Rearch And Application Of Astragalus Polysaccharide Liposome Immunopotentiators

Astragalus polysaccharide (APS), as one of the traditional Chinese medicine, has the function of improving the effect of immune system, anti-tumor, anti-oxidation, antibiotics and anti-canncer. The liposome has been studied widely in recent years as the carrier of drugs for its advantaged superiority, such as targeting or slow releasing, no immunogenicity, low toxicity, improved bioavailability and so on.In this research, astragalus polysaccharide liposome (APSL) were selected as experimental materials to optimize the preparation conditions of APSL, determined the immunological adjuvant activity and mechanism of immunoenhancement in vitro and in vivo and were applied to inoculate with Newcastle disease and swine fever. The purpose of the research is to combine the physiological function of APS with the immune enhancement of liposome to develop the new immunological adjuvant and improve the immune effect of animal vaccine.Experiment 1. Preparation conditions optimization of astragalus polysaccharide liposome To optimize the preparation conditions of astragalus polysaccharide (APS) liposome, APS liposome was prepared by membrane distribution-micromembrane extruding method. L9 (34) orthogonal experiment was adopted to optimize the preparation conditions which was designed with three factors of lecithin to drug ratio, lecithin to cholesterol radio and ultrasonic time, and two indexes of encapsulation efficiency and drug-loading rate. The encapsulation efficiency of APS liposome was assayed by protamine method, the shape and particle size were observed by transmission electron microscope. The results showed that the optimized preparation conditions were as follows:the ratio of lecithin to drug was 10:1, the ratio of lecithin to cholesterol was 8:1 and ultrasonic time was 20 min. The results dedicated that the APS liposome prepared under the optimized conditions had following advantages:high encapsulation efficiency and drug-loading rate, uniform shape and particle size, and good reproduction quality.Experiment 2. Determination of immune-enhancing activity in vitro of astragalus polysaccharide liposome The proliferation effects of APSLon chicken T and B lymphocytes was determined by MTT method, taking APS and blank liposome as control. The results showed that APSL could significantly promote T lymphocyte proliferation singly or synergistically with PHA at 125-15.625 μg·mL-1, and the efficacy was significantly better than those of APS and blank liposome at 125-31.25μg·mL-1. APSL could significantly promote B lymphocyte proliferation singly or synergistically with LPS at 125-15.625μg·mL-1, and the efficacy was significantly better than those of APS and blank liposome at 31.15μg·mL-1.These results suggested that APS and liposome could synergistically enhance the cellular immunityExperiment 3. Determination of immune-enhancing activity in vivo of astragalus polysaccharide liposomeTwo hundred and ten 14-day-old chickens were randomly divided into 7 groups and vaccinated with Newcastle disease vaccine except for blank control (BC) group, repeated vaccination at the age of 28 days. At the same time of the first vaccination, the chickens in five experimental groups were intramuscularly injected respectively with three dosed of APSL, APS and BL, in vaccine control (VC) and BC groups, physiological saline. On days 7,14,21 and 28 after the first vaccination, the blood was sampled for determination of lymphocyte proliferation (same to 2.4.1.) and serum hemagglutination inhibition (HI) antibody titer. On days 14,21 and 28 after the first vaccination,6 chickens were sampled randomly from each group for determining the concentrations of interferon-_ (IFN-_), interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-10 (IL-10) in serum by Enzyme-linked Immunosorbent Assay (ELISA).The results that APS and liposome could synergistically enhance the humoral immunity, cellular immunity and cytokine secrety, and the medium dose was the best.Experiment 4. The effect of astragalus polysaccharide liposome on mRNA expression of cytokines in chickens According to Primer Premier Software, five pairs of RT-PCR primers of IFN-γ、IL-2、IL-4、IL-10 and β-action were designed. The chicken peripheral lymphocytes were cultivated in adding APSL at three concentrations. After cultivation of 36 h, the cells were collected and total RNA was extracted. The expression of cytokine mRNA was determined by fluorescence quantitative RT-PCR assay. The results showed that APSL could improve the transcription level of IFN-γ、IL-2、IL-4、and IL-10mRNA, and were better than APS and BL at 125、62.5 and 31.25μg·mL-1. It might be one of the immune enhancement mechanisms that APSL could promote the animal immunity.Experiment 5. Application of astragalus polysaccharide liposome in ND immunization Four hundred 14-day-old chickens from random two fowl farms were randomly divided into 8 groups,2 parallelisms,50 per group, and vaccinated with ND vaccine except for blank control (BC) group, while repeated vaccination at 28-day-old. At the same time of the first vaccination,2 polysaccharides groups were intramuscularly injected respectively with 2 mg·mL-1 APSL and APS 0.5 ml, control groups in non-adjuvant control (NA) and blank control (BC) groups, physiological saline, respectively, once a day within three successive days. On 7th,14th,21st,28th day after the first vaccination, the change of serum HI antibody titer was determined. The results showed on 7th,14th,21st,28th day after the first vaccination, antibody titer APSL group was the highest which was higher than those groups significantly. These results indicated that APSL could significantly improve the immune response of ND vaccineExperiment 6. Application of astragalus polysaccharide liposome in swine plague immunization Four hundred 30-day-old graces from random 2 piggeries were randomly divided into 8 groups,2 parallelisms,50 per group, and vaccinated with swine fever vaccine except for blank control (BC) group. At the same time 2 polysaccharides groups were intramuscularly injected respectively with 2 mg·mL-1 APSL and APS 2 ml, control groups in non-adjuvant control (NA) and blank control (BC) groups, physiological saline respectively. On 10th,20th,30th day after vaccination, the change of serum HI antibody titer was determined. The results showed on 10th,20th,30th day after vaccination, antibody titer APSL group was the highest which was higher than those groups significantly. These results indicated that APSL could significantly improve the immune response of swine fever vaccine...