【作者】 周立；

【导师】 王业富；

【作者基本信息】 武汉大学 ， 生物医药工程， 2011， 博士

【摘要】 日本血吸虫病是一种分布最广泛的威胁公共健康的人畜共患寄生虫病,在包括中国在内的74个发展中国家流行。传统的检测日本血吸虫的方法主要是依靠镜检法检测粪便中日本血吸虫的虫卵,这种方法也被作为我国确诊日本血吸虫病的金标准。但是这种方法在血吸虫病低度流行区以及在经过化疗治疗后的情况下的灵敏度并不令人满意,因为日本血吸虫的虫卵是圆型的而且跟曼氏血吸虫的虫卵相比并没有那么显眼。即使病人在得病期间,从粪便中排出的虫卵分布不均匀加上排出量的波动性,这些都会影响结果的准确性。考虑到从尾蚴感染宿主到其完全成熟发育到成虫这个过程需要近一个月时间,以检测虫卵的方式确诊血吸虫病会有严重的滞后性。此外,在免疫学诊断方面,通过检测循环抗原的方式检测血吸虫在血吸虫病低度流行区以及在化疗治疗后的情况下灵敏度依然很低,而检测抗血吸虫抗体的方法在其他寄生虫感染存在时会有交叉反应,缺乏特异性。近年来,以聚合酶反应法为基础的检测方法在血吸虫的诊断中显示了它高特异性和灵敏度。但是这种方法需要对扩增的产物进行诸如电泳检测等的后续处理,不但浪费时间也容易因为交叉污染导致假阳性结果。考虑到现有的临床诊断方法、预防策略都不理想,WTO建议找寻出一种新型的策略和方法来控制血吸虫病。本文一方面建立了一种新型的快速、灵敏、特异性的诊断日本血吸虫的方法,为我国血吸虫诊断提供了新思路,建立更有效的方法；另一方面也构建了能诱导性表达的转基因小鼠,为后续找寻研发抗日本血吸虫的疫苗以及研究日本血吸虫免疫逃逸的机制打下基础。针对日本血吸病早期患者粪便和血液中日本血吸虫DNA的检测所建立的一种快速、高效超灵敏的实时诊断新技术。荧光定量PCR的方法通过以封闭反应管模式在扩增的同时进行检测的途径能够避免气溶胶引起的假阳性结果。此外,不需要后续的下游检测分析,节省了用常规凝胶电泳观察实验结果的时间。近期Wichmann运用荧光定量PCR的方法针对血吸虫诊断有了一项新的突破,能够通过针对血吸虫基因组中多拷贝基因为靶检测位点设计引物和探针,以提高检测的灵敏度,从血吸虫感染的小鼠和人的血清中检测到曼氏血吸虫核酸。临床的数据也证实该方法在对曼氏血吸虫和埃及血吸虫检测有着可靠的灵敏度和特异性。但是Wichmann的文章中也提到他们的方法的灵敏度在血吸虫的不同物种之间会有波动,而他们用于检测的靶序列还未在日本血吸虫中得到证实。我们的技术以实时荧光定量PCR为平台,以日本血吸虫18S核糖体基因为靶序列能够检测到最低10fg的日本血吸虫基因组DNA,这比常规PCR灵敏度高大约100倍。我们能够在感染后1周从小鼠血清中检测到日本血吸虫DNA；在感染后4周从小鼠粪便中检测得到阳性,这比传统的病变学检测方法提前了一周时间。该技术也能够推广应用到能够引起人感染的亚洲其他血吸虫的检测。从仅仅由雄性血吸虫感染的小鼠实验组得到的结果,虫体的DNA仅仅能在感染后的前4周检测到、这个结果说明感染后前4周的血吸虫DNA来源于降解的虫体,感染4周后的血吸虫DNA来源于新产生的卵。本课题所建立的荧光定量检测方法灵敏度高、特异性强,适用度广,能够用于日本血吸虫的早期感染诊断,并能用来监控低感染区传染病的流行情况。针对于外界病原体的入侵,建立起有效地免疫应答对于宿主防御和生存是非常重要的。IL-22起源于T细胞,主要由Th17细胞产生,能够参与机体与外界环境直接接触处的防御。在气道黏液细胞表面(主要是气管、支气管),肠胃道区域以及皮肤起到健全防御屏障和调节伤口修复的作用。已知IL-22能够在机体受到寄生虫入侵时能发挥保护作用,故我们建立了基于Tet-off/on诱导表达系统的在肺部中特异、高效表达IL-22的转基因小鼠,借此我们用来找寻一种防止血吸虫感染或者强化现有疫苗的方法。更多还原

