張鴻民臺灣大學：食品科技研究所陳裕鏞Chen, Yu-YawnYu-YawnChen2007-11-272018-06-292007-11-272018-06-292004http://ntur.lib.ntu.edu.tw//handle/246246/56329Abstract Wolfiporia cocos gilbertson (WCG) [the sclerotium of Poria cocos (Schw.) Wolf], an oriental fungus termed as Fu-Ling in Chinese, has been widely used as a Chinese traditional herbal medicine, such as a tonic food for health promotion, for centuries. In the present study, a neutral polysaccharide fraction from WCG (WCGPS) was isolated by cool-water extraction and the subsequent ethanol precipitation. The effect of WCGPS on the proliferation and differentiation of human leukemic cells, U937 and HL-60, was investigated in vitro. Results showed that the conditioned medium prepared with WCGPS of 15 mg/mL- stimulated human blood mononuclear cells (WCGPS-MNC-CM) exhibited an potent activity in suppressing the proliferation of U937 and HL-60 cells by up to 87.3% and 74.7%, respectively. Furthermore, WCGPS-MNC-CM treatment induced about 66.6% of the U937 cells and 49.4% of the HL-60 cells to differentiate into mature monocytes/macrophages, which also markedly express surface antigens of CD11b, CD14, and CD 68. The differentiated U937 and HL-60 cells displayed physiological functions such as phagocytosis and respiratory burst. However, neither WCG-PS alone nor normal MNC-CM showed similar effects. When consider both of the results from DNA ladder test and MTT test together, it demonstrated that the minor part of leukemic cells without differentiation underwent the apoptosis procedure. Interestingly, the levels of interferon (IFN)-g and tumor necrosis factor alpha (Geneva-Popova and Murdjeva 1999) in MNC-CM prepared without WCGPS treatment were very low, however, they were substantially increased up to about 41 and 10 folds, respectively, in WCGPS-MNC-CM. Antibody neutralization experiments of the WCGPS-MNC-CM were performed and the results revealed that the growth-inhibiting and differentiation-inducing activities of PSPS-MNC-CM were mainly due to the elevated cytokines, especially IFN-g and TNF-a. Therefore, these two cytokines acted synergistically on inhibiting tumor cell growth and inducing differentiation of the targeted U937 and HL-60 cells. The molecular weight of WCGPS was approximately 160 kDa, as estimated by gel permeation chromatography. Results revealed that WCGPS was capable of inhibiting proliferation and inducing differentiation of human leukemic U937 and HL-60 cells by stimulating cytokine production from human peripheral blood mononuclear cells. It suggests that WCGPS is a biological response modifier, instead of a cytotoxic reagent, and may be potential to be an adjuvant in cancer therapy.Fuling (Poria cocos, PC) is a traditional Chinese medicinal herb with mild adverse pharmacological effect. The gentle physiological effects and multiple medical uses make PC as one of the most useful ingredient in the prescriptions of Chinese traditional medicines. The crude extracts of PC have been found to stimulate the human peripheral blood mononuclear cells (PBMNC) to secret many cytokines. The conditioned medium (CM) exerted the inhibitory activities to the expression of HBsAg in Hep G2A2 cells that are stably transfected with HBV DNA. The bioactive fractions were obtained from cool-water extracts by a 80 % ethanol precipitation and a series of separating procedures, such as cation-exchange chromatography, anion-exchange chromatography, and then, gel filtration chromatography. The most active fraction with which CM was prepared can significantly inhibit the expression of HBsAg in Hep G2A2 cells. The relative expression of HBsAg in Hep G2A2 cell line was about 43.3 % when the CM was stimulated with this fraction at a dosage of 30 mg/mL. However, obvious cytotoxicity of this fraction to Hep G2A2 cells was observed. This active fraction was estimated to be a neutral polysaccharide fraction with a molecular weight of about 160 kDa. By the same separating procedures, the other active fraction (II) was obtained from the supernatant of cool-water extracts of PC by 80 % ethanol soluble portion, and was characterized to be an anionic polymer with a molecular weight of about 28 kDa. The fraction (II) termed as PC-P28. Of note, a remarkably inhibitory activity (about 38 %) to the expression of HBsAg in Hep G2A2 cells was observed when cells were cultured with the CM prepared with this PC-P28. Importantly, PC-P28 exhibited no or slight cytotoxicity to Hep G2A2 cells when the CM was stimulated with a level of PC-P28 lower than 5 mg/mL. CM prepared with this active PC-P28 exhibited high productions of IFN-γ and TNF-. PART ONE Antiproliferative and Differentiating Effects of Polysaccharide Fraction from Fu-Ling (Poria cocos) on Human Leukemic U937 and HL-60 Cells Abstract 01 List of Abbreviations03 CHAPT 1. Introduction to leukemic cells, BRMs, and immunomodulation 1.1 Fuling [Poria cocos (Schw.) Wolf] (Polyporaceae) 04 1.2 The potential anti-tumor activities of Fuling 06 1.3 Immune system and tumor 07 1.4 Leukemia 1.4.1 The characteristic of HL-60 leukemia cell as target cells15 1.4.2 The characteristics of U937 leukemia cell17 1.5 Effect of interferon-g (IFN-g) on the antitumor20 1.6 Role of INF-g in the differentiation of leukemia cells21 1.7 The differentiation activity of IFN-g may exert synergistically with other cytokines or factors23 1.8 The restriction of cancer therapy by IFN-g alone 25 1.9 Effect of TNF-a 26 1.10 The restriction of cancer therapy by TNF-a alone 27 1.11 The role of TNF-a in the differentiation of leukemia cells 28 1.12 Does TNF-a have the therapy potential for the patients with myeloid leukemia ? 