甲真菌病组织切片及溶甲涂片中真菌的凝集素结合形式

1、    甲真菌病组织切片及溶甲涂片中真菌的凝集素结合形式        【摘要】目的探讨甲真菌病组织切片、溶甲涂片中真菌的凝集素结合形式，并检测其与培养中相应真菌凝集素结合形式是否等同。方法对已分离鉴定致病菌的72例甲真菌病的病甲组织切片、溶甲涂片，用ABC法对12种凝集素进行组化染色，并与已知培养中的相应菌种进行比较。结果44例甲组织切片、28例溶甲涂片内7个种、属的真菌与其培养中的相应真菌呈现相同的凝集素结合形式，仅铁锈色小孢子菌豌豆凝集素的结合形式与培养中不同，从而表明

2、真菌表面的糖蛋白作为凝集素受体的结构颇为稳定，在经过标本制备处理后仍保持种、属的特点。溶甲涂片方法还具有阳性率高和省时的优点。结论甲组织切片、溶甲涂片内真菌的凝集素结合形式与培养中相应真菌具有相同的凝集素结合形式，有助于甲真菌病病原菌的鉴定。【关键词】甲真菌病组织学真菌凝集素染色Study on the Lectin Binding Patterns of Fungi in Histological Section and in Nail Lysate from Patients with OnychomycosisYANG Guoling，CUI Chunhua，LIN Xiran，et a

3、l.（Department of Dermatology, First Affiliated Hospital, Dalian Medical University, Dalian 116011）【Abstract】 Objective To study the lectin binding patterns of fungi in histological sections and in smears of sodium hydroxide lysed nail from the patients with onychomycosis. Methods Using 12 biotinyl l

4、ectins and avidin? horseradish peroxidase(ABC method), the binding patterns of the fungi in the histological sections and nail lysates from 72 cases of onychomycosis were studied and compared with their counterparts in the published reports. Results All the lectin binding patterns of 7 fungi in the

5、histological sections and nail lysates from onychomycosis patients showed identical with their counterparts in the culture, except that Microsporum ferrugineum stained positively with pisum sativum agglutinin in tissues while negatively in the culture. The nail lysate method revealed advantages of h

6、igh positive rate and time? saving procedure. Conclusion The lectin binding patterns of the fungi in histological sections and nail lysates appear identical with their cultured counterparts and may be helpful in mycological identification in causal fungi in onychomycosis. 【Key words】 Onychomycosis H

7、istology Fungi Lectin staining 凝集素具有识别特异糖蛋白并与之结合的能力，而真菌细胞表面结合糖的成分因菌种而有所差异，故特定的凝集素结合形式可能成为菌种鉴定的指标之一。林俊萍等1和郭伟等2曾分别报道了培养中的14种皮肤癣菌和13种常见条件致病菌与14种凝集素的结合形式，显示凝集素与真菌的结合形式在一定程度上具有属，乃至种的特点。本研究在此基础上，对已经分离鉴定出致病菌的甲真菌病组织切片及溶甲涂片中的真菌进行凝集素组化染色，考察其结合形式与培养中相应真菌的凝集素结合形式是否等同，以探讨是否有助于甲真菌病的病原学诊断。材料与方法（一）对象：为72例具有典型临床表现、K

8、OH直接镜检阳性，真菌培养已鉴定出病原菌的甲真菌病。（二）材料：1试剂及来源：生物素标记凝集素和链霉亲和素（sa）为美国Vector公司产品。其中12种凝集素包括：刀豆凝集素（ConA）、扁豆凝集素（LCA）、豌豆凝集素（PSA）、麦胚凝集素（WGA）、花生凝集素（PNA）、蓖麻凝集素（RCA）、双花藕豆凝集素（DBA）、槐凝集素（SJA）、西非单叶豆凝集素（BSA1）、荆豆凝集素（UEA1）、菜豆凝集素E及L（PHAE及PHAL）。二氨基联苯胺（DAB）为美国Sigma公司产品D5637。2已分离鉴定出菌种的44例甲组织切片及28例溶甲涂片包括：红色毛癣菌23例，铁锈色小孢子菌4例，絮状表皮

9、癣菌2例，念珠菌属16例，曲霉属11例，青霉属3例，镰刀菌属3例，念珠菌属曲霉属3例，念珠菌属红色毛癣菌4例，曲霉属红色毛癣菌2例，念珠菌属青霉属1例。表144例甲组织内及28例溶甲涂片真菌的凝集素结合形式红色毛癣菌(n=23)铁锈色小孢子菌(n=4)絮状表皮癣菌(n=2)念珠菌属(n=16)曲霉属(n=11)青霉属(n=3)镰刀菌属(n=3)念珠菌属曲霉菌属(n=3)念珠菌属红色毛癣菌(n=4)曲霉属红色毛癣菌(n=2)念珠菌属青霉属(n=1)ConA<"2 (99 bytes)" src="/med/cano/201003/201003171754102

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13、6 16>WGA<"2 (99 bytes)" src="/med/cano/201003/20100317175410267" 16 16><"2 (99 bytes)" src="/med/cano/201003/20100317175410267" 16 16><"2 (99 bytes)" src="/med/cano/201003/20100317175410267" 16 16><"2 (99 bytes)

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19、175410267" 16 16><"2 (99 bytes)" src="/med/cano/201003/20100317175410267" 16 16>注：表示与培养中真菌具有不同的凝集素结合形式结果44例甲组织切片和28例溶甲涂片中真菌的凝集素结合形式见表1。同一种真菌在组织内与溶甲涂片中凝集素结合形式完全相同。红色毛癣菌、铁锈色小孢子菌、絮状表皮癣菌、念珠菌属、曲霉属、青霉属及镰刀菌属的7个种属ConA、PSA均呈阳性反应，其余10种凝集素的结合形式则各不相同，显示这些真菌在甲组织切片和溶甲涂片中与凝集素结合形式具有属间特点及部分种间特点（见14）。10例分离出混合种属者，其凝集素结合形式呈现叠加的特点。讨论结果表明，