明带抗原免疫引起卵巢功能紊乱的一种值得深入研究的途径。更多还原

【Abstract】 Aim: To evaluate the mucosal immune response, antifertility effect and pathological change of ovarian induced by intranasal and vaginal immunization with zona pellucida DNA contraceptive vaccine, and explore the feasibility of overcoming ovary dysfunction, which was often caused by immunization of zona pellucida antigen, and improving contraceptive efficacy by inducing mucosal immunity using chitosan/pVAX1-pZP3α nanoparticles. Methods: Chitosan/pVAX1-pZP3α nanoparticles was prepared and administered into each experimental mouse on weeks 0,3 and 6 by intranasal and intravaginal respectively, whereas chitosan without DNA was given to control. Serum and vaginal washes samples collected every two weeks were used to determine anti-pZP3 IgA and IgG response by ELISA. On week 10, these female mice were mated with male mice and were checked for pregnant. On week 13, the mice were killed and ovarian serial section was prepared for microscopy. For study expression of the antigen of zona pellucida DNA vaccine in vivo, recombinant plasmid pEGFP-N1-pZP3α was constructed. Tracheas, lungs were moved from mice intranasally immunized by chitosan/pEGFP-N1-pZP3α nanoparticles and vaginas from vaginal group on day 2, 4, 6, 8, 10 and frozen section was prepared for fluorescence microscopy. Results: Anti-pZP3 IgA antibody response could be detected in serum and vaginal washes, intensity and lasting time varying over a wide range among different mouse. 8 mice (8/10) in intranasal group and 5 mice (5/7) in vaginal group didn’t fall pregnant while the control did. Anti-fertility was relative to anti-pZP3 IgA antibody level in serum when mating mice. There was no difference about ovarian histology among unpregnant, pregnant experimental mice and control mice. No pathological changes were found in ovaries of immunized mice. Since day 4 after given plasmid pEGFP-N1-pZP3α, faint fluorescence detected in some epithelial cells of tracheas, lungs and vaginas indicated DNA expression in these tissues. Conclusion: Intranasal and vaginal immunization with chitosan/pVAX1-pZP3α nanoparticles could induce mucosal immune response ingenital tract and inhibit fertility without any effect on ovarian structure in mice. Inducing mucosal immunity would be a promising regimen for study of developing a safe zona pellucida contraceptive vaccine.更多还原