Experimental Study Of The Immunological Activity Of Astragalus Polysaccharide Regulation Of Bcg

Objective:To identify the regulatory effect on APS on the immunocompetence of BCG vaccine.METHODS:1.grouping According to the weight of KM mice,animals were randomized into four groups:the normal control group,the BCG group,the BCG+APS group and the APS(preimmunization)+BCG group.In the same day,Mice from the APS(preimmunization) +BCG group were immunized with APS s.c.,injection was performed once daily for 3 days on both sides of the subiliac area of the mouse in a volume of 50μl for each side.2.vaccination On the next day of preimmunization completion,vaccination was performed as follows:mice from the normal control group were immunized with PBS-TW80(0.05%) s.c on both sides of the subiliac area of the mouse in a volume of 50μl for each side;mice from the BCG group were immunized with BCG s.c on both sides of the subiliac area of the mouse in a volume of 50μl for each side;mice from the BCG+APS group were immunized with mixed liquor(BCG+APS) s.c on both sides of the subiliac area of the mouse in a volume of 100μl for each side;mice from the APS(preimmunization)+BCG group were immunized with BCG s.c on both sides of the subiliac area of the mouse in a volume of 50μl for each side.3.Cell culture Spleen cells were harvested from 2 animals(3 weeks and 5 weeks postvaccination) prior to infectious challenge.Cells were cultured in the presence of heat-killed BCG for 48 h at 37℃.Antigen-specific production of IFN-γ,was measured in culture supernatants by specific enzyme linked immunosorbent assay(ELISA) methods.4.Pulmonary BCG challenge and the correlated experiments To establish a model of pulmonary mycobacterial infection,anesthetized mice were challenged intratracheally with BCG at 5 weeks postvaccination.At 1 week postchallenge,the following experiments were done:(1) Eyeballs of anesthetized mice were picked to collect blood serum and whole blood.(2) All BAL samples were collected and spun in a microcentrifuge at 4,000 rpm for 1 min at 4℃.After that,supematants were removed and stored at -84℃for cytokine analysis.Cell pellets were resuspended in 500μl of PBS,and total cell counts were determined.Then,the film preparation of cells were made and stained with Wright's stain for differential cell.(3) Lungs were fixed in 10%formalin,tissues were then embedded in paraffin,cut into 4- to 5-μm-thick sections,and stained with hematoxylin and eosin.Other tissue sections were subjected to Ziehl-Neelsen staining,which is specific for mycobacteria.(4) the subgroups of T cell in peripheral blood were measured by flow cytometer(FCM).RESULTS:1.Spleen Cell culture and the measurement of IFN-γAt 3 weeks postvaccination, the WN-γlevel of the BCG+APS group was higher than that of mice from other groups(P＜0.05),but the IFN-γ,of the BCG+APS group decreased to the level of the normal control group(P＜0.05) at 5 weeks postvaccination.2.The numbers of total leukocytes and differential immune cell subsets in BALF At 1 week postchallenge,there was a greater number of lymphocytes in APS(preimmunization)+BCG group than that of mice in BCG+APS group(P＜0.05); however,the numbers of macrophages in normal control group was the highest among all the four groups(P＜0.05).3.the measurement of T cell subgroups in peripheral blood At 1 week postchallenge,the percentage of CD4~+ T of the BCG+APS group was higher than that of mice in APS(preimmunization)+BCG group(P＜0.05).4.the concentration of IFN-γin peripheral blood and BALF There was no statistical significance among all the four groups in IFN-γ,of BALF,but the IFN-γ,of the BCG group was higher than that of mice in APS(preimmunization)+BCG group(P＜0.05) in peripheral blood.5.Pathology change of the lung issue According to the size,quantity and range of the granulomas,the extent of the granulomas of lung issue was valued:the BCG group and the BCG+APS group were the most significant,the APS(preimmunization)+BCG group was the second;the normal control group was the slightest.6.the BCG residue of the lung issue The BCG residue was not found in the lung issue of APS(preimmunization)+BCG group,and the other three groups had different levels of BCG residue.Conclusion:1.APS as vaccine adjuvant can enhance the immune activity of BCG.2.The research indicates that the preimmunization s.c.with APS to the vaccination spot can reduce high-dose BCG-induced pulmonary pathological lesion in BCG-vaccinated mice,and control the immune response in infected local area,with a improved elimination potency to BCG.These observations support a new vaccination strategy.