| Ganoderma lucidum polysaccharide (GLP) is a biological active polysaccharide isolated from the Chinese herbal medicine of Ganoderma. GLP is one of the most effective ingredients in Ganoderma and has attracted a great deal of attention in the biomedical area at home and abroad. Numerous studies reported that GLP could markly promote lymphocyte proliferation and improve the expression of cytokines of humans and mices so as to enhance the immunity in vivo and in vitra. This reminded us of the value of GLP as a new-type immune adjuvant in the domestic animal and poultry industry. Therefor, we studied the adjuvanttivity of GLP on chicken peripheral lymphocytes proliferation and on the expression of IL-6 gene and IFN-γ gene in vitro, and researched the adjuvanticity of GLP for Newcastle disease vaccine in vivo to offer theoretical evidence for exploiting new type immunopotentiators.1. The acute toxicity study of GLPObjective:To evaluate the acute oral toxicity of GLP on rat. Methods:Pre-experiment, 12 healthy SD rats were randomly divided into three groups, male and female were equal. Respectively, the rats were orally administrated with 500 mg·ml-1,100 mg·ml-1,20 mg·ml-1 of GLP by 1 ml/100 g, one times a day, and observed the mental state and mortality of rats for 7 days. Formal experiment, according to the results of Pre-experimen, ten rats(male and female were equal)were orally administrated with 500 mg·ml-1 of GLP by 1 ml/100 g, one times a day. Then we observed the mental state and mortality of rats. Results:Pre-test and repeated test showed no rats died. A female rat appeared short diarrhea in group of 500 mg·ml-1 and turned for the better soon. There were no damage to the heart, liver, spleen, lung, kidney after dissecting. The oral administration had reached 5000 mg·ml-1. Conclusion:According to the classification table of chemical acute toxicity, GLP was classified as a practically non toxic substance, so GLP application security.2. The effects of GLP on chicken peripheral lymphocytes proliferation in vitroObjective:To evaluate the effects of GLP on chicken peripheral lymphocytes proliferation in vitro through MTT assay. Methods:The maximal safe concentrations was detected. According the maximal safe concentrations, and absorptive capacity of the body, GLP was dissolved into 7 concentrations from 35.156μg·mL-1 to 2250 μg·mL-1. Blood samples were collected from heart of two-month-old chickens for the preparation of lymphocytes. Then seven concentrations of GLP without ConA or with ConA (the final concentration reaching to 10 μg·mL-1) were added into lymphocytes for 48 h. Briefly,20 μL of MTT (5 mg·mL-1) was added into each well at 4 h before the end of incubation. The absorbance at 490 nm (A490 value) of each well was measured by microliter enzyme-linked immunosorbent assay reader and background was subtracted at 630 nm. Results:The A490 values of GLP at 1125～70.3125 μg·mL-1 groups were significantly larger than that of cell control group (p <0.05), the A490 values of GLP at 35.156～17.578μg·mL-1 groups were no significant larger than that of cell control group (p>0.05). Conclusion:GLP could significantly enhance lymphocytes proliferation singly or synergistically with ConA.3 The effects of GLP on IL-6 mRNA and IFN-γmKNA of chicken peripheral lymphocytes in vitroObjective:To evaluate the effects of GLP on IL-6 mRNA and IFN-y mRNA of chicken peripheral lymphocytes in vitro. Methods:The preparation of lymphocytes as above, the resulting pellet was re-suspended to 1×107 mL-1 with RPMI-1640 media. Then the concentrations of GLP at 450,225,112.5μg·mL-1 and at 450,112.5,28.125 μg·mL-1 with ConA (the final concentration reaching to 10 ug-mL"1) were added into. After 12 h of incubation at 37℃,5%CO2 incubator, the lymphocyte was collected to for RNA isolation for RT-PCR. Results:The IFN-y mRNA level of GLP at 112.5μg·mL-1 dose group was significantly higher than that of ConA group (p< 0.05). The IL-6 mRNA level of GLP at 450 and 225μg·mL-1 dose group was significantly higher than that of ConA group (p< 0.05). Conclusion:GLP could promote IFN-y mRNA and IL-6 levels in peripheral lymphocytes.4. The adjuvanticity of GLP for Newcastle disease vaccine in vivoObjective:To evaluate the the adjuvanticity of GLP for Newcastle disease vaccine in vivo. Methods:17514-day-old Roman chickens were assigned to 7 groups randomly. The chickens except blank control (BC) group were vaccinated with Newcastle disease vaccine, repeated vaccination at 28 days old. At the same time of the first vaccination, the chickens in experimental groups were orally administrated respectively with the GLP at 100 mg·kg-1, 50 mg·kg-1,25 mg·kg-1,12.5 mg·kg-1,6.125 mg·kg-1 5 concentrations, in vaccination control (VC) and BC group with physiological saline, once a day for three successive days. On days 7,14,21 and 28 after the first vaccination, the peripheral lymphocytes proliferation and serum ND antibody titer and Immune organ index were determined. Results:Throughout the test period, the chickens in each group had no adverse reactions and no morbidity and mortality. The peripheral lymphocytes proliferation and antibody titers at 25 mg·kg-1 groups of GLP were significantly larger than that of VC group and BC group (p<0.05) at a plurality of time points and GLP could promote the development of the spleen and bursa numerically at all of the doses. Conclusion:GLP was safe on chickens and could enhance the immune effect of Newcastle disease vaccine. |
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