| Hepatoma carcinoma is one of the most common and most dangerous malignant tumor in China. At present the risk of hepatoma carcinoma was rising, the mortality rate of cancer deaths was in the front in China. High-risk groups have tens of millions, but many patients when treatment is already late, what its reason is that the lack of specificity of high early diagnostic methods. AFP is well-known the biological markers of hepatoma carcinoma, closely related with cell number and the differentiation of the cancer cells, but there is some information shows that,20%～30%of HCC patients’AFP detection for negative, or is still in a relatively low levels of the state. In addition, the AFP heighten can also be seen in other disease (such as:malignant teratoma, liver hemangioma, chronic hepatitis and liver cirrhosis, etc.). Although at present a lot of hospital combine ultrasonic inspection with AFP can greatly improve hepatoma carcinoma’s detection rate, but there are about20%of patients with hepatoma carcinoma be misdiagnosised that missed the best treatment time. Therefore, to seek for new of liver cancer sensitive specific serum marker, and looking for new laboratory diagnosis model, is the clinical to solve problems.The isobaric tags for relative and absolute quantitation (iTRAQ) was made by the application of biological systems research a peptide ABI in vitro markers. Research proves that, iTRAQ marker technology is the study of the cell or tissue’s different physiological condition, more normal and pathological state protein group of similarities and differences between an effective way. Looking for and found that different from normal physiological state of specific protein markers disease, it help to clarify the disease pathogenesis, to disease prevention, diagnosis, prognosis and curative effect of monitoring have a positive effect, and help as the new targets for drug clinical development.Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry(MALDI-TOF/MS) is a new soft Ionization organic matter spectrum that recently developed, in recent years has become the detection and identification of peptides, polysaccharide, nucleotide, protein, polymer, glycoprotein, and a variety of polymer synthesis of powerful tools, it is also the protein study group the most powerful tool.Our topic research objective in application iTRAQ markers and MALDI-TOF-TOF biological mass spectrometry technology identification hepatoma carcinoma patients serum protein expression differences, identification for the potential serum marker Clusterin in the hepatoma carcinoma cells SMMC-7721and normal liver cells HL-7702, hepatoma carcinoma organizations and cancer adjacent tissues. Chapter one:Comparison of acetonitrile、ethanol and chromatographic column to Eliminate High-abundance Proteins in Human SerumObjective To compare the effects of acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human14removal to Eliminate High-abundance Proteins in Human Serum.Methods Elimination of serum high-abundance proteins, such as albumin and IgG, were performed with acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human14removal.Results Grey value analysis from1-DE figure, serum after processed by acetonitrile method, multiple affinity chromatography column Human14removal method and ethanol method, the grey value of albumin respectively by the original serum the19into157.2,40.8,8.2; From2-DE analysis that from the multiple affinity chromatography column Human14method, the protein points noticeable increased, compared to the original serum added137;processed by acetonitrile method and ethanol method, although protein point reduced, but the low abundance protein point were showed.Conclusions The acetonitrile precipitation could eliminate the vast majority of high abundance proteins in serum and gain more proteins of low molecularweight, ethanol precipitation could eliminate partly of high abundance proteins in serum, but low abundance proteins were less harvested, And multiple affinity chromatography column Human14method can effectively removed the high abundance proteins, and keep a large number of low abundance proteins. Chapter two:Identification for the potential serum marker Clusterin using mass spectrometry and the verification testObjective To identify the serum differentially expressed proteins of hepatoma carcinoma and verify mRNA and the following protein expression levels in cells and tissues of hepatoma carcinoma.Methods The serum differentially expressed proteins were surveyed in20participants with hepatocellular carcinoma and20healthy people by iTRAQ labeling and LC-MALDI-TOF/TOF MS. Real-time RT-PCR was used to detect different mRNA expression in hepatoma carcinoma cells and healthy hepatocytes, as well as in30pairs of hepatoma carcinoma and adjacent normal tissues. Western-blot was used to determin the clusteri expression in hepatoma carcinoma cells.Results There were51proteins that be significant differently expression between the hepatoma carcinoma group and healthy control. Among them, serum level of clusterin was lower in hepatoma carcinoma patients than that in healthy people by mass spectrometry analysis. The mRNA level of clusterin was20times lower in hepatoma carcinoma cell than that in healthy hepatocyte, and was2.38times lower in hepatoma tissues than that in adjacent normal tissues. The OD values was8.06and27.81in hepata carcinoma cells and normal hepatocytes (P<0.01), respectively,the expression was obviously low in hepatoma carcinoma cell. Conclusions The expresion of clusterin decreased in serum of hepatoma carcinoma patitens, hepatoma carcinoma cells and tissue, the relationship with hepatoma carcinoma was worth further studying. |
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