AKP染色鉴别组织内死活血吸虫虫卵的条件探索与效果观察  

Exploring the effectiveness of and optimal conditions for alkaline phosphatase staining to determine viable or dead eggs in tissue

作　　者：王芳[1] 曾庆仁[2] 喻鑫玲[3] 周军[4] 薛伟世[1] 张祖萍[2] 李岳生[3] 何永康[3] 贺美[2] 李闻文[1] 

机构地区：[1]中南大学湘雅医学院检验系,湖南长沙410013 [2]中南大学湘雅医学院细胞与分子生物学实验中心 [3]湖南省血吸虫病防治所诊断中心 [4]中南大学湘雅三医院中心实验室

出　　处：《中国病原生物学杂志》2011年第5期336-339,F0004,共5页Journal of Pathogen Biology

基　　金：中央高校基金前沿研究计划重点项目(No.2010QZZD027);湖南省科技项目(No.2010FJ3109);中南大学研究生创新项目基金资助课题(No.2010ssxt273)

摘　　要：目的探索碱性磷酸酶(AKP)染色条件,观察其对宿主组织内死活血吸虫虫卵的鉴别效果。方法采用偶氮偶联法和NBT/BCIP两种AKP酶染色方法对血吸虫感染后第45 d鼠(未治疗组10只)和经吡喹酮治疗后3个月鼠(治疗组10只)的肝、肠组织内完整虫卵,冰冻切片虫卵和石蜡切片虫卵进行比较检测和分析;收集12份有近、远期变性虫卵的血吸虫病患者肠黏膜活检标本,进行虫卵染色,计数虫卵阳性数,评价两种AKP染色法对血吸虫死卵和活卵鉴定的效果。结果两种AKP染色方法对未治疗组小鼠组织内完整虫卵染色仅半数阳性,其阳性反应主要取决于完整虫卵是否裸露。未治疗组小鼠肠组织石蜡切片和冰冻切片偶氮偶联染色法阳性率分别为97.72%和92.47%,NBT/BCIP染色法阳性率分别为97.65%和98.49%;肝组织两种染色法的阳性率分别为81.17%、86.55%和74.36%、95.32%。在冰冻切片中,肝、肠组织偶氮偶联检测的敏感性均低于NBT/BCIP法;治疗组两法的阳性率均为0。12份临床标本,偶氮偶联法检出1例阳性,NBT/BCIP法检出3例阳性。结论 NBT/BCIP AKP染色法可用于鉴别组织切片中血吸虫虫卵的死活,但在实际应用时尚有待于进一步探索活检肠黏膜组织的处理方法。Objective To explore different conditions for alkaline phosphatase（AKP） staining and observe the effectiveness of AKP staining at identifying viable or dead eggs in order to provide grounds for its use in clinical tests.MethodsLiver and intestinal tissue from 10 mice 45 days after infection with Schistosomiasis japonica（Sj） and 10 mice treated with praziquantel（PZQ） for three months after Sj infection were prepared as frozen sections and paraffin sections and subjected to mechanical trituration and a strong base to free eggs.A kit for AKP staining using azo coupling（AKP1） and a kit using NBT/BCIP（AKP2） were used to detect free schistosoma eggs in tissue and sections from experimental animals and specimens from 12 patients with degenerate mature eggs.Eggs were stained,the number of stained eggs was counted,the percentage of stained eggs was calculated,and the effectiveness of the two methods in differentiating between viable eggs and dead eggs was assessed.Results Both methods of AKP staining stained about 50% of eggs from infected mice,and the percentage of eggs stained was determined by whether eggs were completely free from tissue.The percentage of stained eggs in paraffin sections from untreated mice was 97.72% and that percentage in frozen sections was 92.47% according to AKP1 staining.That percentage was 97.69% in paraffin sections and 98.49% in frozen sections according to AKP2 staining.Statistical analysis revealed that AKP1 staining was more sensitive than AKP2 staining when examining paraffin sections of liver.There were no differences between the two methods when examining paraffin sections of the intestine.AKP2 staining was more sensitive than AKP1 staining for sections of both the liver and intestine.The percentage of stained eggs was zero for the treatment group.AKP1 staining stained 1 egg and AKP2 staining stained 3 eggs in specimens from the 12 patients,so NBT/BCIP staining was more effective.Conclusion The NBT/BCIP method of AKP staining of tissue sections is a useful tool to d

关 键 词：血吸虫 日本 虫卵 碱性磷酸酶 血吸虫病患者 肠黏膜 

分 类 号：R383.24[医药卫生—医学寄生虫学]