| BACKGROUDHow to identificate benign and malignant pleural effusion has been always an important issue for clinicians, tuberculosis and cancer were the common diseases which caused pleural effusion in our country, while the main cancer was lung adenocarcinoma. Principles of treatment of lung cancer and tuberculosis is completely different, nearly 20% of the pleural effusion caused by tuberculosis and adenocarcinoma were still still difficult to identify after by the full clinical examination. Therefore, to find the tumor maker and indicators of inflammation response caused by tuberculosis and to investigate the value of combined detection were became a research hotspot in recent years.PDZ-binding-kinase/T-LAK cell-originated protein kinase (PBK/TOPK) is a kind of new silk-threonine kinase, belongs to the family of MAPKK molecules, activated and phosphorylated in mitosis, with anti-apoptosis. Fujibuchi T found the high expression not only related to the malignant transformation of tumor, but also could promote the malignant transformation.In our previous study on PBK/TOPK protein expression in lung carcinoma on the chip with 760 disks, we found that the expression of PBK/TOPK are higher in lung adenocarcinoma cell, which suggests PBK/TOPK may have significant cnilical value on differential diagnosis between lung adenocarcinoma and tuberculosis. At present, studies on the expression of protein PBK/TOPK in pleural fluid and its value of identificate primary lung adenocarcinoma and tuberculosis pleural effusion were not reported.TTF-1 is a tissue specific nuclear transcription factor, with 38000 as relative molecular mass, which selectively expressed in alveolar typeⅡcells, the bronchial epithelial cells, thyroid follicular cells, thyroid parafollicular cells and embryonic brain certain areas. Although TTF-1 specifically expressed in lung cancer and thyroid cancer, but thyroid cancer associated with pleural effusion was few, so TTF-1 is found a more ideal tumor marker for detection of lung cancer pleural effusion, especially for determining primary lung cancer pleural effusion.SPA is one of the major components of lung surfactant proteins; Shijubo N first proposed that by detecting the SPA expression of patients with pleural effusion could identify primary lung cancer, which confirmed that SPA was a valuable immunohistochemical marker for primary lung adenocarcinoma. For the formation mechanism of SPA in pleural effusion, Saitoch H thought that the expression of SPA in pleural effusion mainly correlated-with the number of cells in pleural cavity which could synthesize and secret SPA, while the lung adenocarcinoma cells and part squamous carcinoma cells could synthesize and secret SPA, so the expression of SPA in primary lung adenocacinoma pleural effusion was higher than in tuberculosis pleural effusion. Detecting SPA protein in pleural effusion is important for identificating lung cancer and tuberculosis.A549 and GLC-82 cell lines are two kinds of human lung adenocarcinoma cell lines which most commonly used to study the development, diagnosis and treatment of lung cancer. Although A549 and GLC-82 both are human lung adenocarcinoma cell lines, however the differences and the similarities based on the morphology and immunocytochemistry is still unknown. The research was designed to find the answers.OBJECTIVE AND SIGNIFICANCETo explore the different expression of protein PBK/TOPK, TTF-1, SPA in primary lung adenocarcinoma and tuberculosis pleural effusions, and the diagnosis value of above three indicates when they were used to distinguish the primary lung adenocarcinoma pleural effusions from tuberculosis pleural effusions singly or combining with biochemical tests.METERLAILS AND METHODSI. Identify and Quantitative analysis in morphology and immunophenotype on cell lines A549 and GLC-82 from human lung adenocarcinoma andExperimental materials:A549 and GLC-82 cell lines stored in liquid nitrogen by the Department of Pathology.Experimental Methods:HE staining for cell-seeded coverslips and cell block, and Papanicolaou stain for Cell-seeded coverslips were respectively applied to observe the morphology of cells. Immunocytochemistry based on the sections of cell block was used to examine the expressions of CK7, CK8, CK5/6, CK19, SPA, TTF-1, PBK, Ki67, P53 in A549 and GLC-82 cells. The differences and the similarities of A549 and GLC-82 cells were compared on morphology and protein expression by Image pro-Plus soft ware and Leica Q500 MC Image analysis system. Ⅱ. Quantitative analysis of the expression of immunophemotype PBK/TOPK, TTF-1 and SPA in cell lines A549 and GLC-82 from human lung adenocarcinomaExperimental materials:A549 and GLC-82 cell lines stored in liquid nitrogen by the Department of Pathology.Experimental Methods:Immunocytochemistry based on the sections of cell block was used to examine the expressions of PBK/TOPK, TTF-1 and SPA in A549 and GLC-82 cells. The PU value of the expressions of PBK/TOPK, TTF-1 and SPA were detected by Leica Q500 MC Image analysis system, based on which the relationship between the PU value and the cell lines would be analysised.