【作者】 季哲；

【导师】 潘迎捷； 唐庆九；

【作者基本信息】 南京农业大学 ， 微生物学， 2007， 博士

【摘要】 灵芝(Ganoderma lucidum(curtis:Fr.)P.Karst.)在我国已有悠久的应用历史,可用于多种疾病的治疗。灵芝的化学、药理和临床等现代研究也证明灵芝具有抑制肿瘤、保肝、调节血压、降血糖、抗病毒、延缓衰老、抗炎等多种作用,其中灵芝的抗肿瘤作用更是近年来的热点。由于灵芝的化学成分十分复杂,难以得到纯化合物进行抗肿瘤活性及其作用机制的研究,限制了灵芝产业的深入发展。本研究室运用分离纯化与体外筛选模型相结合的方法,分别得到提高免疫功能的活性成分GLIS和诱导细胞凋亡的有效部位GLAI,本论文在前人工作的基础上对它们的作用机制进行探讨,为将来的进一步研究和开发建立了基础。1.灵芝多糖GLIS的免疫刺激活性研究灵芝多糖GLIS是经过多步色谱分离纯化得到的糖蛋白组分,以前的研究发现GLIS可以激活淋巴细胞,并能够特异性地刺激淋巴B细胞增殖,增加NK细胞的活性。本论文在前人工作的基础上,探讨了GLIS对小鼠巨噬细胞株RAw264.7、小鼠骨髓来源巨噬细胞(BMMs)和小鼠淋巴细胞(MSLs)的免疫调节作用,并对激活巨噬细胞的作用机理进行了研究。GLIS处理小鼠巨噬细胞株RAW264.7后,发现其形态发生了变化,如体积变大,并且形成伪足;同时发现GLIs能刺激RAW264.7细胞呼吸爆发,产生大量活性氧,并刺激RAW264.7细胞释放NO,两项实验结果证明GLIS可激活免疫巨噬细胞的氧依赖杀伤机制;GLIS还刺激RAW264.7细胞增加IL-1β、IL-12p35和IL-12p40细胞因子mRNA的表达,而细胞因子TNF-α、IL-6和IL-18 mRNA的表达则基本无变化;另外还发现其分泌TNF-α、IL-1β和IL-12细胞因子的量明显增加。GLIS处理小鼠骨髓来源巨噬细胞(BMMs)后,也发现其体积变大,并且形成伪足;同时GLIS也能刺激BMMs呼吸爆发,产生大量活性氧;并刺激BMMs释放NO,结果与RAW264.7细胞株实验相同;GLIS刺激BMMs后相关细胞因子的表达也增加,在mRNA水平,IL-6、IL-12p35和IL-12p40明显增加,TNF-α、IL-1β和IL-18也有所增加。在蛋白水平,TNF-α、IL-1β和IL-12三种细胞因子的表达明显增加。GLIS还可以可刺激淋巴细胞增殖,并且可刺激其呼吸爆发。研究结果显示GLIS通过激活巨噬细胞氧依赖杀伤系统及刺激其表达细胞因子达到全面激活免疫功能进行抗肿瘤的目的。2.灵芝三萜GLAI诱导肠癌细胞凋亡作用研究灵芝三萜GLAI是通过活性跟踪经多步分离纯化从灵芝子实体中分离得到的一个有效部位,对GLAI的理化性质研究发现,其三萜含量为62.8%;HPLC检测发现GLAI含有8个峰,其中有两个峰确定为灵芝酮二醇和丹芝醇A。GLAI对多种肿瘤细胞株具有抑制活性,包括小鼠淋巴癌L1210细胞株、人淋巴癌K562细胞株、人肠癌SW620细胞株和人乳腺癌MCF7细胞株。GLAI处理SW620细胞后,其细胞形态出现明显的变化,细胞变圆、皱缩,体积变小,并可观察到凋亡小体的产生;经过Hoechst 3258荧光染色和annexin V-FITC/PI双染流式细胞仪检测,证明SW620细胞发生了凋亡:研究还发现,GLAI处理SW620细胞后,凋亡关键酶Caspase-3的活性明显增高;同时发现,随着药物浓度和作用时间的增加,凋亡抑制基因BCL-2 mRNA的表达,逐渐减少;而凋亡促进基因BAX mRNA的表达随之增加;另外发现,凋亡重要相关因子P53蛋白的表达随着药物浓度的增加而增加,而凋亡抑制蛋白XIAP则随着浓度的增加而减少。研究结果显示GLAI能够通过激活Caspase-3酶途径诱导肠癌细胞SW620凋亡,同时通过上调凋亡促进因子的表达,下调凋亡抑制因子的表达达到诱导SW620细胞凋亡的效果。综上所述,灵芝的抗肿瘤作用一方面是通过灵芝多糖激活免疫细胞,通过提高免疫系统的能力来达到消除肿瘤的目的;另一方面是通过灵芝三萜诱导肿瘤细胞凋亡,从而达到消除肿瘤的效果。因此灵芝的抗肿瘤作用可能是多途径共同作用的结果。更多还原

