| Asteosarcoma is the first morbility in tumor of osseous system, It often happen children and youngsters.lt has very high malignancy and very low survival rate. In the early (EnnekingⅡA), Asteosarcoma patient have no severity clinical situation, so most patients are diagnosed at advanced stage disease (Enneking 11B/Ⅲ) and the five-year survival rate is only 20%-50%. about half of patients have pulmonary metastasis and the five-year survival generally 10%-15%, so, Asteosarcoma has a severity threaten to the health of children and youngsters.On the other hand, If it was diagnosed and have a surgery in the early phase,the five-year survival rate can improve to 80%, If we can improve the diagnosis rate from 25% to 75%, It can decrease about half of the death. To improve the diagnosis of early stage, it have very important social and economic value.The happen and development of Asteosarcoma is very complicated course. Mult-factorial alterations are involved in Asteosarcoma. Hovever, to study to the mechanism of Asteosarcoma often static and focal, It only conclude single gene or protein research, to think about the abnormity gene and protein expression from jug-handled.It is very difficult to grasp the change of the relative gene and protein of Asteosarcoma. So,this method have limitations on complic and dynamic tumor desease. Moreover,many research stay on gene and transcription levels, it has no develop to the level of protein, the level of gene can not anser many question about the level of protein expression and the time of protein expression, the modify of translation and the question interaction among protein. Therefore, It is important to elucidate the incidence and develop of tumor in protein level and to search the intimate relative protein mark. It provides the facility that the achievement of human genome project(HGP) combined with the proteome research becoming a hot spot in the post-genome era.Aim of the study: To optimize the two-dimensional gel electrophoresis (2-DE) and computer image analysis technique and apply it in the proteome research of the osteosarcoma, increase the data resolution and repetitive probability of the experiment, and explore a better experimental method to support further study of osteosarcoma proteome. With the Optimized Two-dimensional Gel Electrophoresis—Image Analysis Technique and SELDI Technique,we can detect blood serum protein expression map of health people and asteosarcoma patient,to establish marked differertial proteins. and compare and analysis the 2-DE images of proteome with asteosarcoma and the nomal tissue,to find the differential expressed protein in protein level. Analyses on differentially expressed protein is to have a foundation for 2-DE maps of asteosarcoma.Methods: The protein of osteosarcoma tissue was extracted. Immobilized pH gradient was taken as the key factor and link in the isoelectric point focus. For instance, sample handle, electrophoresis parameter and gel concentration are get on with a series of optimization. 2-DE was further adopted to separate the total protein of normal team and of osteosarcoma team. Then, after argentum tinct, the 2-DE atlas was analyzed by an optimized computer image analysis software. The 2-DE protein expression spectrums of serum of osteosarcoma were obtained and compared the spots. With the MALDI—Tof—MS the 2-DE maps were analyzed, then search the maps in SWISS-PRO database for maked proteins and verify the reliability of PMF. The 2-DE maps of osteosarcoma and normal tissue were obtained with good repetitivity and comparativity,The differentially expressed proteins were picked out to digest in gel and then identified by MS.Results: This study established and optimized the 2-DE methods, and the computer assisted image analysis system was also established. Repetitive experiments shows that in three experiments in a row, the amount of protein spots of the same team sample deviates from the relative standard as following (the average of variation coefficient %): Osteosarcoma and the normal team is 22.83±9.79 and 21.63±9.93 respectively. (the range of variation coefficient %): Osteosarcoma and the normal team is 3.72-58.90 and 2.54-54.74 respectively. The isoelectric point and molecular weigh of the same protein spots in three experiments deviates from relative standard as following: 8.53%±5.17%, 12.86%±5.18% and 11.17%±8.86% respectively. Therefore, better resolution and repetitive 2-DE atlas were obtained.Satisfactory 2-DE maps including resolution and reproducibility were obtain. These were used to compare the proteomic difference associated with serum of Osteosarcoma and nomal peple. Of which 20 significantly difference spots were identified. With 13 spots increased and 7 spots decreased in quantity in Osteosarcoma. 13 spots were analyzed by PMF, 6 spots were analyzed by LC-MS/MS,They all have positive result, p8 spot have no result. The correlative function of differential protein spots were explore in Osteosarcoma.To compared the Osteosarcoma tissue and nomal team,our studies find that there is no significantly difference in hige molecular weight. There are 39 difference spots were identified and these spots represent 25 protein, in these spots, 10 spots increased and 10 spots decreased and 2 spots expressed only in Osteosarcoma, The mobility of three spots have changed.Conclusions: 1.The optimized 2-DE and image analysis technique is a better method to support further study of osteosarcoma proteome.2.There are 20 spots differentially expressed in sprum of Osteosarcoma and nomal man, these 10 identified proteins are mainly related with Ca2+ metabolism and cell signal transduction and so on.3. Compared with osteosarcoma and nomal tissue,our studies find differentially proteins related with cell signal transduction and apootosis,metabolism enzyme, cell defence protein and proteins which have a function in sustain cell backbone, energy metabolism and so on,when these proteins have changed in quanlity,there may be an effect on the development of osteosarcoma.
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