【作者】 张莘莘；

【导师】 谢明勇； 聂少平；

【作者基本信息】 南昌大学 ， 食品科学与工程， 2014， 博士

【摘要】 黑灵芝多糖PSG-1是从黑灵芝子实体中提取的多糖组分，主要由木糖、岩藻糖、甘露糖、半乳糖、葡萄糖等组成。大量研究表明，该多糖具有抗氧化、提高免疫力、心肌保护、抗糖尿病、抗肿瘤、抗衰老等作用，但其抗肿瘤作用机制方面的研究甚少，本研究旨在探讨PSG-1的抗肿瘤作用及其分子机制，从而为其开发为一种新型药品或功能食品提供有价值的理论依据。本研究分别从体外和体内两种途径研究PSG-1的抗肿瘤作用及机制。本课题在现代分子生物学理论及技术的指导下，应用流式细胞术(FlowCytometry, FCM)、蛋白印迹(Western Blot)、酶联免疫(Enzyme LinkedImmunosorbent Assay, ELISA)及逆转录-聚合酶链式反应(ReverseTranscription-Polymerase Chain Reaction, RT-PCR)等技术，分别从整体、细胞、分子、基因水平，将PSG-1对荷瘤小鼠的抑制肿瘤生长作用及作用通路进行了深入研究。通过整体和离体两种途径进行研究和探讨，具体研究如下：在体外研究中，将不同浓度的PSG-1分别直接作用于S180、CT26肿瘤细胞，通过MTT法测定PSG-1对上述两种肿瘤细胞的增殖抑制作用，结果发现：在体外，各剂量浓度的PSG-1对S180、CT26细胞的增殖抑制率无显著性差异；这说明PSG-1对S180和CT26肿瘤细胞无直接抑制增殖作用，同时也无细胞毒性。另外，用PSG-1预处理小鼠腹腔巨噬细胞，实验结果证明：腹腔巨噬细胞吞噬杀伤S180和CT26肿瘤细胞的能力与PSG-1浓度呈剂量依赖性增强。说明PSG-1能有效地活化腹腔巨噬细胞，提高其吞噬杀伤能力。在体内实验中，首先分别采用S180、CT26肿瘤细胞株建立荷瘤小鼠模型，一周左右后，接种处长出80~90mm3大小的肿瘤说明造模成功。然后将小鼠进行随机分组，每天灌胃或注射给药一次。2周后，将部分小鼠首先进行眼眶取血，然后处死，分离出肿瘤和各组织器官；其余部分小鼠用于提取腹腔巨噬细胞和脾细胞实验，测定细胞相应的功能活性。结果显示：与对照组相比，PSG-1组小鼠肿瘤抑制率随剂量升高逐渐增加，脾指数、胸腺指数也显著增加，差异性显著(P<0.05)。此外，PSG-1组小鼠血清中和腹腔巨噬细胞分泌的细胞因子和一氧化氮（NO）均呈剂量依赖性增加；巨噬细胞的吞噬能力也有效增强，此外PSG-1还显著提高了T、B淋巴细胞的增殖能力。说明PSG-1在体内具有很强的抑制肿瘤生长的效果，并且能通过改善荷瘤小鼠的免疫功能，间接地起到抗肿瘤作用。为了明确PSG-1抗肿瘤活性的分子机制和作用途径，本实验分别从不同的角度做了深入的研究和探讨。通过分离纯化得到荷瘤小鼠的腹腔巨噬细胞，然后分别加入anti-TLR4抗体、p38MAPK阻断剂SB203580和NF-κB特异阻断剂pyrrolidine dithiocarbamate (PDTC)，来探讨PSG-1抗肿瘤作用的信号路径。结果显示：anti-TLR4抗体能部分阻断PSG-1诱导的促腹腔巨噬细胞分泌TNF-α的作用，说明TLR4可能是PSG-1在细胞膜上的作用受体之一。特异性阻断剂SB203580能完全阻滞PSG-1诱导的提高腹腔巨噬细胞产生TNF-α的作用；此外，PDTC也能够显著地抑制PSG-1诱导的TNF-α的分泌。为进一步验证，本研究运用Western-blotting法测定了TLR4、NF-κB、IκBα以及pp38等蛋白的表达情况。结果显示，各组荷瘤小鼠中，PSG-1组腹腔巨噬细胞的膜蛋白TLR4表达显著增加，核蛋白NF-κB表达水平也呈剂量依赖性升高；而细胞质中IκBα的蛋白表达量逐渐降低，并且磷酸化蛋白pp38表达也显著增强，与对照组相比，具有显著性差异。实验结果提示PSG-1通过作用于腹腔巨噬细胞膜上的TLR4受体，将信号传递进入细胞，然后激活NF-κB和p38MAPK信号通路，促进TNF-α的分泌，强化机体免疫力，间接地发挥抗肿瘤的作用。另外，本研究通过流式细胞术检测了各组荷瘤模型小鼠的肿瘤细胞的凋亡程度。测定结果显示PSG-1能显著地诱导荷瘤小鼠肿瘤细胞的凋亡。通过ELISA活性试剂盒检测发现PSG-1能显著提高荷瘤小鼠肿瘤细胞中的cAMP、PKA、AC活性，下调DG、cGMP、PKC的活性；Western Blotting法检测PKA和PKC蛋白的表达，结果显示PSG-1显著地促进了PKA蛋白的表达，下调了PKC的蛋白表达量。我们推测，PSG-1可以通过激活cAMP/PKA信号途径，下调DG-PKC信号通路来诱导并促进肿瘤细胞凋亡，从而抑制抗肿瘤的生长。采用流式细胞术观察各组荷瘤小鼠肿瘤细胞的线粒体膜电位变化和ROS的产生，发现PSG-1能有效地促进线粒体膜电位的降低和ROS的产生，Western Blotting检测细胞色素C、凋亡相关蛋白Bax、Bcl-2和p53的表达，结果显示：PSG-1显著地提高了促凋亡蛋白Bax和p53的表达水平，减少了了抑制凋亡蛋白Bcl-2表达量，同时还促进了线粒体中细胞色素C释放到细胞质中。此外，通过检测Caspase-9,-3的活性发现，PSG-1有效地活化了Caspase-9,-3。可见，PSG-1能够通过激活线粒体凋亡通路，实现诱导肿瘤细胞凋亡的作用，从而发挥抗肿瘤活性。综上所述，本研究证实了PSG-1在体内具有很强的抗肿瘤和免疫调节作用，其作用机制可能是通过活化免疫器官，激活免疫细胞及免疫信号通路，来提高机体免疫力；通过激活G蛋白信号转导通路和线粒体凋亡通路，从而促进肿瘤细胞凋亡，最终实现其较强的抗肿瘤作用。更多还原

【Abstract】 Ganoderma atrum polysaccharide (PSG-1) was extract from the mycelia ofGanoderma atrum. Sugar analysis revealed that PSG-1was composed of glucose(Glc), mannose (Man), galactose (Gal) and galacturonic acid (GalA) in molar ratioof4.91:1:1.28:0.71. Numerous studies demonstrated that PSG-1has antioxidant,immunomodulatory, myocardial protection, antidiabetic and anti-aging properties. Alot of researchers have studies the antitumor activity of PSG-1and made remarkableprogress, but the exact mechanism is still unclear. It is, therefore, necessary for themechanism to be studied further.This