A Study On Associated Pleural Fluid Proteins Of Lung Tuberculosis And Lung Adenocarcinoma By Comparative Proteomic Analysis

Research backgroundPleural fluid(PF) of lung tuberculosis and lung adenocarcinom is the most common two kinds of pleural fluid.At present,the diagnosis of PF,mostly uses the multi objective examination generalized analysis method.But each kind of target's sensitivity and the specificity are different.The nearly 20%hydrothorax of tuberculars and the cancerous pleurisy cause,was still very difficult to distinguish after the overall check in clinical.It is a nodus in clinic to how to distinguish the two kinds of PF.SDS-PAGE is under electric field function,according to the protein subunit's molecular weight size to separate protein.SDS-PAGE nowdays has been used in the seperation of urine and blood,and distinguishing correlated diisease by contrasting electrophoresis straps.There are no reports on SDS-PAGE used to separate PF.Pleural fluid contains plasma protein and protein comed from lung.PF of lung tuberculosis and lung adenocarcinom also contain definited proteins coming from each diseae.The previous research was related to the change of some kind of protein in the two kinds of PF.There are no comparative study on total proteins in the two kinds of PF.The protein group's study development has provided the very good technology platform for the body fluid research.Protiomics research on body fluid such as blood plasma,blood serum,cerebrospinal fluid,urine,saliva is developting vigorously.At present,the group's study research regarding the body fluid protein is on the rise. There are many group study researches on the blood plasma or the blood serum protein.There are very few reports on the hydrothorax protein group study both in domestic and in foreign. Although hydrothorax ingredient is similar with the blood plasma,still many differences is present,because of thoracic cavity diseases having their self's feature.The protein group's study research on hydrothorax will deepen the understanding on the chest cavity diseases.The Raman spectral analysis method has the non-destructive merit,and is the important molecular structure analysis method.Surface-enhanced Raman scattering (SERS) technology may research arrangement orientation and structure of elements contact surface and surface adsorption member for example biological macromolecule deoxyribonucleic acid and protein and so on.SERS technology may use in analyzing directly the structure condition of biology member in the aqueous phase,and needs the sample amount very few.Raman spectroscopy is already applied widespreadly in domains on chemistry,material,environment,archaeology as well as biomedicine,obtained the rapid development in many domains.In tumor research, Raman spectrum may use in pathology tissue slice,cell research,and blood sample research.There are no reports on discrimination of PF by Raman spectral analysis.Research goalThis research aims to display the difference between pulmonary tuberculosis hydrothorax and the lung adenocarcinoma hydrothorax on the protein level,and establish the foundation for differential diagnosis research of both.Methods1.160 examples of hydrothorax was collected in South hospital,from July,2007 to October 2008,of which 45 examples are hydrothorax of pulmonary tuberculosis and 47 examples are hydrothorax of lung adenocarcinoma.Each example passes through the organization pathology,the cellular pathology or the union clinical material confirmation.The protein density of each example hydrothorax was determined by the BCA method,and compared by the statistics.2.The separation of hydrothorax protein in pulmonary tuberculosis and the adenocarcinoma of lung using SDS-PAGE electrophoretic techniques:28 examples of pulmonary tuberculosis hydrothorax and 36 examples of adenocarcinoma of lung hydrothorax was used.Each example was separated hydrothorax the protein by 5% concentration rubber and the different density(6%,8%,10%,12%,15%) separation rubber of SDS-PAGE,tested the Mas light blue dyeing,and analyzed the 10% separation rubber gelatin image using AlphaEaseFC Stand Alone image analysis software.3.The comparative proteome analysis of hydrothorax of pulmonary tuberculosis and lung adenocarcinoma:After respectively mixed 45 pulmonary tuberculosis and 47 adenocarcinoma hydrothorax,centrifugating to eliminate cell components,removing the high abundance albumin and IgG,removaling desalts by the acetone precipitation, determinating protein density by the Bradford method.Two kinds of hydrothoraxes of the same qualities were carried on the two-dimensional electrophoresis separately. Discovering the difference protein particles by the ImageMaster software analysis, then identifying these difference proteins by the mass spectrum appraises.4.Comparison of SERS atlas of pulmonary tuberculosis and adenocarcinoma hydrothorax:searching the difference of Laman peak in SERS atlas of pulmonary tuberculosis and adenocarcinoma hydrothorax,and carting on the diagnosis experiment appraisal.Results1.Protein density of pulmonary tuberculosis hydrothorax and adenocarcinoma of lung hydrothorax by BCA method respectively is(68.77±14.41) mg/ml and (54.92±10.24) mg/ml.Protein density of pulmonary tuberculosis hydrothorax is higher than the lung adenocarcinoma of hydrothorax(P＜0.05),and the difference has statistics significance.2.The SDS-PAGE atlas indicated that,protein content of 50KD-60KD molecular weight scope in pulmonary tuberculosis hydrothorax is higher than that in the adenocarcinoma of lung hydrothorax.3.Afer separation by 2-DE and identification by MS,the two kinds of hydrothorax proteins screens six different protein particles.The content of Apolipoprotein D, Ficolin-3,Serotransferrin,Complement C4-B in lung adenocarcinoma hydrothorax is higher than pulmonary tuberculosis hydrothorax.The content of plasma retinol-binding protein,the Alpha-1-antitrypsin in pulmonary tuberculosis hydrothorax is higher than the lung adenocarcinoma hydrothorax.4.Raman spectrum atlas of pulmonary tuberculosis hydrothorax and lung adenocarcinoma hydrothorax is consistent basicly.Raman intensity in pulmonary tuberculosis hydrothorax is higher than lung adenocarcinoma hydrothorax;Taking the appearance of Raman peak(1282.74cm^-1) as the diagnosis target of lung adenocarcinoma hydrothorax,the diagnosis sensitivity is 68.75%,peculiarity is 84.61%,standardized masculine predicted value is 70.25%,standardized negative predicted value is 79.52%,standardized error diagnostic rate is 25.98%,standardized accuracy is 74.02%.ConclusionsThere are obviously difference between pleural fluid of lung tuberculosis and lung adenocarcinoma on protiomics:1.Protein density of pulmonary tuberculosis hydrothorax is higher than that of lung adenocarcinoma hydrothorax,and the difference has statistics significance.2.Protein content of 50KD-60KD molecular weight scope in pulmonary tuberculosis hydrothorax is higher than that in the adenocarcinoma of lung hydrothorax,and the difference has statistics significance.3.The content of Apolipoprotein D,Ficolin-3,Serotransferrin,Complement C4-B in lung adenocarcinoma hydrothorax is higher than pulmonary tuberculosis hydrothorax; the content of plasma retinol-binding protein,Alpha-1-antitrypsin in pulmonary tuberculosis hydrothorax is higher than the lung adenocarcinoma hydrothorax.4.Raman spectrum may be used to identify pleural fluid of lung tuberculosis and lung adenocarcinoma.Raman intensity in pulmonary tuberculosis hydrothorax is higher than lung adenocarcinoma hydrothorax.The appearance of Raman peak(1282.74cm^-1) may be taken as the diagnosis target of lung adenocarcinoma hydrothorax.The diagnosis sensitivity is 68.75%,the peculiarity is 84.61%.