Study On Immunological Regulation Effect And Injection Of Polysaccharide Fromtermitomyces Albuminosus (Berk.) Heim

Termitomyces albuminosus (Berk.) Heim. is a kind of famous domestic fungus in Liangshan province in Sichuan. It is liked by many people for its abundance nutritional ingredient and delicious. The extraction,purification and biologic activity of Termitomyces albuminosus polysaccharide(TAP) have been reported a little at home and abroad, and there are no reports on animal health care products or immunologic adjuvant of TAP. In this dissertation,immunological regulation effect and mechanism in vitro or vivo of TAP were researched,and injection of TAP was prepared, it would provide evidence for clinical application as a immunologic adjuvant. The details are divided into six parts as follows:1.To isolate and purify TAP with an economical,safe and efficient process,we chosed the best hot water diffusion technology through single factor test on extraction temperature, dosage liquor ratio,extraction time and frequency.The protein in TAP was removed by freeze thawing and Sevage method,pigment was removed by H2O2and hydrone was removed by dialysis.TAP was identified as polysaccharide through α-naphthol ethanol method and methanol method.The saccharinity of TAP was analyzed through sulphuric acid-phenol method. The results showed that the best extracted technology was in80℃, dosage liquor ratio was1:20,extracted1h and twice.Extracted time was decreased but yield of TAP was increase by hot water extraction combined with20kHz ultrasonic wave. Purified TAP was beige lip lipstick polysaccharide,its yield was6.69%and saccharinity of TAP was92.17%.2. The influence of TAP on T cellular immune function and immune enhancement mechanism in mouse was observed.The proliferation effect on splenic lymphocyte of mouse was observed through MTT method.The cell factors as IL-4、IL-2and IFN-γ were detected by ELISA;T lymphocyte subgroups were analyzed by flow cytometry (FCM).The expression of above cytokines was detected by RT-PCR.The results showed that,splenic lymphocyte of mouse was activated and proliferated with only TAP,TAP cooperated with ConA or LPS in vitro.Compared with ConA group,25～100μg/mL TAP could significantly promote1L-4、IL-2and IFN-γ secretion of splenocyte in72h (P<0.01),and after treated with25μg/mL TAP in48h,CD4+%(P<0.01) of splenic cell and CD4+/CD8+(P<0.05) were all depressed,but CD8+%(P<0.05)was raised,CD3+%was raised by50μg/mL TAP (P<0.05).IL-4、IL-2and IFN-y mRNA expression were significantly raised by50μg/mL TAP(P<0.01),and they were kept with higher expression level than those of ConA group in72h.TAP could notablely enhance cellular immune function,T lymphocyte was the target cell of TAP.3. This experiment was conducted to study immunoregulatory effect of TAP and approach to its mechanism in normal and immunosuppressive mouse caused by Cyclophosphamide(CTX).Astragalus polysaccharides(APS)was used as positive control drug.The immunity indices such as macrophage phagocytosis,immune organ indices, serum hemolysin,T lymphocyte subgroup,cell factor,lymphocyte proliferation and immune organ pathological changes were tested.The results showed that20mg/kg TAP could increased hemolysin content (P<0.01),macrophage phagocytosis (P<0.01) and immune organ indices (P<0.01or P<0.05) in nomal and immunosuppressive mouse.Compared with model group,CD4+/CD8+was notably depressed by20mg/kg TAP (P<0.01),the content of IL-2、IFN-γ were respectively increased312.3%(P<0.01) and88.38%(P<0.01),IL-4was apparently degraded (P<0.01) by20mg/kg TAP. Lymphocyte proliferation in vitro was notablely enhanced and immune organ pathological changes were abated by20mg/kg TAP (P<0.01).Compared with APS,TAP had the similar or better immunoregulatory effect. The research provided that the immunosuppression caused by CTX could be antagonized by TAP.The humoral and cellular immunity function were enhanced,and unstuck immune function was corrected by TAP in immunosuppressive mouse.4. The preparative method,quality control and stability of TAP injection were investigated in the test.TAP injection was prepared through filtration sterilization method in sterile environment.The quality requirements of injection,such as content of tannin,PH, bacteria,pyrogen and microparticle of TAP injection were detected.The content of polysaccharide was detected by sulphuric acid phenol method,and monosaccharide was detected by Fehling’s reagent method. The influence of highlight and hyperpyrexia to TAP injection were observed,and the expiration date of TAP injection was estimated through classics constant temperature method.The results showed the detected quality indexes were consistent with requirements of injection,content of polysaccharide was99.2～99.9%, monosaccharide was not discovered in TAP injection,TAP could be degraded by highlight or hyperpyrexia.The stability of TAP injection was better when it was stored away from light in25℃and its expiration date was4.3y.5. The safety of TAP injection was detected through acute and chronicity toxicity test,local stimulation test,sensitivity test and hemolysis test. Results showed,the LD50of TAP injection in mouse couldn’t be detected within162.5～6000mg/kg injected dose.In chronicity toxicity test, paradoxical reaction on physiology or behavior and immune organ pathological changes of mouse didn’t be observed within25～100mg/kg injected dose.The TAP injection couldn’t cause eye conjunctiva and muscle inflammation,sensitivity or hemolysis reaction.TAP injection could be subcutaneously or intramuscularly injected without side effect.6. The influence of TAP injection to immunity effect of vaccine was observed in the test.Astragalus polysaccharides (APS) was used as positive control drug.The Foot and Mouth disease (FMD) antibody in piglets were detected by indirect hemagglutination test,and the content of leucocyte (PBL,neutrophil,monocyte and lymphocyte in peripheral blood were detected by animal cytoanalyze.New castle disease (ND) antibody in chicken were detected by hemagglutination inhibition test,and the immune organ indexes were detected.Results showed the antibody of FMD and above blood physiological indexes in piglets were increased during58d after first immuneby high dose TAP injection(P<0.07),its effect gradually weakened after that time.The antibody of ND and immune organ indexes in chicken were increased by middle dose TAP injection (P<0.01).The immune enhancement effect of middle or high dose TAP injection was better than it of APS injection with the same dose.Above all,TAP was a favourable immunoregulator and had an antagonistic effect to the damage induced by CTX.TAP injection could be subcutaneously or intramuscularly injected with common vaccine,it was safe and effective as a new immunological adjuvant.