Ⅲ. Expression and differential diagnosis significance of PBK/TOPK, TTF-1, and SPA in pleural effusions due to lung adenocarcinoma and tuberculosisExperimental Materials:This study was retrospective,94 cases of pleural effusion which met the diagnostic criteria were collected from Nanfang Hospital, from September 2009 to October 2010 including:56 cases of lung cancer, with an average age of 59.0±12.2 years,38 cases of tuberculous pleural effusion, with an average age of 44.9±20.2 years.Experimental Methods:Collect the clinical data. The total protein level of each pleural fluid was quantified using the Bicinchoninic acid. Western blotting and ELISA were used to detect the expression of PBK/TOPK, TTF-1 and SPA. Receiver operating characteristics (ROC), the parallel and serial test were applied to analysis the accuracy of PBK/TOPK, TTF-1, and SPA to distinguish lung Aden carcinoma from tuberculosis pleural lesions. IV. Differential diagnosis significance of PBK/TOPK, TTF-1, SPA combining with biochemical examination in pleural effusions due to lung Aden carcinoma and tuberculosisExperimental Materials:See III (Expression and differential diagnosis significance of PBK/TOPK, TTF-1, and SPA in pleural effusions due to lung adenocarcinoma and tuberculosis).Experimental Methods:Collected the pleural effusion biochemical tests of patients including:adenosine deaminase (ADA), lactate dehydrogenase (LDH), C-reactive protein (CRP), glucose (Glu), white blood cell count (WBC). Then combining with PBK, TTF-1, and SPA test results, to explore the differential diagnosis value of the single and combined detection in lung adenocarcinoma and tuberculous pleural effusion. Receiver operating characteristics (ROC), the parallel and serial test were applied to analysis the best threshold and the best combination of diagnostic indicators to distinguish lung adenocarcinoma from tuberculosis pleural lesions.RESULTSI。Identify and Quantitative analysis in morphology and immunophenotype on cell lines A549 and GLC-82 from human lung adenocarcinoma and1. The morphometry results show that the values of cell area, nuclear area, cytoplasm area, cell and nuclear shape factor PE, cell shape factor ARc of GLC-82 cells are larger than those of A549, but the values of nuclear/cytoplasm ratio, nuclear area density, nuclear shape factor ARn, RFFn and irregular index of cell and nuclear shape of A549 cells are larger than those of GLC-82. There were significant differences to all the tested morphological parameters between the cells of A549 and GLC-82 (P<0.002) except cell perimeter and RFFn (P>0.05). 2.CK7 and CK8 were positive in A549 and GLC-82 cells with diffuse membrane and cytoplasm expression, while CK5/6 and CK19 both were negative in both two cells lines. Ki67 showed diffuse expression in nuclei of A549 and GLC-82, and with higher level in GLC-82. P53 showed scattering expression in nuclei of A549 and GLC-82. The expression level of CK7, p53 and PBK were higher in A549 cells than that in GLC-82, but CK8 and Ki67 were higher in GLC-82 cells than that in A549 cells (P=0.001).Ⅱ. Quantitative analysis of the expression of immunophenotype PBK/TOPK, TTF-1 and SPA in cell lines A549 and GLC-82 from human lung adenocarcinomaPBK showed diffuse expression in membrane and cytoplasm of A549 and GLC-82 with higher level in A549. Both of SPA and TTF-1 were negative in A549 and GLC-82 cells. The PU value of PBK/TOPK was higher in A549 cells (9.18±0.66) than that in GLC-82 (4.77±2.31) (P=0.000).Ⅲ. Expression and differential diagnosis significance of PBK/TOPK, TTF-1, and SPA in pleural effusions due to lung adenocarcinoma and tuberculosis1. The total protein levels in the pleural fluids were significantly higher than the patients with lung adenocarcinoma (20.14±5.90mg/ml) compared to the patients with tuberculosis (18.02±5.54mg/ml) (P<0.05).2. Western blot analysis showed that the expression of PBK/TOPK appeard at 43KD. The IOD value of pleural PBK/TOPK tested by Gel-Pro Analyzer was 319.23±127.84 in 56 patients with lung adenocarcinoma pleural effusions and significantly higher compared to the value of 192.34±136.53 found in 38 tuberculous pleural effusions(P=0.000). Based on the Receiver operating characteristic curves and diagnostic tests, it was found that 204.23 as a diagnostic threshold to distinguish lung adenocarcinoma from tuberculosis, the sensitivity, the specificity and the accuracy is respectively 83.7%,66.7% and 76.6%.The standardizing accuracy, positive predictive value and negative predictive value is respectively 75.2%,71.5% and 80.4%.3. The expression of TTF-1 in pleural effusions tested by Elisa was 955.43±915.74pg/ml in 49 patients with lung adenocarcinoma pleural effusions and significantly higher compared to the value of 267.22±257.05pg/ml found in 36 tuberculous pleural effusions (P=0.000). Based on the Receiver operating characteristic curves and diagnostic tests, it was found that 