【作者】 张淑萍；

【导师】 侯玉英；

【作者基本信息】 山西医科大学 ， 病原生物学， 2002， 硕士

【摘要】 刚地弓形虫(Toxoplasma gondii)被认为是人类眼部疾病的一个重要致病原因。近年的研究证明，眼部弓形虫感染呈现上升趋势。视网膜脉络膜病灶可能来源于先天性或后天性感染，病变可发生于感染的急性或慢性期。通常认为健康成人，后天性弓形虫感染表现为亚临床型，且形式多样。临床证实有多种眼病与弓形虫感染相关。 多数弓形虫视网膜脉络膜炎的诊断是通过眼部检查结合血清学试验获得的，对于非典型病例，检测眼部液体中一种异常的弓形体抗体反应，分离出病原体或病理学方法均可建立眼弓形虫感染的诊断。酶联免疫吸附试验(Tox-ELISA)是目前常用的临床检测弓形虫特异性抗体的血清学方法。聚合酶链反应(Tox-PCR)是近年发展起来的一种特异性诊断方法，通过对特异病原体(Tox)DNA扩增来证实病原体感染的快速诊断手段。 实验目的：本研究旨在通过对临床眼病病例和实验小鼠进行分析，了解眼病弓形虫(Tox)感染的情况，探讨眼病与弓形虫感染间的关系；建立小鼠急性弓形虫感染的动物模型，观察Tox眼病的病理变化；为临床建立方便快捷的弓形虫感染诊断方法。 方法： 1、以住院治疗眼病患者120例及健康成人150例为对象。眼病分类：内源性葡萄膜炎80例，中心性渗出性视网膜脉络膜炎(简称“中渗”)30例，先天性脉络膜缺损6例，渗出性视网膜脱离4例。对照组(健康成人组)由在校大学生90人和农场工人60人组成，有猫、狗等动物接触史：眼病患者17例，健康成人76例。应用Tox-IgM-ELISA和Tox-PCR对120例眼病患者和150例健康成人的血液标本进行分析。用x~2检验进行统计学分析。 2、健康成年昆明小鼠30只，雌雄各半，无菌条件下行腹腔接种纯化弓形体RH株活虫苗0.1ml(Tox含量4×10~3／ml)，饲养三周后处死小鼠，摘取双眼，取一眼进行Tox-PCR检测，另一眼用10％甲醛固定，石蜡切片，常规HE染色，光镜下观察视山冠穿医科大学不页d二学位论文网膜玻璃体结构改变。 结果: 120例眼病患者中，血清Tox一IgM阳性感染率n.67% (14/120)，健康对照组150例中阳性Tox一IgM感染率4%(6/150)，二者比较x性4.65，尸<0.05，差异有显著性。眼病患者不同病种间血清Tox一IgM阳性率比较，中渗患者阳性率26.67%(8/30)，葡萄膜炎阳性率8.33%(6/80)，二者比较x2一5.59，尸<0.05，差异有显著性。健康成年组间血清学Tox一IgM阳性感染率比较，大学生组2.22%(2/90)，工人组6.67%(4/60)，二者比较x性0.86，尸>0.05，二者间无显著性差异。 血清Tox一PCR检测结果:眼病患者14例Tox一工gM阳性者PCR结果均为阳性，健康对照组6例T。x一IgM阳性者Tox一PCR阳性5例，两者相符率83.3%。经统计学x’检验，xZ一5.86，尸<0.02，差异有显著性。 实验鼠眼组织均浆Tox一PCR检测结果为阳性，眼病理切片示视网膜脉络膜各层组织无坏死性改变，视网膜内层与色素上皮层分离，各层内有大量炎细胞浸润，视网膜神经纤维层内见大量新生血管形成，部分突入玻璃体腔，引起玻璃体炎症反应和增殖反应，玻璃体可见炎症细胞和红细胞。 结论: 1、本组资料眼病患者的弓形虫感染明显高于健康成人，眼病中“中渗”患者较葡萄膜炎患者的Tox感染率高，证实本地区部分眼病的发生与弓形虫感染有关，应引起临床的高度重视。 2、血清Tox一IgM一ELISA法可作为急性眼Tox感染敏感、简便的诊断方法，可用于后天性弓形虫感染的人群普查;Tox一PCR则可作为眼弓形虫感染的特异性指标，快速、易行、结果确切，能为临床治疗提供依据，并进一步对治疗效果进行监测。 3、本研究建立了小鼠眼弓形虫急性感染模型，用PCR法证实了弓形虫感染后有小部分病原体进入眼组织，直接引起眼组织的病理改变，刺激引起局部炎症细胞聚集。急性鼠眼弓形虫感染的病理改变以视网膜脉络膜炎症反应和增殖性反应为主，炎症细胞可浸润视网膜各层及玻璃体，病理改变符合葡萄膜炎表现。 弓形虫感染的眼部表现形式多样，与宿主的免疫状态直接相关，本研究分析了眼病与弓形虫急性感染间的关系，发现眼病中山1穿医科大学不页士学位论文有弓形虫的感染，且“中渗”的弓形虫感染率高于其它眼病，需进一步加大临床样本，进行验证。实验鼠眼的弓形虫原位PCR将有待解决眼弓形虫感染的定位难题。更多还原

