本文選題：肺炎支原體 + DNA損傷　； 參考：《浙江大學(xué)》2012年博士論文

【摘要】：第一部分肺炎支原體感染相關(guān)致病機(jī)制研究 背景： 肺炎支原體(Mycoplasma pneumoniae, MP)是Eaton等首次于1944年從一位原發(fā)性非典型肺炎患者的痰標(biāo)本中分離發(fā)現(xiàn)的。它是全球各年齡段呼吸道感染的常見病原體,可引起上呼吸道感染、氣管炎、支氣管炎、細(xì)支氣管炎、社區(qū)獲得性肺炎等,并可能與支氣管哮喘的發(fā)生和急性發(fā)作相關(guān)。MP感染一般呈自限性過程,預(yù)后良好,但近年來越來越多報(bào)道發(fā)現(xiàn)重癥與難治性MP肺炎呈上升趨勢,部分患兒甚至出現(xiàn)嚴(yán)重致命并發(fā)癥。MP主要通過粘附宿主細(xì)胞、胞內(nèi)定殖、細(xì)胞毒作用及免疫反應(yīng)等方式致病,但其確切的致病機(jī)制包括肺外表現(xiàn)的發(fā)生機(jī)制仍有待進(jìn)一步研究闡明。 我們實(shí)驗(yàn)室前期研究證明MP感染A549細(xì)胞后能誘導(dǎo)活性氧自由基(Reactive oxygen species, ROS)形成,而ROS又進(jìn)一步誘導(dǎo)了DNA損傷。那么MP在不同細(xì)胞系是否具有同樣的DNA損傷效應(yīng)?另外,MP的粘附作用被認(rèn)為是MP致病機(jī)制中很重要的因素,粘附作用與ROS的產(chǎn)生是否存在關(guān)聯(lián)?此外,前期研究還發(fā)現(xiàn)MP感染A549細(xì)胞后引起了細(xì)胞骨架蛋白波形蛋白的上調(diào),波形蛋白與MP致病機(jī)制又有什么關(guān)系? 鑒于現(xiàn)有的資料,本研究擬通過以下方面進(jìn)行MP感3染相關(guān)致病機(jī)制研究：MP感染誘導(dǎo)不同細(xì)胞系造成DNA損傷的差異；MP誘導(dǎo)A549細(xì)胞產(chǎn)生ROS與粘附作用的關(guān)系；可能影響MP感染后A549細(xì)胞中波形蛋白表達(dá)改變的信號轉(zhuǎn)導(dǎo)通路；以及波形蛋白表達(dá)對MP感染A549細(xì)胞生理應(yīng)激過程的影響。方法：1.通過免疫熒光法檢測yH2AX和堿性彗星實(shí)驗(yàn)檢測DNA損傷。 2.流式細(xì)胞術(shù)檢測ROS水平。3.免疫熒光觀察波形蛋白量的變化。 4.蛋白質(zhì)印跡法檢測MAPK通路相關(guān)分子表達(dá)量的改變,熒光探針法檢測胞內(nèi)Ca2+水平。 5.siRNA干擾波形蛋白表達(dá)后研究對MP感染A549細(xì)胞的生理功能的影響。 結(jié)果： 1.A549細(xì)胞經(jīng)MP處理(10CFU/cell)4h后經(jīng)免疫熒光檢測yH2AX焦點(diǎn)無增加,而MP處理12h與24h后γH2AX焦點(diǎn)形成顯著增加。HEK293細(xì)胞經(jīng)MP處理4h后有一個(gè)非常快速的增加,而隨之的12h與24h后yH2AX焦點(diǎn)并無增加。彗星實(shí)驗(yàn)提示A549細(xì)胞經(jīng)MP處理6h和12h后均存在明顯的拖尾現(xiàn)象。 2.去粘附能力的MP與未處理的MP均能誘導(dǎo)A549細(xì)胞產(chǎn)生ROS,但兩者誘導(dǎo)ROS產(chǎn)生的能力比較差異無統(tǒng)計(jì)學(xué)意義。 3.免疫熒光觀察到MP感染A549細(xì)胞后波形蛋白的熒光強(qiáng)度增強(qiáng)。 4.MP感染A549細(xì)胞后胞內(nèi)ERK1/2. p-ERK1/2、p38、p-p38、Ca2+水平均無變化。 5.siRNA干擾波形蛋白后細(xì)胞的形態(tài)和生存率不受影響,但MP感染后誘導(dǎo)的yH2AX焦點(diǎn)形成減少。 結(jié)論： 1.MP可在A549細(xì)胞和HEK293細(xì)胞誘導(dǎo)產(chǎn)生DNA損傷,但產(chǎn)生的程度和時(shí)間不同,提示不同細(xì)胞系對MP感染所誘導(dǎo)的DNA損傷的效應(yīng)存在差異。 2.MP誘導(dǎo)A549細(xì)胞產(chǎn)生ROS與是否粘附無關(guān)。 3.MP感染A549細(xì)胞后誘導(dǎo)波形蛋白上調(diào),MAPK (ERK1/2及p38)及Ca2+/CaM-CaMK Ⅱ信號轉(zhuǎn)導(dǎo)通路不參與MP誘導(dǎo)波形蛋白上調(diào)的過程。 4.波形蛋白可能影響了MP誘導(dǎo)A549細(xì)胞產(chǎn)生DNA損傷的過程。 第二部分不同呼吸道標(biāo)本中肺炎支原體分子診斷的研究 背景： MP常引起非特異性的呼吸道癥狀,且對p-內(nèi)酰胺類抗生素治療無效,所以正確及時(shí)的診斷就極為重要。MP的實(shí)驗(yàn)室診斷目前標(biāo)準(zhǔn)不一,尚缺乏敏感且特異的診斷方法。MP感染的診斷方法主要有培養(yǎng)、血清學(xué)方法以及PCR方法。其中血清學(xué)方法和PCR方法是臨床上檢測MP感染的主流手段。鼻咽吸出物(nasopharyngeal aspirate, NPA)和肺泡灌洗液(bronchoalveolar lavage, BAL)均能作為PCR方法的標(biāo)本。有關(guān)比較使用BAL和NPA作為PCR標(biāo)本的靈敏度和特異度的文獻(xiàn)很少。故本研究中我們擬評價(jià)不同的呼吸道標(biāo)本(NPA和BAL)在熒光實(shí)時(shí)定量PCR診斷兒童MP感染中的價(jià)值,并求尋找到有助于診斷MP肺炎(MPP)的臨床特征。 方法： 1.收集406例住院的肺炎患兒的NPA和BAL標(biāo)本進(jìn)行熒光實(shí)時(shí)定量PCR檢測MP的DNA,并采集外周血檢測MP特異性IgM。 2.復(fù)習(xí)和整理所有患兒的病例記錄,收集其中相關(guān)實(shí)驗(yàn)室數(shù)據(jù),包括血常規(guī)、CRP、免疫球蛋白、T細(xì)胞亞群等。 結(jié)果： 1.共101例患兒診斷為MPP (24.9%). MPP.患兒年齡中位數(shù)為4.1歲,非MPP患兒年齡中位數(shù)為2.4歲,差異有統(tǒng)計(jì)學(xué)意義(p0.001)。 2.MPP與非MPP相比,相關(guān)實(shí)驗(yàn)室數(shù)據(jù)包括白細(xì)胞計(jì)數(shù)、中性粒細(xì)胞數(shù)目、免疫球蛋白(除了血清IgM)、T細(xì)胞亞群、BAL細(xì)胞學(xué)計(jì)數(shù)差異均無統(tǒng)計(jì)學(xué)意義。 3.BAL-PCR陽性患兒BAL巨噬細(xì)胞比例低于BAL-PCR陰性患兒(p=0.003),而BAL中性粒細(xì)胞比例較高(p=0.007)。 4.采用NPA與BAL作為PCR的標(biāo)本的各項(xiàng)診斷指標(biāo)相比差異均無統(tǒng)計(jì)學(xué)意義：NPA的靈敏度、特異度、陽性預(yù)測值、陰性預(yù)測值分別為78.6%,63.4%,39.8%和90.6%。BAL的靈敏度、特異度、陽性預(yù)測值、陰性預(yù)測值分別為70.3%,58.7%,36.0%和85.6%。 結(jié)論： 1.年齡是有助于診斷MPP的臨床特征。 2.BAL的PCR檢測有助于判斷局部炎癥情況。 3.由于NPA與BAL在PCR方法診斷MP上各項(xiàng)診斷指標(biāo)均接近,而且收集NPA標(biāo)本無創(chuàng)、經(jīng)濟(jì),故NPA作為檢測MP的標(biāo)本要優(yōu)于BAL。
