【作者】 刘静；

【导师】 叶林柏；

【作者基本信息】 武汉大学 ， 微生物学， 2005， 博士

【摘要】 从发酵得到的灵芝菌丝体中分离得到一种多糖成分GLP,并对它的细胞毒作用、抗病毒活性及抗肿瘤作用进行了研究。采用热水抽提和乙醇沉淀的方法从灵芝菌丝体中得到灵芝多糖的粗制品,再用离子交换和分子筛的方法从多糖中分离得到5个不同的峰的灵芝多糖,经含量测定证明第二个峰所含有的灵芝多糖占总的多糖含量的42%,分子量测定表明该峰的多糖的分子量约为30～50Kda,硫酸—酚试验和Lowry-Folin试验分别检测多糖和蛋白质的含量分别为86.4%和8.3%,多糖与蛋白质的比例为10.4:1,命名该峰的多糖为GLP。 采用台盼兰和MTT法检测了GLP对体外培养的非洲绿猴肾细胞Vero细胞的毒性作用。结果表明,GLP对Vero细胞无毒,即使GLP浓度高达2000μg/mL,GLP对细胞也没有任何毒副影响,细胞的活力仍可达到98%以上。 GLP能够抑制单纯疱疹病毒Ⅰ型（herpes simplex virus type 1,HSV-1）和单纯疱疹病毒Ⅱ型（herpes simplex virus type 2,HSV-2）感染Vero细胞并抑制病毒感染引起的细胞病变。CCID₅₀试验表明,GLP能强烈地抑制单纯疱疹病毒Ⅰ型和单纯疱疹病毒Ⅱ型感染细胞。在病毒感染细胞前将GLP和疱疹病毒混合孵育1小时,然后再感染Vero细胞,GLP抑制单纯疱疹病毒Ⅰ型和单纯疱疹病毒Ⅱ型的EC₅₀(50%effective concentration,EC₅₀)值分别为15.37和16.75μg/mL;如果GLP不预先与病毒孵育而和病毒同时加入到Vero细胞中,这时GLP抑制单纯疱疹病毒Ⅰ型和单纯疱疹病毒Ⅱ型的EC₅₀值分别为17.22和18.91μg/mL;但是在单纯疱疹病毒Ⅰ型和单纯疱疹病毒Ⅱ型感染Vero细胞2小时后再加入GLP,这时GLP抑制单纯疱疹更多还原

