| Liposome is an artificial phospholipids vesicle with double membranea. Liposome is a kind of the immunoadjuvants, mainly improving antigens transportion and submission. And main advantagement of liposome is that liposome membrane possesses a compatibility with the cell membrane, and is easy to input drugs and antigen ingredients on or internal with liposome into the cells. In addition, the liposome can slow release drug, reduce toxicity and side effect of drug and so on. Many researches found that lots of the traditional Chinese herbal medicine or traditional Chinese herbal medicine ingridients are not solubility in water or quick metabolism in vivo so as to not sustain longer drug action. That drugs are encapsulated with liposome, not only can solve the above problems, but also can play the advantagements of drugs and liposome. Base on all above mentioned, this study is to prepare the EPSL by membrane distribution-supersound method and the optimal prescription for preparation of EPSL was selected with orthogonal test design; and the immunological adjuvant activity of EPSL was researched including the activity of peripheral T lymphocyte proliferation, antibody titers and secretry and mRNA of cytokines. The aim of this study is to develop high-effect and low-toxicity newtype of immunological adjuvant. The details were divided into five parts as follows:Experiment1Optimization on preparation conditions of Epimedium polysaccharide liposome To optimize the preparation conditions of EPSL. EPSL was prepared by membrane distribution-supersound method. In preparation of EPSL test, an orthogonal L9(34) test was used for optimizing the preparation conditions of EPSL, with four factors of ratio of drug to lipid, ratio of soybean phospholipid to cholesterol, ultrasonic time and water bath temperature and two indexes of encapsulation efficiency and drug-loading rate. The encapsulation efficiency of EPSL was determined by protamine method. The results showed that the optimal preparation condition of EPSL was that ratio of drug to lipid, ratio of soybean phospholipid to cholesterol, ultrasonic time, and water bath temperature were1:30, 4:1,40℃and10min respectively. The optimal formulation and preparation had prepared EPSL with the property of high encapsulation efficiency and drug-loading rate.Experiment2Effect of EPSL on lymphocyte proliferation in chickens in vitro The effects of EPSL and EPS on growth and proliferation of the peripheral lymphocyte were compared by MTT method. EPSL and EPS were diluted to eleven concentrations with RPMI1640. The peripheral lymphocyte in96-well plates were exposed to drugs at series of concentrations, four wells each concentration, to be observed the results after48h. In order to study EPSL and EPS on their effect of the peripheral lymphocyte proliferation, at five concentrations were added into the peripheral lymphocyte in vitro, and cultured48h. The peripheral lymphocyte proliferation was determined with MTT method. These results showed that their maximal safety concentrations falled down and all of EPSL and EPS could promote the lymphocyte proliferation, at62.5-3.906μg·mL-1, EPSL could stimulate the lymphocyte proliferation singly or synergistically with PHA both could promote the lymphocyte proliferation, at the same time, at31.25μg·mL-1and3.906μg·mL-1, EPSL singly or synergistically with PHA could stimulate the lymphocyte proliferation which had better effect than EPS.Experiment3Effects of EPSL on mRNA Expression of cytokine in chickens in vitro In order to study the action mechanism of EPSL on immunological adjuvant activity, the effect of EPSL on expression of IL-2, IL-4and IFN-y mRNA was determined taking EPS as control. The chicken peripheral lymphocyte were cultivated in adding EPSL at three concentrations. After cultivation of36h, the cells were collected and total RNA was extracted. The expression of IL-2, IL-4and IFN-y mRNA was determined by real-time fluorescent quantitation PCR assay. The results showed that EPSL could improve the expression level of IL-2, IL-4and IFN-y mRNA, and were significantly better than EPS at62.5μg·mL-1and31.25μg·mL-1. At62.5μg·mL-1, the action of EPSL was the strongest, at31.25μg·mL-1, the action of EPSL was stronger and in a dose-dependantmanner. It might be one of the immune enhancement mechanisms that EPSL could promote the secretion of IL-2, IL-4and IFN-γ.Experiment4Effect of EPSL on immune response of ND vaccine in chickens In order to compare the immune-enhancing actions of EPSL and EPS in vivo, the effects of EPSL and EPS on immune response of ND vaccine in chickens were determined. Two hundred and ten14-day-old chickens were divided randomly into7groups and vaccinated with New castle disease vaccine except for non-vaccinated (NV) group, repeated vaccination at28-day-old. At the same time of the first vaccination, the chickens in three experimental groups were intramuscularly injected respectively with EPSL, at high, medium and low dose, taking EPS as control, in vaccine control groups and blank control groups, physiological saline. On days7,14,21and28after the first vaccination, the blood samples were collected from cardiopuncture for determination of peripheral T lymphocyte proliferation in vivo by MTT method, brachial vein for determination of serum HI antibody by β-micro-method. The results showed that, EPSL at suitable dose and some time points significantly promoted T lymphocyte proliferation and raised antibody titer. These results indicated that EPSL could significantly improve the adjuvanticity and drug action of EPS, and its high dose possessed the best efficacy.Experiment5Effects of EPSL on cytokine levels of serum in chickens In order to further study the immunological adjuvant activity of EPSL. Two hundred and ten14-day-old chickens were randomly divided into7groups and vaccinated with New castle disease vaccine except for non-vaccinated (NV) group, repeated vaccination at28-day-old. At the same time of the first vaccination, the chickens in three experimental groups were intramuscularly injected respectively with EPSL, at high, medium and low dose, taking EPS as control, in vaccine control groups and blank control groups, physiological saline. On days14,21and28after the first vaccination, the blood samples were collected from brachial vein for determination of the concentrations of IL-2,IL-4and IFN-γ in serum by double antibody sandwich method. The results showed that, EPSL at suitable dose and some time points could significantly increase concentrations of cytokine, and EPSLH possessed the best efficacy. |
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