study was to explore the antitumor activity and mechanism through in vivoand in vitro experiments. According to the modern molecular theory and technology,Flow Cytometry, Western Blot, Enzyme Linked Immunosorbent Assay and ReverseTranscription-Polymerase Chain Reaction were used. The impact of PSG-1on thetumor growth and signaling pathways were investigated from the perspectives ofintegral level, cellular level and molecular gene level, respectively. Specificresearches are as follows:Firstly, PSG-1resistant the S180or CT26tumor cells had been establishedsuccessfully in vitro. MTT method was used to observe the effects of PSG-1on thetumor cells proliferation. The results showed that PSG-1has little effect on theproliferation of tumor cells. It is suggested that PSG-1has no cytotoxicity on thetumor cells directly. However, PSG-1-primed macrophages exhibited a highertumoricidal activity than untreated macrophages in vitro. This finding indicted thatPSG-1could stimulate the peritoneal macrophage and increase the phagocytosisfunction.In vivo, S180or CT26tumor cells were implanted subcutaneously into righthind groin of the mice and the tumor-bearing mice model was established. Aftertumors developed to approximately80-90mm3, the mice were randomized into fivegroups and administrated with PSG-1or5-fluorouracil (5-Fu). On Day15, the micewere sacrificed. Tumors, thymus and spleens were extirpated. The blood samples of mice in each group were centrifuged and the peritoneal macrophages andsplenocytes were collected for the corresponding detections. The results showedPSG-1had significant inhibitory effects on the growth of tumor in mice. The thymusand spleen indexes were significantly increased compared with control group. Thecytokines in the serum and peritoneal macrophages were increased dose-dependently,as well as NO production. Phagocytosis ability of macrophage from the mice treatedwith PSG-1increased significantly. In addition, there was a marked increase in theCon A-or LPS-induced proliferation of splenic cells in PSG-1treated groups. In vivoassay, we believed that PSG-1has the potent antitumor activity, which could notonly inhibit the tumor growth markedly, but also enhance the immunity of the host.Different concentrations of PSG-1could increase the production of TNF-α fromtumor-bearing mouse peritoneal macrophages. Anti-TLR4antibody, specific p38MAPK blocker SB203580or specific NF-κB blocker PDTC was added into thecultures to investigate the signaling pathway of PSG-1. The results showed thataddition of anti-TLR4antibody markedly, although not complete, decreased theproduction of TNF-α stimulated by PSG-1. However, specific p38MAPK inhibitorSB203580and NF-κB blocker PDTC could absolutely abrogate the stimulatoryeffect of PSG-1on the TNF-α production by macrophages from tumor-bearing mice.We also explored Western-blotting to check the expressions of TLR4, NF-κB p65,IκB and pp38related proteins. The results showed PSG-1caused a relative increasein TLR4expression. The