华南地区肉芽肿性小叶性乳腺炎的细菌学研究

发布时间：2018-06-21 21:41

本文选题：肉芽肿性小叶性乳腺炎 + 细菌　；参考：《广东药科大学》2017年硕士论文

【摘要】：肉芽肿性小叶性乳腺炎(granulomatous lobular mastitis,GLM),是一种发生于乳腺小叶、以肉芽肿形成为主要特征的乳腺慢性炎性疾病。通常表现为乳房肿物,伴有疼痛,皮肤呈现出红肿、溃疡、窦道等急慢性炎症反应,影像学和临床上很容易与乳腺癌,特别是炎性乳腺癌相混淆。因此,对GLM的鉴别诊断和对症治疗是预防与治疗乳腺疾病的一个难题。目前对GLM的病理表现和疾病的发展进程认识不足,对该病的病因和发病机制也缺乏深入了解,导致临床上对GLM的治疗带来盲目性,误诊误治的病历并不少见,对患者的身体与身心健康带来严重的影响。本实验拟对华南地区GLM患者的脓液标本和组织标本为研究对象,试图找出引起该地区GLM的潜在致病菌,并且对其进行鉴定和分析,并结合文献分析不同地区GLM的致病菌的异同。利用细菌培养、革兰氏染色和细菌16S rDNA序列测定的技术鉴定华南地区GLM的潜在致病细菌,通过系统发育分析的方法,构建细菌特定的系统发育树,从而确定细菌的分类地位。本实验有望为GLM的病因和发病机制提供一定的理论依据。方法:选择2014年6月至2016年6月于中山大学孙逸仙纪念医院乳腺科门诊就诊及住院的华南地区肉芽肿性小叶性乳腺炎患者,在手术室无菌条件下取得的32份标本,其中脓液标本14例,组织标本18例。将收集到的14例脓液标本分别接种于血琼脂平板培养基,在37℃恒温,5%CO2培养箱倒置培养48 h以上。若培养呈阳性,则进行革兰氏染色。将培养呈阳性的细菌和收集到的18例GLM患者的组织标本进行总DNA的提取,采用细菌特异性的引物,PCR扩增细菌16S rDNA序列,克隆建库后进行序列测定,测序结果跟NCBI基因数据库进行比对,利用MEGA软件进行系统发育分析,确定细菌的分类地位。结果:对华南地区14例GLM患者的脓液标本进行细菌培养,两天后发现只有4例标本能够在培养基上长出菌落,脓液的细菌检出率为28.57%。长出的菌落细小光滑,将细菌进行革兰氏染色,在油镜下观察到细菌呈杆状,且呈紫色,可初步判定为革兰氏阳性杆菌。将测序得到的细菌16S rDNA序列与NCBI基因数据库进行比对,其中两例与数据库上C.kroppenstedtii的相似度达到99.9%;另外两例分别与数据库上M.abscessus和P.avidum的相似度也高达100%。采用邻接法(Neighbor-Joining,NJ)计算及构建系统发育树进行系统发育分析,从分子系统发育水平上支持了这四例细菌的分类地位。对华南地区18例GLM患者的组织标本进行HE染色,发现GLM周围有乳腺导管扩张症,导管内有炎性渗出物与分泌物,周围可见淋巴细胞、浆细胞浸润。分别提取18例GLM组织标本的总DNA,全部提取成功,然而只有两例标本能够成功PCR扩增细菌16S rDNA序列,对16S rDNA序列进行克隆建库,测序。将测序得到的细菌16S rDNA序列与NCBI基因数据库进行比对,发现其中的一例与国际基因数据库上T.aquatica的相似度达到99.9%;另一例与数据库上C.amycolatum的相似度也为99.9%。应用DNAClub、MEGA软件进行序列分析和系统发育分析,构建细菌的系统发育树,从分子系统发育水平上支持了这两例细菌的分类地位。结论:基于本实验研究的华南地区32例GLM标本,初步确定该地区GLM与细菌C.kroppenstedtii、M.abscessus、P.avidum、C.amycolatum和T.aquatic具有相关性。通过细菌培养和不依赖于培养的分子生物学方法能够对华南地区GLM相关的细菌进行鉴定,总的细菌检出率为18.75%。这些种类的细菌的非特异性感染均可能引起GLM的发生。
[Abstract]:Granulomatous lobular mastitis (granulomatous lobular mastitis, GLM) is a chronic inflammatory disease of the breast that occurs in the mammary lobule and is characterized by granulomatosis. It is usually characterized by breast mass, accompanied by pain, with acute and chronic inflammatory reactions such as redness, ulcers and sinuses. Cancer, especially inflammatory breast cancer, is confused. Therefore, the differential diagnosis and symptomatic treatment of GLM are a difficult problem for the prevention and treatment of breast diseases. At present, the pathological manifestations of GLM and the development process of the disease are insufficient, and the etiology and pathogenesis of the disease are lack of understanding, which leads to blindness in the clinical treatment of GLM and misdiagnosis. The medical records of mistreatment are not rare and have serious influence on the body and physical and mental health of the patients. This experiment aims to study the purulent specimens and tissue specimens of GLM patients in Southern China area, try to find out the potential pathogenic bacteria that cause GLM in this area, and identify and analyze them, and analyze the pathogenic bacteria in different regions of GLM in the literature. Similarities and differences. The potential pathogenic bacteria of GLM in Southern China area were identified by bacterial culture, Gram staining and bacterial 16S rDNA sequencing. By phylogenetic analysis, a specific phylogenetic tree of bacteria was constructed to determine the classification status of bacteria. This experiment is expected to provide a certain theoretical basis for the etiology and pathogenesis of GLM. Methods: 32 specimens of granulomatous lobular mastitis in Southern China area from June 2014 to June 2016 in the Sun Yixian Memorial Hospital of Zhongshan University were selected under aseptic conditions in the operation room, including 14 cases of pus specimens and 18 tissue specimens. 