【Abstract】 Toxoplasma gondii is considered as an important pathogen of human ocular disease. Recent study has proved that the infection of ocular toxoplasma is increasing. Retinochoroidal focus maybe comes from congenital or acquired infection, which occurs at the acute or chronic stage of the infection. In general, the acquired toxoplasma infection in healthy adults is sub-clinical type with different forms, clinical practice shows that many ocular diseases are linked with toxoplasma infection.Most diagnosis of toxoplasma retinochoroiditis is acquired by ocular examination combined with serology test. To some nontypical cases, testing an abnormal toxoplasma antibody reaction in ocular liquid, separating the pathogen or through the pathological way can build the diagnosis of ocular toxoplasma infection. Tox-ELISA is the usual clinical serology method to test the specific toxoplasma antibody at present. Tox-PCR is a recently developed specific diagnostic technique by amplifying the Tox-DNA to test the toxoplasma infection rapidly.Objectives:1)To learn the condition of ocular toxoplasma infection by analyzing clinical cases with ocular disease and mice models, discuss the relation between ocular disease and toxoplasma infection;2)To build animal models of mice acute toxoplasma infection, and observe their pathological changes;3)To provide a convenient and rapid clinical diagnostic techniquefor toxoplasma infection.Methods:l)We studied 120 inpatients and 150 healthy adults. The classifications of ocular diseases: 80 endogenous uveitis, 30 central exudative chorioretinitis, 6 congenital choroidal defect and 4 exudative retinal detachment. The control group composed of 90 college students and 60 farm workers, 17 ocular patients and 76 healthy adults had the history of contacting cat or dog etc. We analyzed the blood preparations of 120 ocular patients and 150 healthy adults by Tox-IgM-ELISA and Tox-PCR. Data were analyzed with x 2test.2)We injected the pured live toxoplasma RH into the abdominal cavity of 30 healthy adult mice(Kunming, half male and half female) in sterile condition, killed them 3 weeks later and extracted both eyes. One eye was performed Tox-PCR test, the other was fixed with 10% Formaldehyde, paraffin section and HE stain. Observed the structural changes of retina and vitreous with light microscope.Results:The sero Tox-IgM test was positive in 11.67% (14/120) patients among 120 cases with ocular disease, 4% (6/150) in the’control group, the difference was significant. (x2=4.65, P<0.05). 26.67% (8/30) positive rate in patients with central exudative chorioretinitis, 8.33% (6/80) positive in patients with uveitis, the difference was significant x 2=5.59,P<0.05). The serology Tox-IgM test was positive in 2.22% (2/90) college students and 6.67% (4/60) workers. The difference was not significant x 2=0.86, />0.05).The results of sero Tox-PCR test: 14 Tox-IgM positive cases with ocular disease were all positive in PCR test, 5 cases were PCR positive in 6 Tox-IgM positive cases among the healthy control group. The difference was significant x 2=5.86,P<0.02).The Tox-PCR test was positive in the experimental mice ocular tissue. The pathological section found that there was no necrosing change in every layer of retina and choroid. The pigment epithelium layer detached from the inner layer of retina, and there was amount of inflammatory cellular infiltration in each layer. Lots of new vascularformed in the retinal nerve fiber layer and some of them stretched into the vitreous body thus induced the inflammation and proliferation, and we could see red cells and inflammatory cells in the vitreous.Conclusion:1)The rate of the toxoplasma infection in this group is much higher than that in healthy adults, and the Tox infection rate in central exudative chorioretinitis is higher than uveitis. It is proved that some ocular diseases are connected with toxoplasma infection, and we should pay much attention to it in clinic.2)The sero Tox-IgM-ELISA is a simple and sen更多还原