289.88pg/ml as a diagnostic threshold to distinguish lung adenocarcinoma from tuberculosis, the sensitivity, the specificity and the accuracy is respectively 91.8%,83.3% and 88.2%.The standardizing accuracy, positive predictive value and negative predictive value is respectively 87.55%,84.6% and 91%.4. The expression of SPA in pleural effusions tested by Elisa was 139.58±39.18ng/ml in 49 patients with lung adenocarcinoma pleural effusions and significantly higher compared to the value of 62.82±25.67ng/ml found in 36 tuberculous pleural effusions(P=0.000). Based on the Receiver operating characteristic curves and diagnostic tests, it was found that 99.4ng/ml as a diagnostic threshold to distinguish lung adenocarcinoma from tuberculosis, the sensitivity, the specificity and the accuracy is respectively 89.8%,91.7% and 90.6%.The standardizing accuracy, positive predictive value and negative predictive value is respectively 90.75%,90.5% and 90%.5. The parallel and serial diagnostic tests results, based on combined analysis of PBK, TTF-1, SPA proteins for identificating lung adenocarcinoma and tuberculous pleural effusion, showed that the best differential diagnosis combination was SPA+ TTF-1, and its serial tests has the highest diagnostic value, the corresponding sensitivity and specificity was 82.4%,98.6%.Ⅳ. Differential diagnosis significance of PBK/TOPK, TTF-1, SPA combining with biochemical examination in pleural effusions due to lung adenocarcinoma and tuberculosis1. The expression of LDH and GLU in lung primary lung adenocarcinoma effusions were both higher than in tuberculous pleural effusions, but the former had significant difference (P<0.05), the latter not (P=0.393). While the:expression of ADA, CRP and the white blood cell count in tuberculous pleural effusions were all higher than in lung primary lung adenocarcinoma effusions (P<0.05).2. Receiver operating characteristics (ROC) analysis for LDH to identificate primary lung adenocacinoma pleural effusion, and for ADA, CRP, WBC to identificate tuberculous pleural effusion showed that the area under the crove was respectively 0.649(P=0.015),0.936(P=0.000),0.781(P=0.000),0.640(P=0.022); The diagnostic threshold of LDH, ADA, CRP, WBC were respectively 243u/l,16.95u/l,8.75mg/l,1500, the corresponding sensitively was respectively 83.9%,89.5%,78.9%, 52.6%, the specificity were 50%,82.1%,60.7%,75%.3. The diagnostic tests result based on combination analysis of PBK/TOPK, ADA and CRP showed that the serial test of PBK and ADA was with the highest diagnostic value, the sensitivity and specialty were found to be 74.9% and 94% respectively. The diagnostic tests result based on combination analysis of TTF-1, ADA and CRP showed that the serial test of TTF-1 and ADA was with the highest diagnostic value, the sensitivity and specialty were found to be 82.2%and 97% respectively. The diagnostic tests result based on combination analysis of SPA, ADA and CRP showed that the combine test of SPA and ADA was with the highest diagnostic value, the sensitivity and specialty of the parallel test were found to be 98.9% and 75.3% respectively, as well as the sensitivity and specialty of the serial test were found to be 80.4% and 98.5% respectively.4. The diagnostic tests result based on analysis of PBK/TOPK, TTF-1 and SPA respectively combining with ADA, CRP showed that the combinations of PBKand ADA, TTF-1 and ADA, SPA and ADA were with good diagnostic values, of which the SPA and ADA was with the best value.CONCLUSIONS1. The morphological differences between the cell line A549 and GLC-82 exist obviously, but the immunophenotype is of some of similarities and differences. Both of SPA and TTF-1 were negative in A549 and GLC-82 cells, Ki67,CK7,CK8,PBK. P53 were positive with different level in both A549 and GLC-82 cells. Recognizing these similarities and differences is useful to help us to identify the cell lines of A549 and GLC-82, and to understand and compare the related research results on A549 and GLC-82.2. PBK/TOPK, TTF-1 and SPA do exit in primary lung adenocarcinoma and tuberculosis pleural effusions, and all of which were with higher level in primary lung adenocarcinoma pleural effusions. Using PBK/TOPK, TTF-1, SPA to identificate to primary lung adenocacinoma pleural effusion, the responding diagnostic threshold were 204.23,289.88pg/ml,99.4ng/ml, the sensitivity were respectively 83.7%,91.8%,89.8%。The best differential diagnosis combination was SPA+TTF-1, and its serial test has the highest diagnostic value, the corresponding sensitivity and specificity was 82.4%,98.6%.3. The diagnostic tests result based on analysis of PBK/TOPK, TTF-1 and SPA respectively combining with ADA, CRP showed that the combinations of PBKand ADA, TTF-1 and ADA, SPA and ADA were with good diagnostic values, of which the SPA and ADA was with the best value. When we applied combined testing, the sensitivity and specialty had been substantially improved, which was significant for clinical application. |
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