【作者】 杨秀红；

【导师】 秦川；

【作者基本信息】 中国协和医科大学 ， 病理与病理生理学， 2007， 博士

【摘要】 严重急性呼吸综合征(severe acute respiratory syndrome,SARS)是本世纪初爆发的由一种新的冠状病毒引起的传染性疾病,主要的靶器官是肺脏,病死率达10%左右。SARS已经淡出人们的生活已有四年多了,目前的研究主要集中在SARS发病机制和抗病毒药物、疫苗的研发上,而这些研究的基础是可靠的SARS动物模型。已有文献报道非人灵长类(恒河猴、食蟹猴、绒猴、非洲绿猴)、小鼠(BABL/c、C57/B6、基因修饰小鼠)、仓鼠、狸子猫、雪貂等均能支持SARS-CoV的复制,其中有些动物显示了不同程度的病理变化,但在临床疾病和病理上仍缺乏与SARS病人的相似性。据研究,SARS-CoV是通过其表面的S蛋白和靶细胞的表面受体血管紧张素转换酶2(ACE2)结合侵入宿主细胞的,这提示人ACE2(hACE2)转基因鼠有可能作为对SARS-CoV易感的动物模型,因此我们进行了hACE2转基因鼠对SARS-CoV易感性的实验研究并利用此动物模型探讨了SARS的发病机制。本研究主要包括以下三个部分的实验内容:1.hACE2转基因小鼠的制备和鉴定:选择小鼠ACE2(mACE2)启动子驱动的hACE2全长编码序列(Coding sequence,CDS)基因片断,通过显微注射的方法,制备携带hACE2基因的转基因小鼠。经PCR检测到4个阳性首建鼠;RT-PCR及Nested RT-PCR结果显示在其中一个Founder的子代鼠的肺、心、肾和小肠有hACE2基因表达;Realtime-PCR检测此Founder子代鼠转基因的拷贝数,结果显示为单拷贝转基因小鼠;Western blot结果显示hACE2蛋白在转基因小鼠的不同组织包括肺、心、肾和小肠均有表达;免疫组化显示,在转基因小鼠的肾脏近曲小管上皮细胞、肺脏小血管内皮细胞可见hACE2蛋白表达:无创性血压监测观察发现,hACE2转基因小鼠的心率、血压与野生鼠相比未见明显差异;病理组织学检查未发现转基因小鼠心、肺、肾等器官组织结构异常。2.hACE2转基因小鼠对SARS—CoV易感性的实验:经鼻腔接种PUMC01株SARS-CoV后,野生型和转基因小鼠接种SARS—CoV后均未出现死亡现象。在接种后的第3天和第7天,RT-PCR和免疫荧光试验(IFA)结果显示,转基因小鼠肺组织上清的病毒载量和病毒滴度明显高于野生型小鼠(P＜0.05);在接种后的第7天,转基因小鼠的感染率明显高于野生型小鼠(P＜0.01);转基因小鼠出现典型的间质性肺炎,而且伴有肾脏、心脏、肝脏、脑组织和胃肠道等肺外组织的损伤。而部分野生型小鼠只在感染后第三天出现轻微的间质性肺炎。通过免疫组化染色,在转基因小鼠的肺脏上皮细胞和血管内皮细胞及大脑神经细胞可发现SARS-CoV抗原;接种病毒后在部分感染小鼠的血清中可检测到SARS-CoV特异性的抗体IgG和IgM,但转基因小鼠抗体阳性率低于野生型小鼠。上述资料表明,hACE2转基因小鼠对SARS-CoV的易感性明显高于野生型小鼠。3.利用hACE2转基因小鼠探讨非特异性的免疫反应在SARS发病中的作用:应用ELISA的方法,观察到转基因小鼠接种病毒后,肺组织上清细胞因子IL-6、TNF-α和IFN-γ明显升高;Western Blot和免疫组化染色检测到转基因小鼠感染SARS-CoV后肺组织一氧化氮合酶(iNOS)表达明显增强,阳性颗粒主要位于肺泡巨噬细胞;比色法测定到转基因小鼠感染SARS-CoV后血浆中NO含量明显增多。综上所述,hACE2在我们制备的转基因小鼠体内部分组织表达,尽管接种病毒后具有缺少病死率的局限性,但hACE2转基因小鼠的易感性明显高于野生型小鼠,病理变化与SARS病人相似。这种模型的建立对于研究SARS发病机制和抗病毒药物的评价具有重要的意义。更多还原

