【作者】 常花蕾；

【导师】 雷林生；

【作者基本信息】 南方医科大学 ， 药理学， 2009， 硕士

【摘要】 大量研究表明金针菇具有抗肿瘤、免疫调节、抗病毒、抗炎、抗疲劳、耐缺氧等功能。金针菇中含有多种有效成分,其中金针菇多糖受到广泛关注,其主要作用表现为免疫调节与抗肿瘤。免疫调节方面的研究证明金针菇多糖（FVP）能促进正常和荷瘤小鼠脾淋巴细胞转化,增强NK细胞活性和增加IL-2的生成,增强正常小鼠腹腔巨噬细胞的吞噬能力,增加正常小鼠的血清溶血素含量。抗肿瘤方面的研究证明金针菇多糖（FVP）对小鼠S₁₈₀肉瘤、小鼠腹水型肝细胞癌H₂₂有抑制作用,对治疗卵巢癌、乳腺癌都有较好疗效。但在细胞因子水平上金针菇多糖如何发挥免疫调节作用和抗肿瘤作用尚不完全清楚,有待深入研究。目的:1.研究金针菇多糖（FVP）对正常小鼠脾细胞产生肿瘤坏死因子-α（TNF-α）、干扰素-γ（INF-γ）、白细胞介素-2（IL-2）的影响,及对脾细胞代谢MTT活力的影响。2.研究金针菇多糖（FVP）对正常小鼠腹腔渗出细胞产生肿瘤坏死因子-α（TNF-α）的影响,及对腹腔渗出细胞代谢MTT活力的影响。3.研究金针菇多糖（FVP）对荷S₁₈₀实体瘤小鼠血清肿瘤坏死因子-α（TNF-α）、干扰素-γ（INF-γ）含量的影响及抑瘤作用。4.研究金针菇多糖（FVP）免疫调节作用和抗肿瘤作用的作用机制。方法:1.取正常小鼠脾细胞,加入金针菇多糖（FVP）,在37℃下培养48h,收集培养上清,用ELISA方法检测其中TNF-α、IFN-γ、IL-2的含量,或者用MTT比色法检测细胞代谢MTT活力。2.取正常小鼠腹腔渗出细胞,加入金针菇多糖（FVP）,在37℃下培养10h,收集培养上清,用ELISA方法检测其中TNF-α的含量,或者用MTT比色法检测细胞代谢MTT活力。3.建立荷S₁₈₀实体瘤小鼠动物模型,空白对照组:每天腹腔注射（ip）无菌生理盐水0.1ml/10g体重,其他组给药体积与此相同;阳性对照组:每天ip灵芝多糖100 mg/kg;FVP低、中、高剂量组:每天分别ip FVP 25、50、100mg/kg;连续给药7d后次日上午,分离血清用于TNF-α和IFN-γ的含量测定。取血后,分离瘤体,称重,并计算抑瘤率和脾脏指数。结果:1.FVP（50,100,200μg/ml）可以增强正常小鼠脾细胞代谢MTT活力,促进脾细胞分泌TNF-α、INF-γ、IL-2,且增强作用随多糖浓度的增加而增强,在一定浓度范围内具有剂量依赖关系。与对照组相比,FVP促进脾细胞分泌TNF-α的作用最为明显。2.FVP（50,100,200μg/ml）对正常小鼠腹腔渗出细胞代谢MTT活力有增强作用,可以显著促进腹腔渗出细胞分泌TNF-α（P=0.000,0.000,0.000）,且有一定的浓度依赖关系。3.FVP（25,50,100mg/kg）三个剂量组对小鼠S₁₈₀实体瘤生长均有抑制作用（P=0.000,0.000,0.000）,且可以增加S₁₈₀实体瘤小鼠的脾脏指数（P=0.000,0.000,0.000）。其中,中、高剂量组可提高荷S₁₈₀实体瘤小鼠血清中TNF-α的水平（P=0.005,0.000）和INF-γ的水平（P=0.047,0.009）,具有一定的剂量依赖关系。4.FVP（200μg/ml）能促进正常小鼠腹腔渗出细胞产生TNF-α,抗TLR4抗体（1和2μg/ml）对这一促进作用有显著的对抗作用（P=0.034,0.010）,但不能完全阻断;然而,p38 MAPK特异性阻断剂SB203580（20μM）和NF-κB特异性阻断剂helenalin（10μM）,可以完全取消FVP促进荷瘤小鼠腹腔巨噬细胞分泌TNF-α的作用（P=0.006,0.003）。结论:1.金针菇多糖促进正常小鼠脾细胞代谢MTT活力和分泌细胞因子TNF-α、IFN-γ、IL-2。2.金针菇多糖促进正常小鼠腹腔渗出细胞代谢MTT活力和分泌细胞因子TNF-α。3.金针菇多糖抑制小鼠荷S₁₈₀实体瘤的生长,并且提高荷S₁₈₀实体瘤小鼠血清中TNF-α和IFN-γ的水平。4.金针菇多糖促进TNF-α产生的信号转导机制可能是作用于小鼠腹腔渗出细胞上的TLR4,然后经p38 MAPK信号转导途径激活NF-κB,后者启动TNF-α基因转录。5.金针菇多糖免疫调节作用和抗肿瘤作用可能与促进正常小鼠产生TNF-α有关。更多还原