【Abstract】 Ganoderma lucidum(GL),called "Lingzhi" in China,has a long history of use for the treatment of many different diseases.Chemical,pharmacological and clinical studies have testified that GL possesses various therapeutic functions including anti-tumor, hepatoprotective,anti-senility,anti-viral,anti-inflammatory,anti-hypertensive and hypocholesterolemic activity.In recent years,the anti-cancer effects of GL have become a major focus of research activity involving this mushroom.However,the chemical composition of GL is very complex,and obtaining purified material for research into the pharmaceutical effects and mechanisms of GL is often difficult.In this study,two compounds(GLIS-Ganoderma lucidum immunity stimulator,and GLAI-Ganoderma lucidum apoptosis inducer) have been isolated from GL fruit bodies and their bioactivities determined.GLIS stimulates immunity and GLAI induces apoptosis of tumor cells.The mechanisms underlying these activities have been investigated in order to establish a basis for future research.1.Immunomodulating activity of GLISGLIS is a proteoglycan isolated from GL fruit bodies using successive chromatographic steps.Previous research has shown that GLIS stimulated the proliferation of lymphocytes,and that most of the activated lymphocytes were B cells.GLIS also stimulates the activity of NK cells.In this study,the mechanism(s) involved in macrophage stimulation investigated using GLIS modulation of RAW264.7 cells(a mouse macrophage-like cell line),bone marrow-derived macrophages(BMMs) and mouse spleen lymphocytes(MSLs).After exposure to GLIS,RAW264.7 cells exhibited altered morphology,increased in size and formed numerous pseudopodia.GLIS also triggered the respiratory burst in RAW264.7 cells,and stimulated the macrophages to produce superoxide anion and NO. GLIS therefore activates the Oxygen-Dependent Kill System of macrophages.GLIS also regulated related cytokine expression at the mRNA level in activated RAW264.7 cells. Interleukin-1β(IL-1β),IL-12p35 and IL-12p40 gene expression was markedly increased within six hours after exposure whereas no changes were recorded in IL-6,IL-18 and TNF-a gene expression.Large increases in IL-1β,IL-12 and TNF-a protein production were also recorded.After exposure to GLIS,BMMs also exhibited altered morphology,increased in size and formed numerous pseudopodia.GLIS also triggered the respiratory burst in BMMs and stimulated the macrophages to produce superoxide anion and NO.Therefore,GLIS also activates the Oxygen-Dependent Kill System of macrophages.GLIS also regulated related cytokine expression at the mRNA level in activated BMMs.IL-6,IL-12p35 and IL-12p40 gene expression was markedly increased,and increases in IL-1β,IL-18 and TNF-a gene expression was also observed.Large increases in IL-1β,IL-12 and TNF-a protein production was also recorded.GLIS stimulated the proliferation of lymphocytes,and induced the respiratory burst in MSLs.The data indicate that induction of the antitumor effect of macrophages by GLIS involves activation of the Oxygen-Dependent Kill System and regulation of related cytokine expression.2.Apoptosis-inducing activity of GLAIA triterpene fraction,GLAI,was isolated from GL fruiting bodies using successive separation steps combined with activity tracking.The triterpene content of GLAI was 62.8%(using ganodermanondial as standard).HPLC revealed 8 peaks including those corresponding to ganodermanondial and lucidumol A.GLAI inhibited the proliferation of several tumor cell lines including L1210(mouse lymphocytic leukemia),K562(human chronic myelogenous leukemia),SW620(human lymph node metastasis,colon carcinoma) and MCF7(human breast adenocarcinoma,pleural effusion).Marked changes in the morphology of SW620 cells occurred following exposure to GLAI.Cells became more rounded and crimped,and many apoptotic bodies were observed.Hoechst 3258 fluorescence staining combined with annexin V-FITC/PI staining confirmed that GLAI induced apoptosis in SW620 cells.Exposure of SW620 cells to GLAI also increased the activity of caspase-3,the key enzyme associated with apoptosis.At the mRNA level, resulted in down-regulation of Bcl-2 gene expression,and also in XIAP protein production (both Bcl-2 and XIAP are associated with apoptosis inhibition).Conversely,increasing GLAI concentrations and exposure times up-regulated expression of apoptosis enhancer Bax gene and production of apoptosis enhancing p53 protein.In summary,multiple pathways are involved in the antitumor activity GL including the stimulation of macrophages by GL polysaccharide to effectively eliminate tumor cells,and the induction of tumor cell apoptosis by GL triterpenes.更多还原