The Establishment And Application Of Detection Technology For Phosphoryprotein In Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors.for more patients with hepatitis and cirrhosis,themotbidity and mortatity of HCC are higher than that of the world average in china,more than 50%HCC worldwide occur in china,which causing 23 million people's death each vear. HCC is the second-leading cause of cancer death and accounting for 18.8% in the death of malignant tumors in china. Currently, the underlying molecular mechanisms of hepatocarcinogenesis that lead to malignant transformation of normal liver cells and metastasis of tumor cells remain unclear. The detection and anticipation of HCC and postoperative recurrence and metastasis majorly depend on AFP and type-B ultrasonic is unavailable to the tumor smaller than 1 cm. So there is a blind are a in diagnosis and it is hard to make an accurate anticipation. Therefor that studing and revealling the exact molecule mechanism of occurrence and development of HCC, exploring novel tumor markers and targets of drug therapy for tumor is important for the cure of HCC.Proteomic becomes one of the most popular researching area of systemic biology. With the development of proteomic and its crossover with other subjects, new technology and researching ways has been found. Those technology gradually play as an important role in modern bio-medical research. Basic researches of tumor steps into an brand new world. It is usually limited our vision by study protein function merely on the quantity change level. Most protein's activities infected by modification and regulation after translation. Sometimes protein modification dramatically changed without any quantity change. The modification of protein phosporylation is the most important way of covalence modification in human body, which participate in most life activities. Those activities include cell proliferation, development and differentiation; cellular structure regulation; cell atrophy; nerve activities; muscle contraction; metabolism; tumor genesis etc. considering about the important meaning of phosphorylation modification, more and more biological scientists star to work hard in order to explore the unknown process of protein phosphorylation and the biological function that mediated by the protein phosphorylation. Understanding the different state of protein phosphorylat-Ion in different physiological situation can make biological signal pathway alternation clear. Furthermore, It can lead us to discover the mechanism of disease genesis. However, the concentration of phosphoryprotein is pretty low and the quantity of phosphoryprotein's chemical site is very little,which remains many challenges for the large scale analyze of phosphoryprotein. So proteomics of phosphoryprotein developing slowly, and its association with hepatic cancer has not been reported all over the world. Now we found some new researching technology during the pre-researching period, Those technology can be used in examine clinical disease, such as hepatocellular carcinoma. It brings a lot of benefits in discovering the mechanism if hepatic cancer genesis and finding the target for cancer diagnosis and treatment.Purpose:1. full-scale and systemic study the specificity and the alternation of phosphopeptide's enrichment by Ti4+-IMAC.2.analyzing the phosphory-Protein expressing profile of both normal liver tissue and hepatic cancer tissue by using the method Ti4+-IMAC combining with LC-MS2-MS3 and database research. Large-scale and high-flux screening of differential phosphoprotein, identifying the phosphorylated sites and protein kinase by using biological information.3. investigate the expression of differential phosphoprotein(PED/PEA-15, XIAP, P27-T187) in tissue of hepatocellular carcinoma and explore its relationship to clinicopathological features of HCC.Methods:1. the standard phosphoprotein(a-casein andβ-casein) wss used as sample,compare and analyze the enrichment effection of Ti4+-IMAC by MALDL-TOF MS.2. analyze the phosphoprotein expressing profile, phosphorylated sites and prorein kinase in both hepatic cancer tissues(12 samples) and normal liver tissue(10 samples) by the method Ti4+-IMAC combining with LC-MS2-MS3 and datebase research,in order to find differential phosphprotein.3. pick up 3 kinds of differential phosphprotein to study the differences between 40 hepatic cancer tissue as well as its nearby hepatic tissue and another 12 normal liver tissue by using IHC,Western blot and RT-PCR.Result:1.the enrichment of phosphopeptide is available and specificity by Ti4+-IMAC.2.240 phosphopeptides,169 phosphorylated sites and 149 phosphprotein including cytoskeletal protein, sigal conduction protein, enzymolysis protein and other unknown proteins were found by the method Ti4+-IMAC combining with LC-MS2-MS3 and datebase research. Further more,20 differential phosphproteins were indentified including cystoskelet-on, cell cycle, Apoptosis and deuto-cell from hepatic cancer comparing with the normal liver tissues.also it is found that the phosphproteins in hepatic cancer were major actived by protein kinase CK2.3. the 3 protein(PED/PEA-15(s-116),XIAP(s-87), P27-T187) were high expressed in Hepatic cancer comparing with nearby hepatic tissue and normal liver tissues by using IHC and Western blot(P<0.05). And The expression of PED/PEA-15(s-116) was significantly associated with pathological grade, clinical stage in HCC(P<0.05), however it was not significantly correlated to other clinicop-athological features(P>0.05). the expression of PED/PEA-15(s-116) and XIAP(s-87) was positive correlation, (r=0.371, P=0.018).Conclution:1. Ti+-IMAC is available to enrichment phosphopeptides for its high specificity and alternative, which can be an ideal material for the study of ph osphoproteomic.2. the method Ti4+-IMAC combining with LC-MS2-MS3, database research and Bioinformatics provides a drastic perspective in Phosphoproteomic, which may be an important tool to explore the pathogenesy of tumor.3. the phosphprotein datebase (149 phosphproteins, phosphorylated sites and protein kinase) and 20 differential phosphproteins may paly an important role in exploring the hepatic cancer pathogenesy and providing a new drug therapy way.4. the expression of PED/PEA-15 (s-116),XIAP(s-87),P27-T187 is the same as MS by IHC and Western Blot,suggesting the high expression of those proteins may be closely realted to the occurrence and development of HCC.