Three-dimensional Tissues Culture Based Screening And Identification Of Serum Marker In The Lung Adenocarcinoma

Background: Lung cancer is the leading cause of death in the world. The 5-year survival rate of advanced lung cancer patients is lower than 10%, whereas that of early patients, which received standard treatment, could exceed 80%. Under present medical condition, early diagnosis and management is the solo way to increase long-term survival rate of lung cancer. For many patients with symptoms are in advanced stage at the time of diagnosis, to diagnose patients without symptoms should depend on candidates screening. Serologic screening is a economic and convenient method, which accepted to people. Because restricts from technology and methodology, there is no obvious significance of serologic screening in diagnosis of the lung cancer. With the advancing of research method, we may achieve new progress in the study of lung cancer serum marker. Which not just of important meaning to the diagnosis of lung cancer, but also do influence on the management of lung cancer. The aim of our study is to screen a serum marker of lung adenocarcinoma probable in significance.Methods: The research was composed of three parts. The first part is about how to construct a rat-tail collagen based three-dimensional platform. Optical microscope, electron microscope, immunohistochemistry and tumor transplantation were utilized to evaluate conditions of the tissues growth. In the second part, Differential proteomics was used to analyze proteins from lung adenocarcinoma tissues and normal lung tissues culture media based on three-dimensional culture model. Then, MALDI-TOF-MS was utilized to identify the discovered differentially expressed proteins. In the third part, the identified differentially expressed proteins were analyzed by Western Blot test and a most distinguishing expressed protein was selected as the research object. ELISA was employed to detect interesting protein concentration in 160 cases serum specimen(from lung adenocarcinoma patients,lung saqumous patients,small-cell lung cancer patients,lung benign tumor patients,cancer patients occurred in other organs and health people). Finally, ELISA results was analyzed by SPSS software to initially elucidate meaning of the protein in the diagnosis of lung adenocarcinoma.Results : On the constructed rat-tail collagen based three-dimensional culture platform, lung adenocarcinoma tissues and normal lung tissues were grew in satisfied condition. By high-flux differential proteomics, four times and three control groups experiments were finished. 12 pieces Coomassie stained and 3 pieces silver stained 2D eletrophotograms were acquired and analyzed by PDquest software. 19 differential proteins were belonged to homo sapient, of which 14 were identified, and 11 non-differential proteins were belonged to bovine. Of all identified differential proteins, Triosephosphate isomerase (Tim),Nucleotide diphosphate kinase A (NDPK A),Annexin A1 (ANXA1),Annexin A2 (ANXA2),Galectin 3,Proapoptotic caspase adapter protein (PACAP),Superoxide dismutase[Cu-Zn] (SOD1) and Glutathione S-transferase P (GSTP1) were in connection with tumor. The proteins with increased expression were selected as study objects. Only Galectin-3,PACAP and SOD1 were increased expression indeed and the PACAP was the highest expression, which proved by Western Blot test. The ELISA detection indicated that the concentration of the PACAP in the non-small-cell lung cancer patients was higher than that in the small-cell lung cancer patients,lung benign tumor patients,cancer patients occurred in other organs and health people(p<0.05). Receiver operating curve suggested that the sensitivity and the specificity was 80.0% and 57.3% separately, when the PACAP attained the ultimate detect efficacy.Conclusion: The growth of lung adenocarcinoma tissues and normal lung tissues in vivo could be reproduced by the constructed rat-tail collagen based three-dimensional culture model in vitro, which may capable of supplying a convenient research platform for relative research. Combining the three-dimensional technique and the proteomics technique is feasible to screening lung adenocarcinoma serum markers, and also gave a new clue to search for other tumor serum markers. PACAP was detected in the serum of patients with lung adenocarcinoma or lung squamous carcinoma, and the concentration in which was significantly higher than that in small-cell lung cancer patients,health people,lung benign tumor patients and cancer patients occurred in other organs. PACAP may be a potential serum marker of the non-small-cell lung cancer.Innovation: In this research, lung adenocarcinoma tissues and normal lung tissues were successfully cultured on the rat-tail collagen based three-dimensional culture platform for the first time. Rat-tail collagen based three-dimensional culture technique and proteomics technique were initially combined to screening serum marker of the lung adenocarcinoma. Based on the platform, highly expression of the PACAP in the serum of lung adenocarcinoma and squamous carcinoma was discovered firstly. It suggested the PACAP may act as a potential serum marker of non-small-cell lung cancer.