【Abstract】 Schistosomiasis is a major infectious disease and a public health concern in many areas in China and other countries. Traditionally, detection of Schistosoma japonicum relied on microscopic detection of Schistosoma eggs in stool specimens which is also considered the golden standard for diagnosing Schistomiasis. However, this method lacks the sensitivity in low prevalence and in post-treatment situations as S. japonicum eggs are round and have fewer conspicuous characteristics than those of S. mansoni. Even when the disease is active, the analyzed samples might not contain eggs due to the random distribution and highly variable shedding of the eggs. In testing Schistosoma an important delay must be considered from the time of parasite exposure because the process of adult worm maturation after cercarial penetration is slow. Furthermore, detection of circulating parasite antigens lacks the sensitivity in the low prevalence areas and in post-treatment cases. Also, detection of anti-Schistosoma antibodies lacks specificity. Recently, several groups have employed more specific and sensitive diagnostic methods, mainly using the polymerase chain reaction (PCR) techniques. However, those PCR protocols require further processing of the amplification products, which is time consuming and prone to false-positive results because of possible cross-contamination. As the currently available clinical diagnostic methods are far from ideal, developing and evaluation of new strategies and tools for the control of Schistosomiasis are recommended by the WHO.In our study, on one hand, we established a highly sensitive assay for early detection of S. japonicum, and provided a new viable way for the detection of Schistosomiasis. On the other hand, we generated a kind of inducible transgenetic mice with specific expression of IL-22on which will help us to develop new vaccine, find an adjuvant to enhance and augment the protection of vaccine against S. japonicum or make a preparation for the further research about the effect and mechanism on the immune evasion of S. japonicumIn this study, we developed a highly sensitive TaqMan real-time PCR assay for the detection of Schistosoma japonicum DNA in mouse feces and serum samples. Compared to conventional PCR, TaqMan real-time PCR assay offers some advantages in minimizing the risk of carrying over contamination in which DNA amplification and detection is in a sealed tube or well. In addition, further down-stream analysis is not required, which shortens the time needed to obtain results by conventional electrophoresis on agarose gel. A major breakthrough was achieved by detection of cell-free S. mansoni DNA in mouse and human plasma using real-time PCR. Subsequently, the data of a clinical trial showed that this detection method had reliable sensitivity and specificity for diagnosing S. mansoni and S. haematobium infection. However, it was mentioned in the Wichmann report that the sensitivity of this assay may vary among Schistosoma species and the target gene has not been formally evaluated for S. japonicum. Our assay was based on the DNA sequence of the S. japonicum18S rRNA gene and was able to detect10fg of S. japonicum genomic DNA, which is100times more sensitive than conventional PCR. We were able to detect the S. japonicum DNA one week post-infection in mouse sera and4 weeks post-infection in feces, which was one week earlier than egg detection by microscopy in feces. This assay was also highly specific for Asian Schistosomes which are causative species of human Schistosomiasis. In single sex male cercariae infected mice, parasite DNA was only detected in the first4weeks post-infection, suggesting that the DNA was derived from decaying worms’corpse whereas the DNA detected in other infected groups was from both decaying worms’corpse and parasite eggs. Therefore we conclude that the established TaqMan real-time PCR assay is a sensitive, specific and convenient method that could be used for the early diagnostic evaluation of S. japonicum infection in humans and for monitoring outbreaks in endemic areas with low prevalence.Developing an effective immune response to an invading pathogen is critical for host defense and survival. IL-22is derived from T cells and preferentially made by Th17T cells. And IL-22participates in host defense at environmental interfaces, including mucosal surfaces of airways (mainly in the trachea and bronchia) and gastrointestinal tract as in the skin where it maintains barrier integrity and regulates wound repair. We hypothesis that IL-22has a role in the process of S. japonicum invading which could help us to find a way to protect from this helminth or enhance our current vaccines. So in our part2study, we have initiated to generate a series of transgenic mice based on Tet-off/on system, and we have got the CC10-rtTA-hGH/TRE-tight-IL-22mice successfully which expressed pretty high concentration of IL-22in the lung, and we also demonstrate here over-expression of IL-22doesn’t resulted in mortality in adult mice as we could inhibit the transgenic expression of IL-22without feeding of doxcycline before they are mature.更多还原