本文選題：肉芽腫性小葉性乳腺炎 + 細(xì)菌�。� 參考：《廣東藥科大學(xué)》2017年碩士論文

【摘要】：肉芽腫性小葉性乳腺炎(granulomatous lobular mastitis,GLM),是一種發(fā)生于乳腺小葉、以肉芽腫形成為主要特征的乳腺慢性炎性疾病。通常表現(xiàn)為乳房腫物,伴有疼痛,皮膚呈現(xiàn)出紅腫、潰瘍、竇道等急慢性炎癥反應(yīng),影像學(xué)和臨床上很容易與乳腺癌,特別是炎性乳腺癌相混淆。因此,對GLM的鑒別診斷和對癥治療是預(yù)防與治療乳腺疾病的一個難題。目前對GLM的病理表現(xiàn)和疾病的發(fā)展進(jìn)程認(rèn)識不足,對該病的病因和發(fā)病機(jī)制也缺乏深入了解,導(dǎo)致臨床上對GLM的治療帶來盲目性,誤診誤治的病歷并不少見,對患者的身體與身心健康帶來嚴(yán)重的影響。本實驗擬對華南地區(qū)GLM患者的膿液標(biāo)本和組織標(biāo)本為研究對象,試圖找出引起該地區(qū)GLM的潛在致病菌,并且對其進(jìn)行鑒定和分析,并結(jié)合文獻(xiàn)分析不同地區(qū)GLM的致病菌的異同。利用細(xì)菌培養(yǎng)、革蘭氏染色和細(xì)菌16S rDNA序列測定的技術(shù)鑒定華南地區(qū)GLM的潛在致病細(xì)菌,通過系統(tǒng)發(fā)育分析的方法,構(gòu)建細(xì)菌特定的系統(tǒng)發(fā)育樹,從而確定細(xì)菌的分類地位。本實驗有望為GLM的病因和發(fā)病機(jī)制提供一定的理論依據(jù)。方法:選擇2014年6月至2016年6月于中山大學(xué)孫逸仙紀(jì)念醫(yī)院乳腺科門診就診及住院的華南地區(qū)肉芽腫性小葉性乳腺炎患者,在手術(shù)室無菌條件下取得的32份標(biāo)本,其中膿液標(biāo)本14例,組織標(biāo)本18例。將收集到的14例膿液標(biāo)本分別接種于血瓊脂平板培養(yǎng)基,在37℃恒溫,5%CO2培養(yǎng)箱倒置培養(yǎng)48 h以上。若培養(yǎng)呈陽性,則進(jìn)行革蘭氏染色。將培養(yǎng)呈陽性的細(xì)菌和收集到的18例GLM患者的組織標(biāo)本進(jìn)行總DNA的提取,采用細(xì)菌特異性的引物,PCR擴(kuò)增細(xì)菌16S rDNA序列,克隆建庫后進(jìn)行序列測定,測序結(jié)果跟NCBI基因數(shù)據(jù)庫進(jìn)行比對,利用MEGA軟件進(jìn)行系統(tǒng)發(fā)育分析,確定細(xì)菌的分類地位。結(jié)果:對華南地區(qū)14例GLM患者的膿液標(biāo)本進(jìn)行細(xì)菌培養(yǎng),兩天后發(fā)現(xiàn)只有4例標(biāo)本能夠在培養(yǎng)基上長出菌落,膿液的細(xì)菌檢出率為28.57%。長出的菌落細(xì)小光滑,將細(xì)菌進(jìn)行革蘭氏染色,在油鏡下觀察到細(xì)菌呈桿狀,且呈紫色,可初步判定為革蘭氏陽性桿菌。將測序得到的細(xì)菌16S rDNA序列與NCBI基因數(shù)據(jù)庫進(jìn)行比對,其中兩例與數(shù)據(jù)庫上C.kroppenstedtii的相似度達(dá)到99.9%;另外兩例分別與數(shù)據(jù)庫上M.abscessus和P.avidum的相似度也高達(dá)100%。采用鄰接法(Neighbor-Joining,NJ)計算及構(gòu)建系統(tǒng)發(fā)育樹進(jìn)行系統(tǒng)發(fā)育分析,從分子系統(tǒng)發(fā)育水平上支持了這四例細(xì)菌的分類地位。對華南地區(qū)18例GLM患者的組織標(biāo)本進(jìn)行HE染色,發(fā)現(xiàn)GLM周圍有乳腺導(dǎo)管擴(kuò)張癥,導(dǎo)管內(nèi)有炎性滲出物與分泌物,周圍可見淋巴細(xì)胞、漿細(xì)胞浸潤。分別提取18例GLM組織標(biāo)本的總DNA,全部提取成功,然而只有兩例標(biāo)本能夠成功PCR擴(kuò)增細(xì)菌16S rDNA序列,對16S rDNA序列進(jìn)行克隆建庫,測序。將測序得到的細(xì)菌16S rDNA序列與NCBI基因數(shù)據(jù)庫進(jìn)行比對,發(fā)現(xiàn)其中的一例與國際基因數(shù)據(jù)庫上T.aquatica的相似度達(dá)到99.9%;另一例與數(shù)據(jù)庫上C.amycolatum的相似度也為99.9%。應(yīng)用DNAClub、MEGA軟件進(jìn)行序列分析和系統(tǒng)發(fā)育分析,構(gòu)建細(xì)菌的系統(tǒng)發(fā)育樹,從分子系統(tǒng)發(fā)育水平上支持了這兩例細(xì)菌的分類地位。結(jié)論:基于本實驗研究的華南地區(qū)32例GLM標(biāo)本,初步確定該地區(qū)GLM與細(xì)菌C.kroppenstedtii、M.abscessus、P.avidum、C.amycolatum和T.aquatic具有相關(guān)性。通過細(xì)菌培養(yǎng)和不依賴于培養(yǎng)的分子生物學(xué)方法能夠?qū)θA南地區(qū)GLM相關(guān)的細(xì)菌進(jìn)行鑒定,總的細(xì)菌檢出率為18.75%。這些種類的細(xì)菌的非特異性感染均可能引起GLM的發(fā)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.
【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R655.8

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