【作者】 王冲；

【导师】 刘维达；

【作者基本信息】 北京协和医学院 ， 皮肤病与性病学， 2014， 博士

【摘要】 近年来,随着骨髓移植,造血干细胞移植以及实质性器官移植等手术的增多,糖皮质激素、免疫抑制剂等药物在临床上的广泛应用,导致深部真菌病发病率呈逐步上升趋势,并且有极高的病死率。烟曲霉和白念珠菌是侵袭性真菌病(invasive fungal disease, IFD)最常见的病原菌。而在国内,孢子丝菌侵袭至皮下导致的孢子丝菌病是我国最常见的皮肤深部真菌病之一。及早诊断和干预是改善深部真菌病预后的前提,但是目前常规诊断方法有一定局限性,造成深部真菌病诊断困难,仅有5.9%-25%的患者在死亡前被确诊为深部真菌病。激光微切割(Laser Cutting Microdissection, LCM)技术的出现和发展,为解决这一临床难题带来契机。本次研究中,我们利用烟曲霉、白念珠菌和申克孢子丝菌感染动物模型,探讨LCM技术联合PCR和巢式PCR (nested PCR, n-PCR)在深部真菌病分子病理诊断中的应用。全文共分三章,各章主要内容简述如下：第一章激光微切割在烟曲霉感染小鼠肺模型分子病理诊断中的应用研究在本章节中,我们通过鼻吸入法构建了烟曲霉感染小鼠肺部模型。然后将小鼠左肺分成三份,一份培养,并将菌落提取DNA作为阳性对照,一份常规HE,PAS染色,最后一份用1：10000稀释Blankphor荧光染色,分别微切割10-20μm长的菌丝单根和3根各20份,然后提取DNA,联合巢式PCR特异性扩增烟曲霉的18S rRNA区,结果发现单根菌丝敏感度为90%,而3根菌丝敏感度为100%,二者无显著性差异(χ2=0.489,P>0.05)；总的敏感度为95%,特异度为95%。较之前的一项LCM联合PCR诊断白鹳侵袭性肺曲霉病(invasive pulmonary aspergillosis, IPA)的研究敏感度高。LCM联合巢式PCR方法在诊断IPA方面敏感度高,特异性好。第二章激光微切割在白念珠菌感染小鼠模型分子病理诊断中的应用在本章节中,我们通过小鼠尾静脉注射白念珠菌混悬液的方法构建了念珠菌系统感染小鼠肾脏模型。然后取小鼠右肾分成三份,一份培养,提取菌落DNA,做阳性对照,一份常规HE,PAS染色,最后一份用1：10000稀释B1ankphor荧光染色,分别微切割1和5个孢子各30份,然后提取DNA,联合n-PCR特异性扩增白念珠菌的5.8SrRNA及其附近的ITS区,单个孢子标本敏感度83.3%(25/30),5个孢子标本敏感度90%(27/30),二者无显著性统计学差异(χ2=0.71,P)0.05);总敏感度为87.7%,特异度为95%。我们的研究结果表明,通过收集单个孢子即可确诊大部分侵及实质性脏器的侵袭性念珠菌病(invasive Candidiasis, IC)病例。因此LCM联合n-PCR在IC诊断方面快速,灵敏,准确,高效。第三章激光微切割在孢子丝菌感染小鼠睾丸模型分子病理诊断中的应用在本章节中,我们通过对小鼠左侧睾丸注射孢子丝菌混悬液的方法构建了孢子丝菌感染小鼠睾丸模型。取小鼠左侧睾丸分成2份,一份培养,一份常规HE,PAS染色,分别微切割1和5个孢子各60份,然后提取DNA,分别PCR扩增孢子菌的几丁质合成酶基因和n-PCR扩增ITS2区。在PCR组,单个孢子标本敏感度66.67%(20/30),5个孢子标本敏感度77.67%(23/30),二者之间敏感度无显著性差异(χ2=0.567,P>0.05)。在n-PCR组,单个孢子标本敏感度70.00%(21/30),5个孢子标本敏感度83.33%(25/30)二者之间敏感度无显著性差异(χ2=0.360,P>0.05)。同时,分别比较单个孢子PCR和n-PCR敏感度,以及5个孢子的PCR和n-PCR敏感度,发现均无显著性差异(χ2=1.000,P>0.05,χ2=0.748,P>0.05),特异度为100%。结果说明孢子数量和不同的核酸扩增方法可能会影响到检测敏感度,虽然结果无显著性统计学差异。其后,LCM收集8例孢子丝菌病临床标本中的单个孢子,分别n-PCR和PCR技术检测。其中3例PCR, n-PCR均阳性,而1例仅n-PCR阳性,因此,PCR和n-PCR I临床标本诊断方面的敏感度分别为37.5%和50%。我们认为这可能与甲醛固定以及PAS染色等标本处理方法影响后续DNA扩增有关。因此,LCM联合PCR或n-PCR诊断石蜡包埋,PAS染色的标本,尤其是回顾性分析的病理组织标本,需要进一步优化条件,提高灵敏度。综上所述,在基于LCM技术检测IFD的过程中,样本的预处理对检测结果影响较大,而不同的菌丝/孢子数量以及不同的核酸扩增方式,可在一定程度上影响检测结果。因此,通过进一步优化标本处理方法,完善分子分析技术,该技术在深部真菌病的分子病理诊断,尤其是疑难病例诊断方面具有较好的临床应用前景和实用价值。更多还原

