附红细胞体感染小鼠的血液学指标检测及药物治疗效果的研究
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摘要
[目的]:检测附红细胞体感染小鼠后的血液学指标变化;探讨附红细胞体的发病机理;观察药物对附红细胞体感染小鼠的治疗效果。[方法]:1.附红细胞体感染小鼠的血液学指标检测:采用了光学显微镜镜检、透射电镜观察以及PCR检测方法,鉴定小鼠感染附红细胞体的模型的建立;测定红细胞膜ATP酶活性、血液常规指标、红细胞超氧化物歧化酶活性、红细胞免疫功能以及一氧化氮含量。2.附红细胞体感染小鼠的药物疗效观察:随机将48只感染附红细胞体的小鼠分成4组,每组12只。第一组为感染组,第二组为感染后不治疗组称为阳性对照组。第三组为青蒿琥酯治疗组,剂量为60mg/kg,第四组为丁胺卡那霉素联合青蒿琥酯治疗组,丁胺卡那霉素的剂量为400mg/kg,青蒿琥酯的剂量为60mg/kg,经静脉注射连续治疗7天。再取12只正常小鼠为阴性对照。每天从感染的小鼠各采血制成鲜血压滴标本,油镜镜检。观察其感染率,测定血液常规指标,在治疗7天后,对血样进行PCR检测。[结果]:鲜血滴片镜检和瑞氏-姬姆萨染色涂片均能清晰地观察到附红细胞体的形态结构,通过透射电镜可以观察到附红细胞体的超微结构,并发现其附着于红细胞表面。根据已有的猪附红细胞体特异性片段设计引物,成功地扩增得到801bp的PCR产物。附红细胞体感染组小鼠的红细胞数量显著低于正常组小鼠的红细胞数量(P<0.01),附红细胞体感染组小鼠的血红蛋白含量、免疫复合物(RBC-C3b)花环率、红细胞的ATP酶含量显著低于正常组小鼠的血红蛋白含量、免疫复合物(RBC-C3b)花环率、红细胞的ATP酶含量(P<0.05),附红细胞体感染组小鼠的红细胞压积、超氧化物歧化酶活性略低于正常组小鼠的红细胞压积、超氧化物歧化酶活性(P>0.05),附红细胞体感染组小鼠的嗜中性粒细胞数略低于正常组小鼠的嗜中性粒细胞数(P>0.05),附红细胞体感染组小鼠的白细胞总数、淋巴细胞数、一氧化氮含量略高于正常组小鼠的白细胞总数、淋巴细胞数、一氧化氮含量(P>0.05),附红细胞体感染组小鼠的红细胞C3b受体(RBC-IC)花环率显著高于正常组小鼠的红细胞C3b受体(RBC-IC)花环率(P<0.05)。经过青蒿琥酯联合丁胺卡那霉素治疗后,附红细胞体感染率逐渐降低,且红细胞已恢复正常;单用青蒿琥酯治疗结束后,红细胞基本恢复正常;阳性对照组在未经治疗后的第7d附红细胞体的感染率为23.08%,与青蒿琥酯联合丁胺卡那霉素组、青蒿琥酯治疗组相比有显著性差异(P<0.05)。经药物治疗后,青蒿联合丁胺卡那霉素组和青蒿琥酯组的红细胞数极显著增加(P<0.01),血红蛋白含量显著增加(P<0.05),各组的红细胞压积相比无显著性差异(P>0.05)。各组小白鼠血液样本经PCR检测,感染组和阳性对照组出现特异性片断,青蒿琥酯组和青蒿联合丁卡组未扩增出特异性片断。[结论]:1.采用鲜血滴片镜检,瑞氏-姬姆萨染色镜检,透射电镜镜检的方法均可作为检测附红细胞体的手段。但光学显微镜镜检存在一定的不足和缺陷。采用PCR检测和透射电镜检测方法可以特异性检测出附红细胞体。2.附红细胞体可以感染SPF昆明小鼠。采用6周龄昆明小鼠作为实验动物,以纯化的人附红细胞体作为感染源攻毒,可以使SPF昆明小鼠感染人附红细胞体,成功地建立了感染模型。3.附红细胞体感染试验小鼠后可引起其红细胞膜结构发生变形,红细胞易于溶解和破裂,造成血液中生理生化指标发生改变。4.青蒿琥酯联合丁胺卡那霉素的疗效较单用青蒿琥酯好,能迅速清除体内附红细胞体,使红细胞的功能和数量迅速恢复正常。
[Objective] To detect the changes of hematological index of mice infected by Eperythrozoon to explore the etiopathogenesis of Eperythrozoon;to compare the therapeutic effects of different drugs treated the infection. [Methods]1. The detection of hematological index of mice infected by Eperythrozoon:①Confirm the establishment of infected model by optical microscope examination, transmission electron microscope observation and PCR assay to amplify distinctive genes;②Evaluate the ATPase activity of erythrocyte membrane, routine hematological index, SOD activity, immune function and NO content of erythrocyte.2.The comparison of therapeutic effects of different drugs treated the infection:12 normal mice were grouped as negative control, and another 48 mice infected by Eperythrozoon were randomly divided to 4 groups(each 12): Group I was mice infected, Group II was treated with nothing as positive control, Group III was with Artesunate(60mg/kg), while Group IV with Artesunate(60mg/kg) associated with amikacin sulphate (400mg/kg).Drugs were injected through intravenous pass for 7 days. In order to evaluate rate of infection, blood samples of mice were collected to make blood slider during the treatment every day, which were tested under oil immersion lens. And the hematological index was detected at the same time.PCR assays were then undertaken to test the blood collected after the treatment. [Results] The