29 1.13 It is risky for the clinic therapy by usage of TNF-a only31 1.14 BRMs are potentially important to act as the fourth modality for the treatment of cancer31 1.15 The potential antitumor activity of BRMs 35 1.16 The therapeutic trials of BRMs 38 CHAPT 2 . Materials and Methods 40 2.1 Materials 40 2.1.1Tools 40 2.1.2 Reagents 43 2.2 Methods 45 2.2.1 Isolation of polysaccharide from Wolfiporia cocos gilbertson (WCGPS) and estimation of molecular weight45 2.2.2 Fractionization by ion-exchange and gel filtration chromatographies46 2.2.3Phenol-Sulfuric Acid Method to determine the content of polysaccharide48 2.2.4 The culture of cell lines 48 2.2.5 Isolation of mononuclear cells and preparation of mononuclear cell-conditioned medium (MNC-CM) 49 2.2.6 Treatment of leukemia 50 2.2.7 Cell proliferation51 2.2.8 Maturation profile 52 2.2.9 Assay for superoxide 53 2.2.10 Assay for Phagocytosis53 2.2.11 Assay for cytokines 54 2.2.12 Assay for differentiation antigens54 2.2.13 Antibody neutralization5 3.2.14 Cell micrographs55 2.2.15 DNA gel electrophoresis56 2.2.16 Statistical analysis56 CHAPT 3. Results 58 3.1 Determination of optimum extraction temperature59 3.2 Determination of incubation period of time for MNC-CM62 3.3 Determination of optimal alcohol volume for precipitation64 3.4 Optimum ratio of MNC-CM to culturing medium and growth inhibition rate of U93766 3.5 DEAE-Sephadex C-25 cation-exchange chromatography68 3.6 Effect of WCG-PS and WCG-PS-MNC-CM5 on leukimea cell proliferation79 3.7 Inhibition of U937 and HL-60 cell growth by WCG-PS-MNC-CM83 3.8 Differentiation-inducing effects of WCG-PS-MNC-CM 86 3.9 Functional changes in U937 cells 91 3.10 Effect of WCG-PS-MNC-CM on the expression of monocytes-associated antigens 98 3.11 Effects of WCG-PS in cytokine level in WCG-PS treated-MNC-CM 102 3.12 Effect of antibody neutralization 107 CHAPT 4. Dissussion113 PART TWO Immunomodulatory Suppression of HBsAg Expression in HepG2 by Poria cocos Abstract 125 CHAPT 5. Introduction to hepatitis B virus , BRMs, and immunomodulation 5.1. Virologic Characteristics of hepatitis B virus 125 5.2. The replication cycle of hepatitis B 128 5.3. HbsAg as index or diagnosis for progression of haptitis B disease 130 5.4.HbsAg as index for hepatocellular carcinoma (HCC) induced by hepatitits B virus34 5.5. Disadvantage of interferon alfa treatments for chronic HBV infection 135 5.6. Disadvantage of lamivudine treatments for chronic HBV infection 137 5.7. The role of cytokines secreted from immunomodulated immune system played in the clearance of hepatitis B virus139 5.7.1. IFN-gamma in the clearance of hepatitis B virus without killing hepatocytes139 5.7.2. TNF-alpha in the clearance of hepatitis B virus without killing hepatocytes 142 5.7.3. The TNF-alpha and IFN-gamma can also exert anti-viral activity by inducing apoptosis of infected cells143 5.8. Immunomodulators from natural products or Chinese medical herb as BRM against the expression of hepatitis B virus146 CHAPT 6. Materials and Methods 151 6.1. Chemicals and reagents 151 6.2. Isolation of polysaccharide from Poria cocos (PC-P28) 151 6.3. Polysaccharide assay153 6.4. Preparation of mononuclear cell-conditioned media (MNC-CM) 154 6.5. Hepatoma cell cultures155 6.6. Assay for the relative HBsAg expression156 6.7. Assay for cytokines157 6.8. Phenol-sulfuric acid method to determine the content of polysaccharide158 6.9. Antibody neutralization158 6.10. Protein assay159 6.11. Statistical analysis159 CHAPT 7. Results161 7.1. Determination of the optimal incubation period of time for MNC-CM 162 7.2. Optimal ratio of MNC-CM to culturing medium for the inhibition of HbsAg expression 164 7.3. Isolation of active compounds from CWEP portion166 7.4. Isolation of active compounds from CWES portion177 7.5. Effects of PC-P28 on cytokine level in MNC-CM 188 7.6. Cytokine levels in PC-P28-MNC-CMs 191 7.7. Anti-cytokine antibody neutralization 193 CHAPT 8. Discussion 195 Reference2103010991 bytesapplication/pdfen-US白血病B型肝炎病毒多醣體調節免疫茯苓Poria cocosLeukemicIFN-gammaTNF-alphaHBsAgpolysaccharideHepG2Immunomodulatory[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56329/1/ntu-93-D89641005-1.pdf