Study On Preparation Of Polysaccharide Of Cistanche Deserticola Liposome And Its Effect On Chicken After Immunization With ND La Sota Vaccine

Polysaccharide of cistanche deserticola(CDPS),extracted from cistanche deserticola,is a kind of active substance with high molecular.The CDPS can enhance the phagocytosis ability of macrophage,increase the value of erythrocytolysin in serum;at the same time,it can facilitate the proliferation of thymic lymphocyte,secretion of IL-2 of lymphocyte from spleen in mice alone and with ConA,PHA together,showed an extensive immune regulating function.If taking CDPS as a immunoenhancer applied to animal directly,there will be some deficients,includ quick metabolism, short action time and high cost.As a type of synthetic targeting vector,slow-release and targeting to endothelial system(RES)are significant characteristics of liposome,it can prolong action time of drugs,and enhance bioavailable efficiency and the intension which drugs orient effect on immune systcm.Based on all above mentioned,the purpose of this study is to prepare the CDPS liposome,as a new type of immunoenhancer,the parameter for preparation of CDPS liposome was selected with homogeneous design.Then the effect of CDPS liposome on chickens after immunization with ND Lasota vaccine was exarned.The function of cellular immunity,humoral immunity and the activity exchange of IL-2,IL-4,IFN-γhave been studied,in order to stduy the possible mechanisms of how CDPS liposome enhance immunologic function.The main contents arc as follows:1.Comparation of three methods to extract polysaccharide from cistanche deserticolaPolysaccharide of cistanche deserticola(CDPS)was extracted with three methods,including hot water,supersound,hot water-supersound and precipitation by alcohol.Protein in the precipitates was removed by Sevag-trypsin.CDPS was identified by molish reaction,iodine-potassium iodide reaction,colorimetry with Fehling's reagent.Polysaccharide in extractive was determined by the anthrone-sulphuric acid colorimetry.Content of pick-up rate was 1.93%,2.28%,6.06%respectively, and the content of polysaccharides in extractive was 65.7%,74.3%,81.2%respectively.The results indicated that hot water-supersound was the most adequate method to extract polysaccharide in cistanche deserticola.2.Study on optimum parameter of preparation of CDPS liposme with homogeneous designThe CDPS liposme was prepared by the reverse-phase evaporation method.Entrapment efficiency,as main index of quantitive evaluation of liposome,was determined by dialysis and anthrone-sulphuric acid colorimetry.Based on principle of homogeneous design and table U^*₆(6⁴), the program of experiment was designed,Four factors,including the ratio of soybean lecithin and chloroform,cholesterol and soybean lecithin,CDPS and soybean lecithin,PH of buffer phosphate on entrapment efficiency was investigated.The results showed the entrapment efficiency was 68.13% and RSD was 2.51%when the four factors were 7%,1:5,1:14,6.6 respectively.In these conditions the PH of CDPS liposome was stable on day 120^thand the entrapment efficiency was 50.15%when preserved at 4℃away from light although the stratification was observed.3.Study on effect of CDPS liposome on La sota vaccine immunized chickenSixty chickens with similar health condition were regarded as the experimental animals,which were randomly divided into 4 groups,with 15 chickens in each group at 20 day-old.Experimental chickens were injected with ND Lasota vaccine when the antibody titer of HI was under 31og2.Thenâ… group were injected with CDPS liposome(concentration:5mg/ml,dose:500μl for one bird),â…¡group were administered with CDPS(concentration:50mg/ml,dose:250μl for one bird),â…¢group were administered with liposome(500μl for one bird),â…£group were injected with saline(dose: 500μl for one bird).Levels of IgG(indirect ELISA),HI titer and serum neutralization test were detected at different times The result of HI suggested that the antibody titer of all groups had risen obviously on the 10^thday after immunization,compared with preimmune.On the 20^thday after immuned,compared with control group,the antibody titer ofâ… group had risen obviously(P＜0.01), it was also slightly higher thanâ…¡,â…¢group.Antibody titer of four groups descented continuously on 30^thday,but the antibody titer ofâ… group was higher thanâ…¡,â…£group(P＜0.05),at the same day,there were no significant deviation betweenâ… group andâ…¢group.The result of indirect ELISA suggested that IgG levels ofâ… group was slightly higher than other groups on 10^thday after immunization,,it was significantly higher than control group on 20^th,30^thday(P＜0.01),it was also significantly higher thanâ…¡,â…¢group on 30^thday,(P＜0.01).IgG levels ofâ… group had risen continuously on 30^thdays,IgG levels of other groups had risen from 10^thday to 20^thday,but had decreased from 20^thday to 30^thday.The result of Serum neutralization test suggested that mortality of chick embryo inâ… group was lower thanâ…¡,â…¢,â…£group.Neutralization index ofâ… group kept the highest in all groups.These results indicated that CDPS liposome,as a new type of immunoenhancer,could boost effective immune response after immunization with ND Lasota vaccine.4.Study on effect of CDPS liposome on activity of T lymphocyte,IL,IFN of La sota vaccine immunized chickenAfter injected ND La sota vaccine,6 chickens,which were randomly obtained from each group were bled and sera from them were detected by MTT assay and acidα-naphthyl acetate esterase test respectly for diterminatoin of lymphocyte transformationt ratio and ANAE⁺ ratio on 15^thday after immunization.On 20^thday,8 immunized chickens were bled and sera from them were tested by ELISA for the determination of the levels of IL-2,IL-4,IFN-γ.These results suggested that lymphocyte proliferation of four groups were no significantly deviation,and ANAE⁺ ratio of lymphocyte of groupâ… ,groupâ…¡,groupâ…¢were no significantly deviation,and ANAE⁺ ratio ofâ…£ lymphocyte of groupsâ… andâ…¡were higher than the control group(P＜0.05),the IL-2 level of groupsâ… andâ…¢had risen obviously,compared withâ…¡andâ…£groups;the IL-4 level of CDPS liposome group kept the highest in all groups,the content of IFN-γwas no significantly deviation amongest groupsâ… ,â…¡andâ…¢;but the IFN-γlevel of CDPS liposome group was higher than the control group,these results indicated there were correlation between effect of CDPS liposome on effective immune response of chicken and activity of T lymphocyte,IL-2,IL-4,IFN-γ.