【作者】 吴雪琼；

【导师】 刘又宁；

【作者基本信息】 中国人民解放军军医进修学院 ， 内科学， 2009， 博士

【摘要】 目的:应用蛋白质谱技术研究活动性肺结核病患者的血清诊断标志物,建立诊断模型早期诊断活动性肺结核;了解潜在标志物的特性及其在肺外结核病血清中的表达情况。方法:应用表面加强激光解吸电离飞行时间质谱技术(SELDI/TOF-MS)和蛋白芯片技术检测下列346例人血清:129例活动性肺结核患者、69例其他呼吸疾病患者、57例结核性胸膜炎患者、4例非结核性胸膜炎患者、19例结核脑膜炎患者、2例非结核脑膜炎患者和66例正常人血清;应用Ciphergen蛋白芯片3.1.1软件比较、分析血清蛋白峰,筛选疾病相关生物标志物;应用Biomarker Pattern 5.0软件建立活动性肺结核的诊断模型。通过反相高效液相色谱(RP-HPLC)分离纯化潜在的生物标志物蛋白,应用基质辅助激光解吸附电离飞行时间质谱技术(MALDI-TOF-MS)分析分离所得的蛋白组分,跟踪目标蛋白,目标蛋白的酶解产物进一步用线性液质联用离子肼串联质谱技术(LC-MS/MS)进行分析,以鉴定潜在诊断标志物蛋白的性质,并SELDI/TOF-MS和蛋白芯片技术进一步分析该诊断标志物在肺外结核病的表达量。结果:(1)129例活动性肺结核血清和135例对照血清蛋白指纹图谱比较,有50个差异蛋白峰(p＜0.01)。以69例其他呼吸疾病血清和66例健康人血清为对照,选择5个蛋白峰(4360、3311、8160、5723、15173 m/z)建立诊断模型1,该模型诊断活动性肺结核的灵敏度为82.95%,特异性为89.63%,准确率为86.36%。以69例其他呼吸疾病血清为对照,选择3个蛋白峰(5643、4486、4360 m/z)建立诊断模型2,该模型诊断活动性肺结核的灵敏度为96.9%,特异性为97.8%,准确率为97.3%。(2)质荷比峰为5643 m/z的蛋白为相对高丰度差异表达蛋白,它在62.8%(81/129)的活动性肺结核患者血清中表达显著增高,在71.9%(97/135)的非结核呼吸疾病患者和健康人血清中表达显著降低。因此,选择5643 m/z蛋白作为潜在的诊断标志物,经RP-HPLC分离、纯化,并利用MALDI-TOF-MS及LC-MS/MS进行鉴定,5643 m/z目标蛋白质是粘蛋白,又称为α-1-酸性糖蛋白。57例结核性胸膜炎患者血清和70例对照血清蛋白指纹图谱比较,有52个差异蛋白峰(p＜0.01)。(3)只有47.4%(27/57例)的结核性胸膜炎患者血清中5643M/Z蛋白表达增高;4例非结核性胸膜炎患者血清中5643M/Z蛋白表达均减少。(4)19例结核性脑膜炎患者血清和68例对照血清蛋白指纹图谱比较,有25个差异蛋白峰(p＜0.05)。73.7%(14/19例)的结核性脑膜炎患者血清中5643M/Z蛋白表达增高;2例原发性脑膜炎患者血清中5643M/Z蛋白表达均减少。结论:(1)蛋白质谱技术标本用量少,可直接快速地检测血清标本,可能成为一种新的结核病诊断方法,通过建立结核病质谱诊断平台服务于临床。(2)通过蛋白质组学技术可有效地筛选、分离、纯化、鉴定结核病特异性标志物。(3)结核特异糖形粘蛋白的表达或粘蛋白糖基化差异分析可能成为一个新的诊断方法用于活动性结核病的诊断和鉴别诊断,为研制新型诊断试剂奠定基础.更多还原

【Abstract】 Objective:To identify the biomarker and develop a diagnostic model for active tuberculosis(TB) using proteomics techniques,and to study the expression of biomarker in the sera from the patients with pulmonary TB and extrapulmonary TB.Method:The proteomic fingerprinting of 346 human sera as follow were analyzed using the surface-enhanced laser desorption ionization time of flight mass spectrometry(SELDI/TOF-MS) and protein-chip technology:129 cases with active pulmonary TB,69 cases with non-TB respiratory diseases,57 cases with tuberculous pleuritis,4 cases with non-TB pleuritis,19 cases with tuberculous meningitis,2 cases with non-TB meningitis,and 66 healthy controls. The peaks were detected and filtrated by Ciphergen Protein Chip(?) Software (version 3.1.1).Using the Biomarker Pattern 5.0 software,a diagnostic model was developed for diagnosis of active tuberculosis.The potential biomarker was separated,purified,analyzed,and identified using reverse phase-high performance liquid chromotography(RP-HPLC),Matrix assisted laser desorption ionisation time-of-flight mass spectrometry(MALDI-TOF-MS) and linear ion chromotograph mass spectrometry/mass spectrometry(LC-MS/MS).Result:(1) Fifty protein peaks in the sera were significantly different between 129 patients with active pulmonary tuberculosis and 135 controls with overlapping clinical features(P＜0.01).Five protein peaks at 4360,3311,8160, 5723,15173 m/z were chosen for the system classifier and the development of diagnosis model 1.The model differentiated the patients with active pulmonary tuberculosis from the controls with a sensitivity of 83.0%,and a specificity of 89.6%.The diagnostic accuracy was up to 86.4%.Three protein peaks at 5643, 4486,4360 m/z were chosen for the system classifier and the development of diagnosis model 2.The model differentiated the patients with active pulmonary tuberculosis from the controls with a sensitivity of 96.9%,and a specificity of 97.8%.The diagnostic accuracy was up to 97.3%.(2) The discrepant protein peak at m/z 5643 was highly expressed in 62.8%(81/129) sera from active TB patients, and lowly expressed in 71.9%(97/135) sera from other respiratory diseases and healthy controls.Therefore,protein at m/z 5643 was screened as a potential biomarker for active TB,separated,purified by RP-HPLC,identified using MALDI-TOF-MS and LC-MS/MS.The potential biomarker was comfirmed as orosomucoid,which is also named as alpha-1-acid glycoprotein.(3) Fifty-two protein peaks in the sera were significantly different between 57 patients with tuberculous pleuritis and 70 controls(P＜0.01).The protein peak at m/z 5643 was highly expressed in 47.4%(27/57) sera from the patients with tuberculous pleuritis,and lowly expressed in 4 sera from the patients with non-TB pleuritis.(4) Twenty-five protein peaks in the sera were significantly different between 19 patients with tuberculous meningitis and 68 controls(P＜0.05).The protein peak at m/z 5643 was highly expressed in 73.7%(14/19) sera from the patients with tuberculous meningitis,and lowly expressed in 2 sera from the patients with non-TB meningitis.Conclusion:(1) The mass spectrometry and protein chip technology need small quantity of samples,can detect directly serum samples.It might be used as a new method for the diagnosis of the active TB.(2) The proteomic technology can effectively screen,isolate,purified,and identified the TB-specific biomarkers.(3) The alpha-1-acid glycoprotein isoforms or the differences in the glycosylation of alpha-1-acid glycoprotein from TB might be used to diagnose the active TB.更多还原