14 cases of pus samples were inoculated to the blood respectively. The agar plate culture medium was incubate at 37 centigrade at 37, and the culture box was upside down over 48 h. If the culture was positive, it was stained with Gram's gram. The positive bacteria and the collected tissue samples of 18 cases of GLM patients were extracted, the bacterial specific primers, PCR amplification of the sequence of 16S rDNA were adopted, and the sequence of 16S rDNA was amplified and the sequence was cloned and the sequence was carried out. The results were compared with the NCBI gene database. MEGA software was used for phylogenetic analysis to determine the classification status of bacteria. Results: 14 cases of GLM patients in Southern China area were cultured for bacterial culture, and only 4 cases were found to grow colonies on the culture medium after two days, and the bacterial detection rate of pus was long out of 28.57%.. The bacterial colonies were fine and smooth, and bacteria were stained by Gram's, and the bacteria were rod-shaped under the oil microscope, and the bacteria were purple. The bacteria were initially determined to be Gram-positive bacilli. The sequence of 16S rDNA sequences obtained by sequencing was compared with the NCBI gene database. The similarity between two cases and the database C.kroppenstedtii was 99.9%, and the other two cases were divided. The similarity between M.abscessus and P.avidum on the database is also high as 100%. uses Neighbor-Joining (Neighbor-Joining, NJ) to calculate and construct phylogenetic tree for phylogenetic analysis. The classification status of the four cases of bacteria is supported from the molecular phylogenetic level. The tissue specimens of 18 cases of GLM patients in Southern China region are stained with HE, and GLM weeks are found. There were mammary ductal dilatation, inflammatory exudates and secretions in the catheter, lymphocytes and plasma infiltrates around them. The total DNA of 18 specimens of GLM tissue was extracted, respectively. However, only two specimens were successful in PCR amplification of the bacterial 16S rDNA sequence, and the 16S rDNA sequence was cloned and sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequenced. Sequencing. Sequencing The 16S rDNA sequence of bacteria was compared with the NCBI gene database, and one of the cases was found to be similar to that of the international gene database. The similarity of the T.aquatica on the international gene database was 99.9%. The similarity of the other case with the database C.amycolatum was also the 99.9%. application DNAClub. The MEGA software carried out the sequence analysis and phylogenetic analysis to construct the phylogenetic tree of the bacteria. The classification status of the two cases of bacteria was supported on the development level of the two cases. Conclusion: Based on this experimental study, 32 cases of GLM specimens in the Southern China area have preliminarily determined that GLM in this area is related to bacterial C.kroppenstedtii, M.abscessus, P.avidum, C.amycolatum and T.aquatic. It is enough to identify GLM related bacteria in Southern China area. The total bacterial detection rate is 18.75%.. The non-specific infection of these bacteria may cause the occurrence of GLM.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R655.8

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