| Lactic acid bacteria (LAB), like other bacteria, are able to produce several types ofpolysaccharides that are classified according with their location relative to the cell. Those that areexcreted outside the cell wall are refered to exocellular polysaccharides or exopolysaccharides(EPSs). They can form an adherent cohesive layer and are called capsular polysaccharides. TheEPSs also can either be loosely attached or be completely excreted in the environment. Thebiological activities have attracted people's interests on physiological functions, such asimmunological activity, anti-tumor activity and anti-anabrosis activity.Exopolysaccharide produced by Lactobacillus delbruecckii ssp.bulgaricus (L.b.EPS) wasinvestigated from three aspects in this paper, which included MTT assay, Giemasa staining method,TEM, Agarose gel electrophoresis, TUNEL assay, FCM, Immunocytochemistry and Real-timePCR techniques.The results were as follows.(1) L.b.EPS could inhibit the cell growth of SGC-79.01 cells.By MTT assay, its inhibitory rate had the correlation with time and dosage. At theconcentration of 40mg·mL-1 for 24h, the inhibitory rate of SGC-7901 cell could reach to 73.81％.Meanwhile, if the SGC-7901 cells were treated with L.b.EPS from 24h to 72h at the concentrationof 10mg·mL-1, its inhibitory rate might change from 52.28 % to 65.37%.(2) L.b.EPS could induce apopiosis of SGC-7901 cells.The results of Giemsa staining method revealed that SGC-7901 cell had morphological.changes with following characteristics: super-aggregation of chromatin, chromatin clumping inapoptotic nuclei, cell body turning round, its shapes decreasing greatly, surface blebbing. By usingTEM, the cell's nucleus becoming shrinking, and the shape being twisted or ruptured by L.b.EPS(40mg·mL-1 for 24h), there were more vacuoles in the cytoplasm, while the control group had theintegrity of the whole body. Agarose gel electrophoresis indicated, only DNA extraction after beingtreated with L.b.EPS at the concentration of 20mg·mL-1 for 24h might lead to DNA ladders on thegel, and DNA ladders were not observed for others. TUNEL assay showed that with theconcentrations (10mg·mL-1, 20mg·mL-1, 40mg·mL-1) increasing, SGC-7901 masccline cells rates in the nuclear zones increased gradually. Apoptosis Index (AI) in L.b.EPS groups was higherthan control groups. AI might reach to 9.84% after being treated with L.b.EPS at the concentrationof 40 mg·mL-1 for 24h. The results of FCM indicated that SGC-7901 cells apoptosis treated withL.b.EPS were dose-dependent. The apoptosis rate might reach to 8.6% at the concentration of 40mg·mL-1. At the salne time, SGC-7901 cells apoptosis treated with L.b.EPS were time-dependentalong with cellular necrosis. The apoptosis rate might reach to 4.2% at the concentration of 10mg·mL-1for 72h.(3) L.b.EPS could regulate the key proteins and genes in apoptosisin SGC-7901 cells.The results of Immunocytochemistry and Real-time PCR techniques indicated that L.b.EPSmight induce SGC-7901 cells apoptosis by bcl-2 down-regulated and bax up-regulated at mRNAand protein levels in vitro.The conclusions were as follows: The results above suggested that apoptosis of SGC-7901cells could be induced by L.b.EPS in vitro. And the decrease of bcl-2 and the increase of bax at themRNA and protein level might be one of the apoptotic mechanisms by which L.b.EPS induced inSGC-7901 cell lines. |
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