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直接PCR法对感染性棘阿米巴角膜炎的诊断价值被引量:5
2013年
背景棘阿米巴角膜炎的致盲率极高,发病初期易误诊为真菌性角膜炎或病毒性角膜炎,常规的临床诊断方法费时较长,特异性差,极易错过治疗的“时间窗”。常规PCR法检测简单、特异、有效,但由于病变标本取材量少,不易提取组织DNA,限制了其临床应用。目的探讨非提取组织DNA的直接PCR法扩增棘阿米巴虫株18SrRNA保守序列在棘阿米巴角膜炎快速诊断中的可行性。方法从山东省眼科研究所暨青岛眼科医院诊治的10例棘阿米巴角膜炎患者的病变角膜中分离10株棘阿米巴虫株,经鉴定均为T4基因型虫株。用直接PCR法分别扩增棘阿米巴原虫、白念珠菌、绿脓杆菌、I型单纯疱疹病毒以及正常人角膜上皮细胞以确定该方法的特异性。将棘阿米巴原虫稀释至不同的浓度以确定直接PCR法的敏感性。采用SPF级6周龄健康雌性BALB/c小鼠构建棘阿米巴角膜炎动物模型,于感染后第1、3、5、7、10、15天获取角膜组织,分别进行直接PCR、实时定量PCR(real—timePCR)、K0H封片镜检和原虫培养鉴定,比较各种方法的有效性和可行性。结果直接PCR法仅能扩增棘阿米巴DNA,其他病原体DNA均未扩增出;在每个获取的棘阿米巴角膜炎样本中最少可检测到10个棘阿米巴原虫。在棘阿米巴角膜炎小鼠模型中,直接PCR法在感染后第1、3、5、7、10、15天的阳性检出率分别为80.0%、90.0%、80.0%、70.0%、70.0%和50.0%,总阳性率为73.3%,高于原虫培养法的31.7%,差异有统计学意义(P=0.005);K0H封片镜检的总阳性率为56.7%,real—timePCR法检测的总阳性率为61.7%,均略低于直接PCR法,差异均无统计学意义(P=0.056、0.172)。结论直接PCR法操作简单,在上述4种方法中对棘阿米巴原虫检测的特异性和敏感性最高,能够快速诊断棘阿米巴角膜炎,尤其对于待检标本少而
袁青宋子成孙士营赵格
关键词:棘阿米巴角膜炎聚合酶链反应
Herpes simplex virus-1 infection or Simian virus 40-mediated immortalization of corneal cells causes permanent translocation of NLRP3 to the nuclei被引量:5
2015年
AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.
Shu-Long WangGe ZhaoWei ZhuXiao-Meng DongTing LiuYuan-Yuan LiWen-Gang SongYi-Qiang Wang
关键词:INFLAMMASOMETRANSLOCATIONKERATITISIMMORTALIZATION
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