| Primary hepatic carcinoma (PHC) is one of the most common malignant tumors in the world. It has some clinical features such as rapid development, high malignant degree, difficult therapy and less survival time. There were nearly 680 000 patients death of PHC in 2007 around the world. It was the third causes of cancer death in the world. In china, the liver cancer incidence and mortality rates were higher than the global due to the large numbers of hepatocirrhosis patients. The patients died from liver cancer in our country accounted 55% of the world population PHC deaths rate per year. And it has no specific drug therapy yet. Therefore, many researchers are dedicated to the research of prevention and cure liver cancer for a long time in order to decrease the incidence and mortality rate.The disproportion of apoptosis played an important role in the tumor development. The tumor development may be inhibited if apoptotic proportion increases of tumor cells being induced apoptosis. Epigallocatechin gallate (EGCG), was a major components of green tea polyphenols extractive catechin, which can inhibit tumor growth and induce cells apoptosis. But higher concentrations of EGCG can caused cell death, so the most effective way of reduce cell injury is by detecting in the early apoptosis of cell and controlling the EGCG dosage.Most studies on the EGCG induced tumor cell apoptosis were evaluation through apoptotic peak (sub G1 peak) measured by flow cytometry (FCM). But sub G1 peak is not specific change in apoptosis. Therefore, this study established a new early apoptosis detection method, PSAB-VA/EL method, to detect early apoptosis in human hepatocelluar carcinoma cell line SMMC-7721 by EGCG.At the same time, the key proteins of apoptosis in human hepatocelluar carcinoma cell line SMMC-7721 induced by EGCG were detected by SELDI-TOF-MS and MALDI-TOF/TOF. The possible mechanism of apoptosis inducing hepatocelluar carcinoma cell line SMMC-7721 by EGCG was investigated.Part OneEstablishment of a PSAB-VA/EL method for detected early apoptosis cellsObjective: To establish an economical, practical and high specificity method for detecting early apoptosis cells, then evaluated this method comparing with Annexin V-FITC/PI method.Methods: 1. The Jurkat cells was treated with different concentrations of EGCG, early apoptosis of Jurkat cells was measured by Annexin V-FITC/PI method, and took it as an apoptosis model; 2. Attempt several different of PSAB antibody and nucleic acid fluorescent to detect early apoptosis model. Results: 1. Jurkat cells were detected by Annexin V-FITC/PI method, with increasing the concentration of EGCG, the proportion of normal cells dropped consistently. While the percentage of apoptotic cells significantly increased, the early apoptotic cells remained at 10%; 2. After comparing PSAB antibody and nucleic acid fluorescent dye, founded that the collocation of PSAB antibody with EL may be used for detecting apoptosis better.Conclusion: 1. The change trends of results were consistent both in SAB-VA/EL and Annexin V-FITC/PI method which detected apoptosis in the percentage of each stage; 2. The PSAB-VA/EL may be a sensitive method used for detecting early apoptotic cells.Part Two The early apoptosis in human hepatocelluar carcinoma cell line SMMC-7721 induced by EGCGObjective: To investigate the changes of apoptosis in hepatocelluar carcinoma cell line SMMC-7721 induced by EGCG.Methods: 1. MTT assay was used to detect the effect concentration of apoptosis of SMMC-7721 cells induced by EGCG; 2. The growth inhibition and early apoptosis of SMMC-7721 cells was measured by flow cytometry using PSAB-VA/EL method (Described in Part One).Results: The best dose of EGCG effecting on induction of early apoptosis in SMMC-7721 cells was 100mg/L, and the early apoptosis rate was up to 48.16%. With increasing the concentration of EGCG (125~175mg/L), early apoptosis was significantly decreased, while the percentage of apoptotic cells was significantly increase.Conclusion: EGCG significantly induced early apoptosis in human hepatocelluar carcinoma SMMC-7721 cells, in a dose-dependent manner.Low dose could induce apoptosis, while large dose could kill cells and cause cell death. Part Three Alteration of proteomic expression in SMMC-7721 cells after induced by EGCG and identifyObjective: The key proteins of apoptosis in human hepatocelluar carcinoma cell line SMMC-7721 induced by EGCG were detected by protein mass spectrometry. To investigate the possible mechanism of apoptosis of hepatocelluar carcinoma cell line SMMC-7721 induced by EGCG.Methods: 1. Proteins coexisting in human hepatocellular carcinoma serum and human hepatocelluar carcinoma SMMC-7721 cells lysate were detected by SELDI-TOF-MS; 2. Proteins existing in human hepatocelluar carcinoma SMMC-7721 cells lysate treated by EGCG was detected by SELDI-TOF-MS; 3. Detection of the key proteins coexisting in human hepatocellular carcinoma serum, SMMC-7721 cells lysate and SMMC-7721 cells lysate treated by EGCG; 4. All the proteins mentioned above were detected by using iTRAQ labeling coupled with MALDI-TOF/TOF.Results: 1. there were 23 proteins coexisting in human hepatocellular carcinoma serum and human hepatocelluar carcinoma SMMC-7721 cells lysate; 2. Comprised with SMMC-7721 cells lysate treated by EGCG, 16 proteins were up-regulated expression and 16 proteins were down-regulated expression in SMMC-7721 cells lysate; 3. Among those proteins, the m/z value of key proteins were 4283.74 Da and 8562.11 Da; 4. The m/z value of highly expressedα-1-Antitrypsin enzymolysis fragment was very close to key proteins 4283.74 Da; The m/z value of low expressed Plasminogen enzymolysis fragment was very close to key proteins8562.11 Da.Conclusion: 1. By detection of human hepatocellular carcinoma serum and SMMC-7721 cells lysate with SELDI-TOF-MS and MALDI-TOF/TOF is helpful to reflect the alteration of low-abundance proteins objectively; 2. The possible mechanism of apoptosis inducing hepatocelluar carcinoma cell line SMMC-7721 by EGCG is concerned with the key proteins ofα-1-Antitrypsin and Plasminogen.
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