茯苓經由調節免疫對抗人類血癌細胞株及肝癌細胞株HepG2 中B型肝炎病毒的HBsAg抗原表現之作用

Immunomodulatory Effect of Poria cocos on Human Leukemic Cell Lines and the Suppression of HBsAg Expression in HepG2

陳裕鏞(Yu-Yawn Chen)

指導教授 ： 張鴻民

國立臺灣大學/生物資源暨農學院/食品科技研究所/博士(2004年)

https://doi.org/10.6342/NTU.2004.10102

摘要

無資料

關鍵字

白血病 ； B型肝炎病毒 ； 多醣體 ； 調節免疫 ； 茯苓

並列摘要

Abstract

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-

並列關鍵字

Poria cocos ； Leukemic ； IFN-gamma ； TNF-alpha ； HBsAg ； polysaccharide ； HepG2 ； Immunomodulatory

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