Apoptosis Induced By Calmette-Guerin-Polysaccharide Nucleic Acid To Chicken Embryo Fibroblasts And Peripheral Blood Lymphocyte In Vitro

Calmette-Guerin-polysaccharide nucleic acid (BCG-PSN) was a kind of biological active substances, which extracted from BCG by hot phenol method and used for the prevention and treatment of chronic bronchitis, cold, etc. In recent years, BCG-PSN was found enhancing immunity, anti-tumor effects with further research and widely used in clinical practice. However, BCG-PSN using in veterinary medicine was rarely reported, especially studying on the deep mechanism of BCG-PSN. In addition, as a mixture of polysaccharides and nucleic acids, the chemical composition of polysaccharides and nucleic acids of BCG-PSN, the individual component effect, the interaction of polysaccharide and nucleic acid were poorly understood, which restricted BCG-PSN using in veterinary medicine severely. To solve these problems, we extracted BCG-PSN and separated components of it. Then we researched apoptosis of chicken embryo fibroblasts and peripheral blood lymphocytes induced by BCG-PSN.The effect of BCG-PSN on apoptosis of the CEF in vitro To evaluate the effect of BCG-PSN on apoptosis of chicken embryo fibroblast (CEF) in vitro, the concentration of apoptotic proteins such as Caspase-3, Caspase-8, Caspase-9and morphological changes after co-cultured CEF with BCG-PSN at different times and concentrations were studied by using MTT method, flow cytometry, spectrophotometry method, micro visual measurement etc. The results showed that the growth activities of CEF presented induction at the beginning and then followed by reduction with increasing BCG-PSN gradually, suggesting that, as an immunomodulator, the CEF activities can be enhanced by appropriate dosage of BCG-PSN. The growth activities of CEF, however, can be reduced by a higher concentration of BCG-PSN. The decreased activities of CEF in vitro characterized by deformated and round shape cells, deeply stained cell nuclear and margination, obvious induction of apoptotic cells; The concentration levels of Caspase-3, Caspase-8, Caspase-9showed significant differences between BCG-PSN treated group and control group with the gradual concentration increasing of the BCG-PSN. The concentration levels of Caspase-3, Caspase-9in CEF in vitro decreased with the higher concentration of BCG-PSN, suggesting that the excessive concentrations of BCG-PSN lead to CEF necrosis directly but apoptosis. The concentration levels of Caspase-3, Caspase-9presented induction at the beginning and than followed by reduction after reacted with increasing BCG-PSN, suggesting that the apoptosis can be induced by controlling the concentration and reacting time of BCG-PSN with some kinds of cells.The effects of BCG-PSN on apoptosis of peripheral blood lymphocytes in vitro To evaluate the inhibiting effect of BCG-PSN, separated from BCG through DEAE-cellulose anion-exchanging chromatography method, on the peripheral blood lymphocytes (PBL) in vitro, the effect of BCG-PSN on apoptosis of PBL in vitro, the concentration of apoptic proteins such as Caspase-3, Caspase-8, Caspase-9were tested by Trypan blue method, flow cytometry, spectrophotometry method, micro visual measurement etc. The results showed that the activities of PBL can be enhanced by BCG-PSN at low dosage and reduced after treated with high concentration of BCG-PSN. After treated with1000μg/mL of BCG-PSN, the number of apoptotic cells increased obviously and the concentration levels of Caspase-3, Caspase-9displayed significant differences between control group and1000μg/mL of BCG-PSN treatment group. The apoptosis and necrosis of PBL can be inhibited with low concentration of BCG-PSN, however, the excessive concentrations of BCG-PSN lead to PBL necrosis directly but apoptosis. Single component, BCG-PSA and BCG-NA, played a less induce on the apoptotic protein levels than BCG-PSN did at the same dosage, suggesting that BCG-PSA and BCG-NA induced apoptotic protein by the matter of combination, and BCG-PSA has stronger function than BCG-NA on the induction of Caspase. The apoptotic protein Caspase-9showed the similar changing tendency to Caspase-3, suggesting that the apoptosis of PBL induced by BCG-PSN in vitro may be concerned to the mitochondrial pathway.