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The Function of Polysaccharides Induce Aopotosis in the Hepatocellular Carcinoma Cells and Enhance Immunoregulation with EHV-1Vaccine

Author: WangGuiBin
Tutor: ZhaoMin
School: Northeast Forestry University
Course: Microbiology
Keywords: Mushroom polysaccharides HCC cell lines Apoptosis Cell cycle Mitochondrial signaling pathway Two-dimensional electrophoresis
CLC: R285.5
Type: PhD thesis
Year: 2012
Downloads: 158
Quote: 0
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Abstract


In the past years, hepatocellular carcinoma (HCC) has become a common malignant tumor worldwide, especially in Asia and Africa. There is evidence suggesting that the incidence of HCC is rising and has become a major health problem, accounting for approximately6%of all human cancers and the third most common in cancer-related deaths. One of reasons for such a high mortality is the invasive behaviour of cancer cells, which results in the metastasis of liver cancer. Cancer metastasis consists of several interdependent processes including uncontrolled cell proliferation, invasion through surrounding tissues, migration to the distant sites of the human body, and adhesion, invasion and colonisation of other organs and tissues. Tumour growth and metastasis also require from pre-existing vessels. Therefore, ihibition of growth, invasive behaviour and cancer cell-mediated angiogenesis will lead to the suppression of cancer metastasis and would further increase survival of liver cancer patients.Phellinus linteus (PL), Ganoderma lucudum (GL) and Auricularia auricula (AA) are basidiomycete fungus, located mainly in Asia and tropical America, where it gained significant recognition as medicinal mushroom in the traditional Oriental medicine. The biologically active compounds isolated from medicinal mushroom are polysaccharides, acidic proteo-heteroglycans with mixed α-and β-linkages, and a (1→6)-branched type (1→3)-glycan. These complex polysaccharides have been detected in a variety of different mushroon species and linked to the immunostimulatory and antitumour activities and stimulated proliferation of T lymphocytes and activated B cells, and induced maturation of bone marrow-derived dendritic cells and the macrophage response. Previously study showed that polysaccharides from Phellinus linteus can induce apoptosis directly in HepG2cells in vitro through the TEM assay and Annexin V-FITC/PI staine and it seems that polysaccharides can induce HepG2cells apoptosis through down-regulating the expression of Bcl-2and up-regulating the expression of Bax. So, this study were evaluated to confirm the ability of polysaccharides to inhibit cell proliferation and induce apoptotic cell death in HepG2and Bel-7404cells and to evaluate the effects of polysaccharides on immune responses in vaccinated mouse. The main results are summarized as follows:(1) The crude polysaccharides of Phellinus linteus were obtained by ethanol precipitation and whose components were detected the components by phenol-sulfuric acid assay, DNS assay and BCA assay. And the crude polysaccharides were purified by DEAE-Sepharose F.F. and Sephacryl-S200. The proliferation rates of lymphocyte of polysaccharide fractions obtained during the isolation and purification were detected in vitro in order to observe their immune activities. The results showed that crude polysaccharides obtained from ethanol precipitation and PLP-B1fraction had the most promoting effects on lymphocyte proliferation.(2) Employing cell proliferation assay, cell viability assay, cell adhesion and invasion assay, the PLP-B1fraction was detected to investigate the inhibition effect of PLP-B1on HepG2cells. The results showed that PLP-B1fraction markedly suppressed the proliferation, adhesion and invasion on HepG2cells.(3) Using cell cycle assay and Annexin V-FITC/PI staine, the PLP-B1fraction to was detected to investigate the effect of PLP-B1on inducing the apoptosis on HepG2cells The results showed that PLP-B1fraction induced the apoptosis and inhibited cell proliferatiom as well as colony formation of HepG2cells through the S-phase cell cycle arrest.(4) The data including antibody titer, the content of CD3+,CD4+and CD8+T cells and pathological section by HE staining were measured to determine the effect of polysaccharides from Phellinus linteus as immune reagents on inducing the immune responses. The results showed that the antibody titers, the number of CD3+cells, the ration of CD4+/CD8+T lymphocytes were significantly higher than those in control group and the higher dosage could improve immunity of the organ ramarkably.(5) Using cell proliferation assay and anchor-independent growth assay, the polysaccharides from Phellinus linteus (PL), Ganoderma lucudum (GL) and Auricularia auricula (AA) were detected to see the inhibition effect of three kinds of polysaccharides on HepG2and Bel-7404cells. The results showed that polysaccharides from Phellinus linteus (PL), Ganoderma lucudum (GL) and Auricularia auricula (AA) markedly suppressed the proliferation on HepG2and Bel-7404cells.(6) Using cell cell cycle assay, Western blot and Annexin V-FITC/PI staine, the polysaccharides from Phellinus linteus (PL), Ganoderma lucudum (GL) and Auricularia auricula (AA) were detected the molecular mechanisms underlying the anti-tumor properties of polysaccharides on HepG2and Bel-7404cells. The results showed that polysaccharides from Phellinus linteus (PL), Ganoderma lucudum (GL) and Auricularia auricula (AA) had an antiproliferative effect on HepG2and Bel-7404human hepatoma cells. Growth inhibition of HepG2and Bel-7404cells by PL, GL and AA is mediated through the induction of apoptosis and G1or S cell cycle arrest by the suppression of AKT activity through the inhibition of AKT phosphorylation at Thr308or/and at Ser473; the activation of Bcl-2family proteins, an increase in mitochondrial cytochrome c and Smac release; enhancement of the expression of p27Kip or p21Cip; and the suppression of the activities of cyclin D1/CDK4and cyclin E/CDK2.(7)2-DE technique was empolyed to separated total proteins and identify the differentials proteins in the groups of HepG2cell and HepG2cell treted with three polysaccharides. Subsequently MS analysis was used to identify proteins in two groups and59points were chosen for more than two-flod expression differences between the two groups. Western blot and Real time PCR were used to detect the expression of mRNA and proteins to verify the results of2-D.

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