【Abstract】 A bioactive fraction （GLP） was extracted from the mycelia of Ganoderma lucidum by hot-water extraction and EtOH precipitation. Using ion exchange and molecular filter methods, we got five different kinds of polysaccharides with different peak values. The second peak was assayed to have 42% of the compound, and its molecular weight is about 30⁵0kDa. The amount of polysaccharide and protein is 84.6% and 8.3%, respectively, determined by suffacate-phenol assay and Lowry-Folin assay. The ratio of polysaccharide and protein is 10.4:1. The peak 2 is named GLP.Trypan blue exclusion method and MTT redution assay verified that GLP had no cytotoxic effects on Vero cells even when the concentration was as high as high as 2000μg/mL. The total cell proliferation number was more than 98% compared with the control group cells.GLP can effectively inhibit the infection of herpes simplex virus type 1 （HSV-1） and type 2 （HSV-2） and prohibit the infected cells from infecting other cells. In order to study the possible mode of action of the antiviral activity of GLP, cells were treated with GLP for 1h before infection, the EC₅0 （50% effective concentration, EC₅0）towards HSV-1 and HSV-2 were 15.37 and 16.75μg/mL, respectively. However, when the cells, not treated with GLP before infection, were added with virus simultaneously, the EC50 towards -HSV-1 and HSV-2 changed to 17.22 and 18.91μg/mL, respectively. Further more, whenthe GLP was added after the cells had been incubated with virus for 2h, the EC50 changed to 52.84 and 61.04, respectively. Although the precise mechanism has not yet to be defined, our work suggested that GLP inhibits viral replication by interfering with the early events of viral adsorption and entry into target cells. Thus, this polysaccharide seems to be a potential candidate for anti-HSV agents.Plaque reduction assay further confirmed that the GLP blocked the infection of HSV-1 and HSV-2. In our study, the number of plaque indeed reduced to 50% when the concentration of GLP was 4.6, 5.4, 25 and 8.4 ug/mL when GLP added before, simultaneously, and after treated with GLP. While the concentration of GLP was up to 45, 72, >100 and 88 ug/mL the number of plaque would reduce to 10% only compared with control when GLP added in different time. When GLP was added after the viruses have infected cells, and remove it after viruses have finished synthesizing the biomacromolecules inside the cells（the whole course may last 16¹8h）, the number of their plaques changed little in comparison with control. The results suggest that GLP has no effect on the biomacromolecules synthesizing, assembling and releasing of virus in cells.Quantitative Real-Time PCR also gained the same results. We found that when the suspension contained 8.0x 105 virons was incubated with GLP for lh before infecting cells, the total viron proliferation number was 7.4x105, and decreased 7% compared with control and detected by Quantitative Real-Time PCR. In other words, 93% of virons are still in the supernatant. If the GLP and virus were added into cells simultaneously, the virus proliferation numbers in supernatant only decreased 10%, and 90% （ 7.2 xlO5） of virus are still in it. However, if the virus infected cells without pretreated with GLP, the number of virons left in the supernatant was only 1.7x 105, about 79% of virons had bound to cells. These results suggested that GLP can hold back the interaction between virus and cells. We can also infer that GLP can interfere with the early events of viral adsorption and entry into target cells, and prevent cells from infecting cells.We use chick embryo to detect the antivirus activity of GLP to influenza virus. GLP did not show any effect on the growth of chick embryo even when its concentration was up to 2000 n g/mL. When the chick embryo inoculated with influenza virus （effective dose was 320- hemagglutination units） and incubated for 72h, can grow normally when the GLP with the concentration of 200 P g/mL were added , the result proved that GLPcan inhibit the proliferation of influenza virus in chick embryo. HI test suggested that GLP could heavily inhibit the Hemagglutination caused by virus infection. In this test, the lowest effective concentration of GLP is 78.13ng/mL. The antiviral activity of GLP is due to its combination with the virus envelope protein.We have also investigated the mechanism of the antiviral activity of GLP extracted from the mycelia of Ganoderma lucidum. MTT test suggested that the proliferation and viability of S-180 cells were not affected by GLP even up to the concentration of 5000ug /ml. The total cell proliferation number was more than 95% compared with the control group cells. The trypan blue exclusion method showed that approximately 97% of cells were still active in the same condition. These results suggested that GLP can not inhibit the growth of mouse S-l 80 cells in vitro.Further more, the inhibitory effects of GLP on the growth of tumor cells were investigated in tumor-bearing mice. After 20 days orally addmistration of GLP, the mice were transplanted intraperitoneally through right axilla with suspension contained about lx 106 mouse S-l80 cells. Seven days later, the mice were orally treated with different concentrations of GLP （lOOmg/kg, 200mg/kg and 400g/kg per day）, and the tumor-inhibited rate was up to 53%, 59% and 58%, respectively. These results indicated that GLP can inhibit the growth of sarcoma S-l 80 in vivo not in dose- independent way.In our study, we also found that GLP can stimulate the enhancement of cytokines such as IL-2 and TNF-a in sera of peripheral blood cells. The result suggested that the IL-2 and TNF-a in sera of peripheral blood cells increased from 1.27ng/mL to 2.88ng/mL, and from 1.05ng/mL to 1.82ng/mL respectively. Whereas the concentration of IL-6 did not change obviously. The data from flowcytometry examination showed that the number of CD/ or CDg+ T cells in peripheral blood from mice treated with GLP was stimulated to a higher level in comparison with the control. No significant change happens to the values of CD4+, which only enhanced 3.47% from 54.80% to 58.27%, while the values of CD8+ were enhanced 21.21% from 24.15% to 45.36% （P<0.05）.These results suggested that GLP inhibited tumor growth via enhancing the cellular immune responses instead of killing the tumor cells directly.更多还原