morphosis of Eperythrozoon can be directly observed by microscopic examination of the blood slider and its Wright-Giemsa stain smear, while ultrastructural organization can be observed by transmission electron microscope. It was shown that Eperythrozoon attached to surface of erythrocyte. And 801 bp product was successfully achieved by PCR assay, which was guided by primers designed according to the distinct fragment of pig Eperythrozoon genome. The erythrocyte Number of Group I was significantly lower than Group negative(P<0.01); The erythrocyte number of Group I was significantly lower than Group negative(P<0.01); and the hemoglobin, ratio of RBC-C3b,ATPase activity of erythrocyte derived from Group I were also significantly lower than Group negative (P<0.05); However the hematocrite and SOD activity of Group I were less than Group negative, the data got no statistical significance(P>0.05),so was neutrophilic granulocyte number(P>0.05). In adverse, white blood cells ,lymphocytes and NO content of Group I were a little more than Group negative, but P>0.05. And the ratio of RBC-IC of Group I was significantly higher than Group negative(P<0.05).The erythrocyte can recover from infection after treating with artesunate associated with amikacin sulphate, whose infection rate was also decreased; With the artesunate alone erythrocyte can almost recover to normal status too(Group III); The infection rate of Group II(positive control) at the 7th day was 23.08%, which was significantly higher than Group III and IV(P<0.05); The erythrocytes and hemoglobin were significantly increased after infection being treated by drugs(P<0.05); Hematocrit index of five groups were statistically the same(P>0.05). The distinct fragment of PCR amplification can be achieved in Group I and Group II(positive control), while it got nothing in Group III and IV. [Conclusion] To verify organism is infected by Eperythrozoon, the way of microscopy examination of blood slider or Wright-Giemsa stain smear or transmission electron microscope examination can be carried out. But it is insufficient of optical microscope examination alone, which PCR assay and transmission electron microscope can be employed to detect the infection specially. 2.the purified Eperythrozoon can infect SPF Kunming mice. The infection model can be successfully established by the way which the 6-weeks Kunming mice being infected by purified human Eperythrozoon. 3. the erythrocyte membrane structure loses its normal shape if the Eperythrozoon is injected to experimental mice for infecting cells, and it gets dissolvable and fragile, which ultimately leads to changes of blood physiology and biochemistry.4.The therapeutic effect of artesunate associated with amikacin sulphate is better than artesunate alone for removing Eperythrozoon more quickly. And the number and function of erythrocyte can recover to normal status more fast if the infection is treated according to the associated-protocol.