[Abstract]:Part one: pathogenesis of Mycoplasma pneumoniae infection
Background:
Mycoplasma pneumoniae (MP), which was first isolated from the sputum specimens of a primary atypical pneumonia in 1944, was found in the sputum specimens of a primary SARS patient. It is a common pathogen of respiratory tract infections in all ages of the world. It can cause upper respiratory tract infection, tracheitis, bronchitis, bronchiolitis, community-acquired pneumonia and so on. .MP infection related to the occurrence and acute attack of bronchial asthma generally presents a self limiting process and has a good prognosis. However, in recent years, more and more reports have found that severe and refractory MP pneumonia is on the rise. Some of the children even have serious fatal complications,.MP mainly by adhesion to the host cell, intracellular colonization, cytotoxicity and immune response. Pathogenicity, but its exact pathogenesis, including the pathogenesis of extrapulmonary manifestations, needs to be further elucidated.
Our laboratory studies have shown that MP infected A549 cells can induce active oxygen free radicals (Reactive oxygen species, ROS), and ROS further induces DNA damage. Then, is MP in different cell lines having the same DNA damage effect? And MP adhesion is considered to be an important factor in the pathogenesis of MP. Is there an association with the production of ROS? In addition, previous studies have found that MP infected A549 cells caused the up regulation of the cytoskeleton protein vimentin, and what is the relationship between vimentin and the pathogenesis of MP?
In view of the existing data, this study intends to study the pathogenesis of MP induced 3 infection through the following aspects: MP infection induces the difference in DNA damage in different cell lines; MP induces the relationship between ROS and adhesion in A549 cells; it may affect the signal transduction pathway of the change of vimentin expression in A549 cells after MP infection; and Effects of the expression of MP on the physiological stress of A549 cells infected with MP. Methods: 1.. DNA damage was detected by immunofluorescence assay and alkaline comet assay.
2. the level of ROS was detected by flow cytometry. Vimentin was measured by.3. immunofluorescence.
4. Western blotting was used to detect the expression of MAPK pathway related molecules, and the intracellular Ca2+ level was detected by fluorescence probe.
5.siRNA interfered with vimentin expression and studied the effects of MP on the physiological functions of A549 infected cells.
Result錛，

本文編號：2054596