levels of p65in nuclear fractions were also significantlyenhanced. But the cytoplasmic protein level of IκBα was decreased. So the IκBαdegradation induced NF-κB activation. Moreover, PSG-1induced the activation ofp38MAPK in a dose-dependent manner. It was suggest that PSG-1treatment mayactivate the phosphorylation of p38in macrophages. In conclusion, these resultssuggest that PSG-1could enhance the immunity, and the antitumor effect of PSG-1may be related to their potentiation of the production of TNF-α in tumor-bearingmice. The signaling mechanism by which PSG-1promotes TNF-α production maybe as follows: PSG-1acts on the TLR4receptors on macrophages, signal throughp38MAPK pathway, and then activate NF-κB. The activated NF-κB in turn initiatesthe release of TNF-α. Flow cytometry methods were used to observe the apoptosis of tumor cells. Theresults demonstrated that PSG-1induced the apoptosis of tumor cells oftumor-bearing mice. Furthermore, PSG-1significantly increased the levels ofintracellular cAMP, PKA and AC in S180cells. In contrast, cyclic GMP (cGMP),DG level and PKC activity were decreased. Similarly, Western blot also gained thesame result. The expression of PKA protein was upregulated, while PKC proteinexpression in PSG-1-treated group was lowered. Hence, we speculated that theantitumor mechanism of PSG-1may be related to the induction of tumor cellapoptosis through cAMP-PKA signaling pathway and down-regulation of DG-PKCsignal pathway.To further investigate the other signaling pathway of PSG-1, the mitochondrialmembrane potential (m) and intracellular ROS production were analyzed by flowcytometry. Experimental results suggested that PSG-1caused a very significantdepletion of mand elevated the level of ROS production. The expressions ofcytochrome c, p53, Bcl-2and Bax were detected by Western blot. The resultsrevealed that Bcl-2expression was down-regulated while Bax and p53expressionlevels were strikingly increased. In addition, level of cytochrome c protein in thecytosolic fraction increased in a dose-dependent manner. In contrast, protein levelcytochrome c decreased in the mitochondrial fraction. These results indicated thatPSG-1led to the release of cytochrome c from the mitochondria to the cytosol.Meanwhile, both caspase-3and caspase-9activities were elevated by administrationof PSG-1in the tumor-bearing mice. These results suggested that PSG-1exhibitsprominent antitumor activity in vivo, at least in part by inducing apoptosis throughthe mitochondrial pathway.In conclusion, this study confirms that PSG-1could suppress the growth oftumor. Multiple pathways are involved in the antitumor activity. One of its functionmechanisms is that PSG-1stimulates the immunocytes and immune organs, and thenactivates the whole body immune system through TLR4-mediated NF-κB andMAPK signaling pathways. Besides, PSG-1also could induce the tumor cellsapoptosis via mitochondria-mediated intrinsic apoptotic pathway or Gprotein-mediated signaling pathways.更多还原