【作者】 张庆华；

【导师】 侯玉英；

【作者基本信息】 山西医科大学 ， 病原生物学， 2004， 硕士

【摘要】 研究目的：通过建立免疫抑制家兔感染弓形虫的模型，观察家兔的发病、血循环中抗体的动态变化，各脏器组织的虫体和抗原的分布及病理学改变，来分析和探讨家兔感染弓形虫后的发病情况、免疫特点及致病现象，较准确、可靠地推导免疫缺损者感染弓形虫后的免疫现象及致病机制，为准确的诊断弓形虫病和抗弓形虫感染的研究提供可靠的理论依据，而且对预防和控制弓形虫病具有重要的理论性指导意义。 研究方法：用酶联免疫吸附试验（ELISA）筛选弓形虫抗体（IgM和IgG）阴性的家兔20只，随机分为正常对照组（A），免疫抑制组（B），接种虫体组（C）和免疫抑制接种虫体组（D）。A组家兔不做任何处理， B组家兔肌肉注射地塞米松（2mg/ 只/日），C组家兔腹腔注射弓形虫（5×104个速殖子/只），D组家兔在接种前48小时肌肉注射地塞米松（2mg /只/日），48小时后腹腔注射弓形虫，观察家兔的体毛、体重、食量、眼睛有无分泌物和眼底的变化；接种后0、5、7、9、12、15、18、22、26、30、34、38、42、46天从家兔耳缘静脉采血，用ELISA法检测弓形虫IgM与IgG含量；给药第50天处死家兔、解剖、摘取眼、脑、肺、心、肝、脾，肉眼观察各脏器组织是否有病变；将各脏器组织印片、吉-瑞氏液染色, 观察速殖子的分布；取固定的肝、脑组织块石蜡包埋、切片、采用免疫组化染色（SABC法），观察弓形虫抗原的分布；取固定的各脏器组织块石蜡包埋、切片、采用苏木素-伊红染色，观察组织的病理学变化。 结果与讨论：家兔的发病情况正常对照组与免疫抑制组家兔未见异常表现，接种虫体组家兔第6天出现体毛疏松，10天后基本恢复正常，整个实验中没有死亡现象。免疫抑制接种虫体组家兔第4天出现体毛疏松，10天后出现脱毛、怠动、厌食、体重下降，腹胀、眼部有分泌物；停药后症状逐渐减轻、趋于正常；第2次重复给药后又出现以上症状，并呼吸快、眼底视乳头轻度充血，并有一<WP=5>只家兔肢体瘫痪、抽风、昏迷而死亡，说明随着发作次数的增多，病变也逐渐加重。从实验中发现接种虫体组家兔感染后是一过性的发作，随着保护性免疫的产生, 症状逐渐减轻、消失，可能是由于体内的虫体转入隐性感染的原因。免疫抑制接种虫体组家兔在两次重复给药都出现明显的病症，说明免疫抑制剂可诱发弓形虫病的发作，症状重且持续时间长，严重者可危及生命，造成死亡，因此有一只家兔出现死亡。2. 家兔弓形虫抗体的动态变化正常对照组与免疫抑制组家兔未接种弓形虫，所以无特异性抗体产生，曲线也无动态变化的现象。接种虫体组家兔的IgM第7天出现，并逐渐上升，15天达高峰，维持于26天后下降，但仍高于初始水平；IgG第12天出现，也逐渐上升，38天达高峰，维持至实验结束。免疫抑制接种虫体组家兔的IgM第5天出现，逐渐上升，12天达高峰，22天下降，38天又出现第二个峰值；IgG第9天出现，逐渐上升，34天达高峰，维持至实验结束。从以上接种虫体的两组家兔观察中都有弓形虫特异性抗体的产生，证实两组家兔均感染成功。在两组的特异性抗体动态变化中，免疫抑制接种虫体组家兔抗体（IgM与IgG）出现较早，而接种虫体组抗体出现晚，两组抗体滴度随感染天数增加而升高。免疫抑制接种虫体组家兔整个抗体的动态曲线比接种虫体组高，而且在两次给免疫抑制剂期间IgM出现两个峰值，是否由于免疫抑制剂降低家兔免疫力，其体内虽然产生抗体但保护性作用弱，对弓形虫的识别能力不强，虫体则在体内大量繁殖，刺激机体又产生相应的抗体，这些抗体是否在维持机体的平衡中起着重要的免疫调理作用有待于探讨。 3. 家兔体内弓形虫速殖子的分布正常对照组与免疫抑制组家兔的腹腔液、组织印片未发现速殖子。接种虫体组家兔的腹腔液无色而清，腹腔液和组织印片可见散在的弓形虫速殖子。免疫抑制接种虫体组家兔为浑浊或血性腹水，腹水和组织印片可见大量的弓形虫速殖子。 <WP=6>4. 家兔体内弓形虫抗原的分布经免疫组化检测，正常对照组与免疫抑制组家兔的肝与脑组织的弓形虫抗原表达为阴性。接种虫体组家兔的肝、脑实质组织的细胞浆内有少量散在的棕黄色阳性抗原表达，免疫抑制接种虫体组家兔的肝、脑实质组织的细胞浆与胞核内均有大量的、聚集成堆的棕黄色阳性抗原表达。说明虫体通过血循环可播散至各脏器组织，若虫体大量繁殖，可引起病变。5. 家兔各脏器组织的病理学改变正常对照组与免疫抑制组家兔均未出现异常病理性变化。接种虫体组的家兔有轻度炎性改变。免疫抑制接种虫体组的家兔肝细胞坏死，脾脏结构破坏，肺泡多数萎陷，心肌纤维断裂伴脂肪变，脑神经元层次不清，眼组织的神经上皮层被破坏、视神经纤维排列紊乱稀疏，表明接种虫体组家兔仅有轻微炎性改变，而免疫抑制接种虫体组家兔为器质性改变。说明免疫正常宿主感染弓形虫后病变是功能性的，而免疫抑制的宿主感染弓形虫后的病变是器质性的。我们通过建立免疫抑制家兔感染弓形虫的模型，观察到家兔在免疫功能抑制状态下感染弓形虫后，可出现弓形虫病、体内抗体（IgM和IgG）有动态变化的现象、各脏器组织也有病理学改变，基本证实了免疫抑制剂不仅降低宿主免疫更多还原