【Abstract】 Invasive fungal disease (IFD) are a growing problem in severely immunocompromised patients suffered from hematologic malignancy, solid organ transplant, hematopoietic stem cell transplant and other critical illness receiving corticosteroids, cyclosporine, and other immunosuppressive treatment. Aspergillus.fumigatus and Candida. albicans is the most common species isolated from patients with IFD. Sporothrix globosa is pathogenic agent of sporotrichosis which is a common subcutaneous infection in China. As fungal species vary in their susceptibility to antimycotic agents, accurate species diagnosis is essential in IA to guide antifungal therapy and improve the prognosis. However, conventional approaches, including microscope, culture and histology, lack efficiency and, sometime inaccuracy in diagnosis. In order to overcome the drawbacks of traditional fungal examination, Laser capture microdissection (LCM) technology has been developed to facilitate diagnosis of IFD. To validate the assays and understand the potential diagnostic utility of LCM in diagnosis of IFD, here, we microdissected and collected hyphal strands or spores directly from tissue of three murine model of IFD, challenged by A.fumigatus, C.albicans and S. globosa respectively, using LCM, and subsequently processed for DNA extraction and PCR/nested PCR-sequencing. The current research was divided into three parts as follows:Chapter one:the Application of Laser Microdissection in Molecular Detection and Identification of Aspergillus fumigatus from Murine Model of Acute Invasive Pulmonary AspergillosisBalb/c mice were infected intranasally with1×108CFU/ml of A. fumigatus. After mice were sacrificed, lungs were harvested and divided into three parts. The first part of the specimens was fixed in90%ethanol, subsequently stained with HE and PAS. The second part was cultured on SDA at30℃to morphological identification and extraction of DNA of the A. fumigatus strain for nested PCR (n-PCR) amplification as positive control. The third part was frozen and sectioned. We microdissected and collected Blankophor-stained20-30μm(25.7±3.0μm) length single or three hyphal strands from tissue crysections of murine model of IPA by LCM, subsequently processed for DNA extraction, and amplify the18S rRNA region of A.fumigatus by n-PCR; subsequently, sequencing, and species molecular identification. In single hypha group,18of20microdissected hyphal strands were identified as A. fumigatus.In three hyphae groups, 20of20microdissected hyphal strands were identified as A. fumigatus. The sensitivity of LCM-n-PCR-sequencing in these two group was90%and100%respectively, there was no significant difference between them (χ2=0.489, P)0.05).And totally sensitivity and specificity was95%and95%.The result of our research was more sensitive than the previous report.Chapter two:The Application of Laser Microdissection in Molecular Detection and Identification of Candida.albicans from Murine Model of Acute Invasive CandidiasisBalb/c mice were infected by inoculation of0.5ml of1×106CFU/ml of C. albicans in the tail vein. After mice were sacrificed, kidney were harvested and divided into three parts for HE/PAS stain, cultured on SDA as positive control, and crysectioned, respectively. We microdissected and collected Blankophor-stained single or five spores from tissue crysections of murine model of IC by LCM, subsequently processed for DNA extraction,and amplify the5.8SrRNA region of C. albicans by n-PCR; subsequently, sequencing, and species molecular identification. In single spore group,25of30microdissected spores were identified as C.albicans.In five spores group,27of30microdissected spores were identified as C. albicans.The sensitivity of LCM-PCR-sequencing in these two group was83.3%and90%respectively, there was no significant different between them (χ2=0.71, P)0.05).And totally sensitivity and specificity was87.7%and95%. The result point to a high sensitivity and specificity of the method in animal mode.Chapter three:the Application of Laser Microdissection in Molecular Detection and Identification of Sporothrix globosa from Murine Model of Acute Invasive Sporotrichosis and clinical specimensBalb/c mice were intratesticularly infected by inoculation of25μl of1×106CFU/ml of S.globosa in the tail vein. After mice were sacrificed, testicle were harvested and divided into two parts for HE/PAS stain, cultured on SDA as positive control, respectively. We microdissected and collected PAS-stained single or five spores from tissue sections by LCM, subsequently processed for DNA extraction, and amplify Chitin synthase gene I by PCR, and the ITS2region by n-PCR, respectively; subsequently, sequencing, and species molecular identification.In PCR group, single spores subgroup,66.67%(20/30)LCM samples was identified as S.globosa,in five spores subgroup,77.67%(23/30)LCM samples were identified as S.globosa,there was no significant difference between them(χ2=0.567, P)0.05). In n-PCR group, the sensitivity of single spore and five spores were70.00%(21/30)and83.33%(25/30)respectively, there was no significant different between them(x2=0.360, P>0.05).The specificity was100%. Meanwhile,we retrospectively detected the8archive skin specimens of patients with sporotrichosis by LCM.We microdissected1spore on PAS stained specimens and then amplification by PCR/n-PCR.Among the8archive specimens,3were positive by both PCR and n-PCR,1specimen was just positive by n-PCR. The result suggest that pretreatment of sample may play a key role in the diagnosis and detection of IFD by assay of LCM in conjunction with PCR/n-PCR.Otherwise, the amount of hyphae/spores and the different amplification method of DNA could influence the result in some degree.In conclusion, we assessed the value of validation of the LCM in conjunction with PCR/n-PCR sequencing method in molecular detection and identification of pathogenic fungi in murine model of IFD. The result pointed to a high sensitivity and specificity of the test in animal model, promising use in the clinically attempts to improve diagnosis of IFD, potentially decreasing the chances of samples contamination or colonization. Moreover, the result of the method concurrently identified the pathogenic fungus to species level which is imperative in association with the selection of sensitive antifungal drug. So, the use of LCM based method could be a powerful tool for development of new assays in diagnosis of IFD.更多还原