【Abstract】 Severe acute respiratory syndrome(SARS),an infectious disease which pathogenic agent was a novel coronavirus,broke out in 2003 and rapidly spreaded to many countries in the world.The main target organ of SARS was lung and the fatality rate was approximately 10%.Animal models provided an important tool for studying SARS pathogenesis and evaluating the efficacy of potential drugs and vaccines.Established models for SARS infection included cynomologus macaques,ferrets,cats, mice,African green monkeys,and Golden Syrian hamsters.Some of them presented lung pathological changes of human SARS infection with different severity.However,most of them lacked comparability to SARS patients on clinical diseases and pathology.The spike proteins of SARS-CoV bind to receptor on host cells,named angiotensin-converting enzyme-2(ACE2),and mediate viral entry.This indicates that a mouse transgenic for human ACE2(hACE2) could serve as an animal model for SARS infection.Thus,we produced hACE2 transgenic mice and observed its susceptibility to SARS-CoV.In addition, we detected changes of some cytokines in the inoculated mice.The main works were included below. 1.The production and identification of hACE2 transgenic mice:The mACE2 promoter driving hACE2 CDS fragments were introduced into the pronuclei of ICR fertilized ova using microinjection.PCR was used to screen for the transgene in the genomic DNA of potential transgenic mice. RNA analysis by RT-PCR and nested RT-PCR showed that hACE2 mRNA was transcribed in the lung,heart,kidney,and intestine of transgenic mice. Levels of transgene DNA in founder3 was a single copy in genome as determined by Realtime-PCR.Western blot indicated that hACE2 protein levels of the lung,heart,kidney,and intestine from transgenic mice were all higher than that from wild-type mice.By Immunohistochemistry,hACE2 protein was detected in epithelial cells of kidney and vascular endothelial cells of lung.The uninjured detection of blood pressure of transgenic mice showed that there was not difference between wild and transgenic mice.No organs abnormalities were found in transgenic mice under microscope.2.The experiment of susceptibility of transgenic mice to SARS-CoV:After inoculated intranasally by PUMC01 strain of SARS-CoV,none of thirty-six challenged wildtype and transgenic mice was dead by one week post-infection.The results of RT-PCR and immunofluoresce assay(IFA) indicated that the replication of SARS-CoV in the lung homogenates from transgenic mice were higher than from wild-type mice by day 3 and 7 post-inoculation.The transgenic mice present severe and typical interstitial pneumonia accompanied by many extra-pulmonary organ damages.The wild mice just showed mild interstitial pneumonia in day 3 post-infection. By immunohistochemistry(IHC),the antigen of SARS-CoV was found in epithelial cells,vascular endothelial cells of lung and cerebral neurocytes of transgenic mice.The specific antibodies were found both in wild and transgenic mice.However,the positive number in transgenic mice is less than that in wild mice.The above results showed that the hACE2 transgenic mice were more susceptible to SARS-CoV than wild mice.3.The study of mechanism of SARS on transgenic mice:By ELISA,we found that levels of TNF-α,IL-6 and IFN-γin the lung homogenates from transgenic mice were increased more markedly than wild mice after they were inoculated with SARS-CoV.The expression of inducible nitric oxide synthase(iNOS) in the lung of infected transgenic mice was higher than that of wild mice by Western blot and IHC.The positive signal was found in macrophages in the alveoli.The level of nitric oxide(NO) increased greatly in serum of transgenic mice.To sum up,hACE2 was expressed in some tissues of transgenic mice. Although it had limitation of the absence of lethality,transgenic mice model were more susceptible than wild mice with resembling to SARS patients in pathology and should still aid the evaluation of anti-SARS-CoV drugs and vaccines,as well as the analysis of SARS pathogenesis by virus detection and systemic pathological studies.更多还原