【Abstract】 Purpose of studyBy via of creating animal model of immunosuppressed rabbits infected with toxoplasma, we observed desease episoding , antibody developing changes, distribution of toxoplasma and antigen in each organ and histopathological features of constitutions in order to analysis and investigate episoding characteristic, immunological features and pathogenic phenomenon when rabbits infected with toxoplasma. So we can deduce immunity and pathogenic mechanism exactly and trustly while immunocompromised persons infected by toxoplasma for exact diagnosis of toxoplasmosis and reasearch of anti-toxoplasma infection,and it is of important themry and instruction significance for preventing and controlling toxoplasmosis.Methods of studyTwenty rabbits whose body weight（BW）was about 2kg and antibody（IgM and IgG）in serum was negative after ELISA detecting were randomly divided into four groups: A group: normal control group, B group: immunosuppressed group injected with DEX, C group: control group infected with T gondii, D group: immunosuppressed group injected with T gondii after injecting desamethasone（DEX）. A group had no any treatment, B group was muscularly injected with DEX （2mg/each/day）, C group was peritoneally infected with tachyzoites of RH strain（5×104/each）, D group was muscularly injected with DEX （2mg/each/day）72 hours prior to inoculation and was peritoneally infected <WP=8>tachyzoites of RH strain（5×104/each）after 72 hours. We observed hairs, body weight, appetite, secretion of eyes and changes of fundus of eyes with funduscope. We collected blood from ear-border vein at the day of 0,5,7,9,12,15,18,22,26,30,34,38,42,46 after inoculation and detected toxoplasma antibody（IgM and IgG）contents in serum with ELISA. We put rabbits to death at the day of 50 after administration, dissected , selected eye, brain, lung, heart, liver, spleen and observed having lesions or not with naked eyes; then printing constitutions on slides, staining in Gimesa-Wright liquid to observe distribution of tachyzoites for light microscopy; embed liver and brain fixed in paraffin and cut sections and detected with immunohistochemistry（SABC method） to observe distribution of toxoplasma antigen for light microscopy;embed constitutions fixed in paraffin and cut sections and stained with hematoxylin-eosin to observe histopathological features of constitutions for light microscopy.Results and discussion1. rabbits episoding During the experiment, appearance and behavior of rabbits of normal control group and immunosuppressed group had no changes. Rabbits of control group infected with toxoplasma presented hairs rarefaction at the day of 6 and basicly presented normal after ten days. There was no dying throughout the experiment; immunocompromised rabbits presented hairs rarefaction at the day of 4 and presented dehairing, activities decreasing, anorexia, BW descending, abdomen exponding, having secretion in eyes; symptom gradually relieved and tended to be normal after drug withdrawal; after administration again, presented above symptom again, respiring urgently, opic papilla hyperemia gently. A rabbit presented limbs paralysing, convulsion, coma and died, which indicated that the more episoding, the severer lesions.We can detected from the experiment that rabbits of control group infected with toxoplasma occurred temporary attack, following protective immunity <WP=9>yielded, symptom gradually relieved and disappeared, probably transformed into subclinical infection. Rabbits of immunosuppressed group injected with toxoplasma presented overt disorder after two administration replicatedly, which indicated immunosuppressive agent can induce toxoplasmosis episoding, and symptoms are severe but last for a long time, the severe person can endanger life to die. 2. antibody developing changes of rabbits in serum Rabbits of both normal control group and immunosuppresed group were not inoculate toxoplasma, so had no specific antibody ,and curves had no developing changes yet. IgM of rabbits injected wi更多还原