【Abstract】 Numerous studies have demonstrated that Flammulina velutipes polysaccharides have anti-tumor,immunomodulatory,anti-virus,anti-inflammation,anti-fatigue and anti-hypoxia effects.Flammulina velutipes have many biological active components and people have paid much attention to Flammulina velutipes polysaccharides（FVP）, whose main functions are immunomodulatory action and anti-tumor effect.The research on immunomodulation has testified that FVP can promote spleen cells transformation in normal and S₁₈₀ tumor-bearing mice,enhance activities of NK cells and the production of interleukin 2,strengthen phagocytosis of peritoneal macrophages in normal mice,and increase the amount of serum hemolysin in normal mice.The study of anti-tumor effect have proven that FVP can inhibit S₁₈₀ sarcoma and H₂₂ ascitic type hepatic tumor,and FVP have favorable effects on ovarian cancer and breast cancer.But the mechanism of immunomodulatory and anti-tumor effects of FVP on cytokines is still unclear.It is,therefore,necessary for the mechanism to be further studied.Objective:1.To study the effect of Flammulina velutipes polysaccharides（FVP） on the production of tumor necrosis factorα（TNF-α）,interferonγ（INF-γ） and interleukin 2 （IL-2） by murine spleen cells,and the effect on spleen cells metabolic activity with methylthiazolyl tetrazolium（MTT） colorimetry assay.2.To investigate the effect of Flammulina velutipes polysaccharides（FVP） on the production of tumor necrosis factorα（TNF-α） by murine peritoneal exudate cells and the effect on peritoneal exudate cells metabolic activity with methylthiazolyl tetrazolium（MTT） colorimetry assay.3.To study the effect of Flammulina velutipes polysaccharides（FVP） on the serum amount of tumor necrosis factorα（TNF-α） and interferonγ（INF-γ） in S₁₈₀ tumor-bearing mice,and the tumor growth inhibition rate.4.To explore the mechanisms of immunomodulatory and anti-tumor actions of Flammulina velutipes polysaccharides（FVP）.Methods:1.Splenocytes from normal mouse were incubated at 37℃in the presence of Flammulina velutipes polysaccharides（FVP） for 48h.ELISA was employed to determine the amount of TNF-α,IFN-γ,and IL-2 in the culture supernatants.And MTT colorimetry was used for the assay of cell metabolic activity.2.Peritoneal exudate cells from normal mouse were incubated at 37℃in the presence of Flammulina velutipes polysaccharides（FVP） for 10h.ELISA was employed to determine the amount of TNF-αin the culture supernatants.And MTT colorimetry was used for the assay of cell metabolic activity.3.Mouse models bearing S₁₈₀ solid tumor were established.The control group was then administered intraperitoneally with sterile physiological saline solution at 0.1ml/10g body weight.The administered volume of other groups was the same as that of control.The positive control group was administered with Ganoderma lucidum polysaccharides（GLP）,at 100 mg/kg intraperitoneally and the low,median and high dose groups of Flammulina velutipes polysaccharides were administered at 25,50,and 100mg/kg intraperitoneally.After 7 consecutive days,the serum samples were collected.Then TNF-αand IFN-γlevels in the samples were measured by using enzyme-linked immunosorbent assay（ELISA）.Then the implanted tumors and spleens were removed and weighed,and the tumor growth inhibition rate and spleen index were calculated.Results:1.FVP（50,100 and 200μg/ml） could promote the metabolic activity of murine splenocytes and increase the amount of TNF-α,INF-γand IL-2 in the supernatants of splenocyte cultures.The effect increased along with the increment of the concentration of FVP was in a concentration-dependent manner in a certain range of concentrations.Compared with control group,the promotion effect on TNF-αlevel appeared to be strongest among the cytokines examined.2.FVP（50,100,and 200μg/ml） could promote the metabolic activity of murine peritoneal exudate cells and obviously increase the amount of TNF-αin peritoneal exudate cells cultures（P=0.000,0.000,and 0.000） in a concentration-dependent manner.3.FVP（25,50,and 100mg/kg） could inhibite the growth of S₁₈₀ solid tumor （P=0.000,0.000,and 0.000）,and increase the index of spleen in S₁₈₀ solid tumor mice （P=0.000,0.000,and 0.000）.The median and high doses of FVP could raise the serum levels of TNF-α（P=0.005 and 0.000） and INF-γ（P=0.047 and 0.009） in the S₁₈₀ tumor-bearing mice in a dose-dependent manner.4.FVP（200μg/ml） promoted the production of TNF-αby murine peritoneal exudate cells.Anti-TLR4 antibody（1 and 2μg/ml） showed marked,although not complete,suppression on this effect（P=0.034 and 0.010）.However,specific p38 MAPK blocker SB203580（20μM） and NF-κB blocker helenalin（10μM） could completely abolish the stimulatory effect of FVP on the production of TNF-αby murine peritoneal exudate cells（P=0.006 and 0.003）.Conclusion:1.FVP can promote the metabolic activity of murine splenocytes and increase the amount of TNF-α,INF-γand IL-2 in the supernatants of splenocytes cultures. 2.FVP can promote the metabolic activity of murine peritoneal exudate cells and obviously increase the amount of TNF-αin peritoneal exudate cells cultures.3.FVP can inhibite the growth of S₁₈₀ solid tumor,and increase the serum levels of TNF-αand INF-γin S₁₈₀ solid tumor mice.4.The signaling mechanism by which FVP promote TNF-αproduction may be as follows,the polysaccharides act on the TLR4 receptors on murine peritoneal exudate cells,signal through p38 MAPK pathway,and then activate NF-κB.The activated NF-κB in turn initiates the gene transcription of TNF-α.5.The mechanism of immunomodulatory and anti-tumor effects of FVP may be related with their potentiation of the production of TNF-αin mice.更多还原