[Objective] To detect the changes of hematological index of mice infected by Eperythrozoon to explore the etiopathogenesis of Eperythrozoon;to compare the therapeutic effects of different drugs treated the infection. [Methods]1. The detection of hematological index of mice infected by Eperythrozoon:①Confirm the establishment of infected model by optical microscope examination, transmission electron microscope observation and PCR assay to amplify distinctive genes;②Evaluate the ATPase activity of erythrocyte membrane, routine hematological index, SOD activity, immune function and NO content of erythrocyte.2.The comparison of therapeutic effects of different drugs treated the infection:12 normal mice were grouped as negative control, and another 48 mice infected by Eperythrozoon were randomly divided to 4 groups(each 12): Group I was mice infected, Group II was treated with nothing as positive control, Group III was with Artesunate(60mg/kg), while Group IV with Artesunate(60mg/kg) associated with amikacin sulphate (400mg/kg).Drugs were injected through intravenous pass for 7 days. In order to evaluate rate of infection, blood samples of mice were collected to make blood slider during the treatment every day, which were tested under oil immersion lens. And the hematological index was detected at the same time.PCR assays were then undertaken to test the blood collected after the treatment. [Results] The morphosis of Eperythrozoon can be directly observed by microscopic examination of the blood slider and its Wright-Giemsa stain smear, while ultrastructural organization can be observed by transmission electron microscope. It was shown that Eperythrozoon attached to surface of erythrocyte. And 801 bp product was successfully achieved by PCR assay, which was guided by primers designed according to the distinct fragment of pig Eperythrozoon genome. The erythrocyte Number of Group I was significantly lower than Group negative(P<0.01); The erythrocyte number of Group I was significantly lower than Group negative(P<0.01); and the hemoglobin, ratio of RBC-C3b,ATPase activity of erythrocyte derived from Group I were also significantly lower than Group negative (P<0.05); However the hematocrite and SOD activity of Group I were less than Group negative, the data got no statistical significance(P>0.05),so was neutrophilic granulocyte number(P>0.05). In adverse, white blood cells ,lymphocytes and NO content of Group I were a little more than Group negative, but P>0.05. And the ratio of RBC-IC of Group I was significantly higher than Group negative(P<0.05).The erythrocyte can recover from infection after treating with artesunate associated with amikacin sulphate, whose infection rate was also decreased; With the artesunate alone erythrocyte can almost recover to normal status too(Group III); The infection rate of Group II(positive control) at the 7th day was 23.08%, which was significantly higher than Group III and IV(P<0.05); The erythrocytes and hemoglobin were significantly increased after infection being treated by drugs(P<0.05); Hematocrit index of five groups were statistically the same(P>0.05). The distinct fragment of PCR amplification can be achieved in Group I and Group II(positive control), while it got nothing in Group III and IV. [Conclusion] To verify organism is infected by Eperythrozoon, the way of microscopy examination of blood slider or Wright-Giemsa stain smear or transmission electron microscope examination can be carried out. But it is insufficient of optical microscope examination alone, which PCR assay and transmission electron microscope can be employed to detect the infection specially. 2.the purified Eperythrozoon can infect SPF Kunming mice. The infection model can be successfully established by the way which the 6-weeks Kunming mice being infected by purified human Eperythrozoon. 3. the erythrocyte membrane structure loses its normal shape if the Eperythrozoon is injected to experimental mice for infecting cells, and it gets dissolvable and fragile, which ultimately leads to changes of blood physiology and biochemistry.4.The therapeutic effect of artesunate associated with amikacin sulphate is better than artesunate alone for removing Eperythrozoon more quickly. And the number and function of erythrocyte can recover to normal status more fast if the infection is treated according to the associated-protocol.
引文
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