Study On Dialectically Assiociated Proteins Of Posthepatitic Cirrhosis And Hepatic Cellular Cancer By Serum Proteomics Analysis

Background:Hepatic cirrhosis (HC) is the advanced stage of many chronic hepar diseases. It is a chronic, diffused and progressive pathological change in liver. Many factors including virus, bacterium, infection by Schistosoma, chronic alcoholism, drugs and bane damage can cause HC. Hepatitis B infection is the most common cause in our country.Hepatoma is a kind of common tumor and hepatic cellular cancer (HCC) accounts for about 90%. Pathogeny of HCC has not been clearly known yet. Hepatitis virus infection which is mainly caused by hepatitis B in mainland of China is considered to be the major cause. There are close relationships among chronic viral hepatitis, posthepatitic cirrhosis and HCC. In some extent, they are considered three different developing phases of the same disease. The determinations of treatment based in pathogenesis obtained through differentiation of symptoms and signs of HC and HCC are hot spots of traditional Chinese medicine. The clinical and experimental studies of determination of treatment based in pathogenesis obtained through differentiation of symptoms and signs of HC and HCC are hot spot in traditional Chinese medicine hepatopathy. The relationships between each pattern of syndrome and its related indices have been tentatively confirmed by multi-perspective and multistrata research of molecular biology, immunology, histopathology and imageology, et al. So far, there has not an widely and generally accepted index which can show the nature of Zheng. There needs further studies.Traditional Chinese medicine puts emphasis on the whole status of bodies at every phase of the disease. Determination of treatment based in pathogenesis obtained through differentiation of symptoms and signs means deducing the causes of a disease, nature of disease and location of disease according to the outer signs of a disease. By approaching syndrom, especially the heterologous proteins expression and modified status of different diseases with same symptome and among different symptome with same disease, syndrom Proteomics guided by the theory of syndrom,applying the method of functional proteome to reveal the all proteins and their character related to the formation of syndrom and to interpret the nature of syndrom in the level of integer proteins expression. Proteins can perform all kinds of functions in vital movement. In fact, every kind of life activities is a kind of movement. They are the combined result of specified proteins appearing in different time or space.When we consider the matter basis which causes the syndrom, the nature of syndrom is a kind of proteins or peptides with the function of messenger molecule. Proteomics research reflects the dynamic evolution process of proteins expression of disease in integrated level. It is extremely similar to the method of determination of treatment based in pathogenesis obtained through differentiation of symptoms and signs in traditional Chinese medicine.Objective:Combining differential diagnosis of diseases and differentiation of symptoms and signs, beginning with two clinically correlative diseases and using proteome techniques, to research and prove the heterologous proteins of each disease and the protein bases of dependability in corresponding syndrom of two diseases. To discover the expression of some heterologous proteins in some diseases or syndrom to find the common nature of syndrom in all kinds of complex etiological factors, to approach the systematic and regular relationship among protein polymorphism, disease grouping and medication in traditional Chinese medicine by comparing the differences of protein ingredients between normal samples and sick, among different diseases with same symptome and among different symptome with same disease.Methods:Patients with type B hepatitis correlated HC and patients with HCC are served as research targets. Guided by traditional Chinese medicine theory, the patients with depression of liver-QI and hepatic and renal yin deficiency are screened out. Applying the commonly used 2D electrophoresis in proteomics and combining MALDI-TOF-MS techniques, the different protein components between normal samples and sick, among different diseases with same symptome and among different symptome with same disease are analyzed.1. Establishing the standard of differentiation of symptoms and signs for classification of syndrome.By four steps of symptom questionnaire design, clinical information collection, factor analysis and clinic retest, this study formulates the standard of differentiation of symptoms and signs for classification of syndrome scientifically and systematically in some range of HCC patients. The standard has achieved pleasant effect in clinic.2. 2-DE separating for serum proteome.The specification in the 2-DE separation of serum proteins has been optimized because of the complicated components of serum proteins and their big differences in concentration. Using both methods of acetone precipitating and salt bridge for dechlorination, 2-DE separation becomes more effective. Increasing the sample amount applying longer narrow-range adhesive tapes, the separation effect of low abundance proteins becomes better. The focusing results of time-phase in electrophoresis have been improved time of 8000V.Using these improved techniques, the original technique platform has been improved and 2-DE separation system become more stable. We tried to remove the abundant albumin and IgG in serum using kit. However, when treated with the kit, the protein speckle separated by 2-DE decreases significantly and a lot of proteins have been lost. Thus, we did not apply this method to our study.3. Gel dyeing and image analyzing.The gel were dyed with "sliver blue". The gel dyeing sensitivity is elevated. At the same time, the interference cause by MS test at later period decreases. We get images of PAGE gel dyed and decolored after transmission scanning. PDQuest-7.1.0 is operated to analyze the gel images in order to get information of expression amount and isoelectric point. Protein which have two times expression amount than others is set to be a heterologous protein. The analyzing results are generated by software then verified by handwork.4. Identifying the heterologous protein speckles using MS and database confirming The peptide mass fingerprint (PMF) test of the heterologous protein speckles is carried out by Gene Research Center of Hong Kong University. By scanning and analyzing the database of National Center for Biotechnology Information, we want to find the proteins which match the PMF and some information about these proteins.Results:1. By analyzing the materials acquired from clinic, four factors which are finely agreeable to clinical pattern of syndrome are extracted. According to the statistics analysis together with clinical check, HCC is classified to five pattern of syndrome. There are stagnationof QI due to depression of the liver, endoretention of damp heat, hepatic and renal yin deficiency, asdthenic splenonephro-yang, blood stasis of hepatic blood. Correlative standards formulate.2. High quality serum proteome 2-DE images have acquired. Applying the reformed serum 2-DE separating techniques, an ideal separation effect has achieved and protein loss is avoided even if the high abundance proteins are not removed.3. 35 protein speckles which have obvious expressing differences have been screened out after 2-DE separation and gel image analysis. They are distributed in different groups. The exact distribution is shown in the second section of the experiment part.4. Analyzed by PMF and detected in the database, 9 difference proteins are identified. They are Apo A-1, T4, a₁-antitrypsin, Vitamin D binding protein, haptoglobin, acid glycoprotein, transferring, metaglobulin, albumin mutant R218p and serum amyloid protein precursor. The exact distribution of each group is shown in the second section of the experiment part.Conclusion:In this study, we first use the syndrom proteomics methods to find the proteome basis of different diseases with same symptome and different symptome with same disease. By establishing the differentiation standard of symptoms and signs according to clinical research, applying 2-DE combined mass identifying technique, analyzing serum proteome of patients with depression of liver-QI and hepatic and renal yin deficiency in HC and HCC, the relative heterologous proteins are separated and identified. These proteins are involved in many kinds of human body activites such as metabolism, immunization, blood clotting, endocrine and cancer cell proliferation, and so on. We discover in this study that:1. To study the nature of Zheng, the principal of combining disease with syndrome should be insisted on. Because different etiological factors may cause the different protein expression in the same pattern of syndrome of different diseases;2. "Different diseases with same syndrome" the diagnosis truly has the proteinfoundation. 3. Research technique of "Different syndromes with same disease" is the way to flit the specific object target of syndrome nature.