Effect Of Exopolysaccharide From Lactobacillus Casei On The Gammadelta T Lymphocytes Of Gut-Associated Tissues In Balb/C Mouse Small Intestine

Lactic acid bacteria exopolysaccharides is widely used in food processing areas, which has many biological characteristics such as anti-tumor, immunomodulatory, lowering cholesterol, regulation of gastrointestinal function. Gut is any food must pass through organ. As a food-borne biological active substance, the immunomodulatory effect of lactic acid bacteria EPS on intestine is particular important. However, the research on regulation of the EPS on the intestine mucosal immune system, T lymphocytes in gut-associated lymphoid tissue and its important subsets of γδ T lymphocytes are not yet fully expanded and reported. Therefore, the subject investigated the effect of lactobacillus casei EPS on the four major gut-associated lymphoid tissue T lymphocytes and analyses the immune regulation of γδ T lymphocytes for exploring the impact of lactobacillus casei EPS on intestinal immune system.The subject mainly contained the following aspects:(1) To extract the EPS from the fermentation broth of the lactobacillus case.(2) To separate four kinds of small intestine gut-associated lymphoid tissue lymphocytes in mouse by using the method of Percoll discontinuous density gradient:intraepithelial lymphocytes (IEL), lamina propria lymphocytes (LPL), Peyer’s Patches lymphocytes (PPL) and mesenteric lymph nodes lymphocytes (MLNL).(3) To test the effect of EPS on T and γδ T lymphocytes proliferation in vitro ConA-induced by WST-1assay.(4) To test the effect of EPS on secreting of ConA-induced T and γδ T lymphocytes secret IFN-y, IL-17and TNF-α in vitro by ELISA assay.(5) After gavage EPS treatment of mice, analyzed the effect of EPS on γδ T lymphocytes in four gut-associated lymphocytes by using the method of flow cytometry; inhibition of γδ T lymphocytes in mice as a negative control, detect the mouse intestinal IFN-y, IL-17and TNF-a.mRNA level by using the method of RT-PCR, test the IFN-γ、IL-17and TNF-a content in mouse peripheral blood by using the method of ELISA for analyzing immune regulation function of EPS on mice γδ T lymphocytes.The results were as follows:(1) Successfully determined the separation filed in the small intestine of mice four gut-associated lymphoid tissue lymphocytes by anatomy, histology and cell morohology observation.(2) Lactobacillus casei EPS could promote the proliferation of T lymphocytes in vitro ConA-induced intestinal IEL and MLNL, and there were significant differences in the control group (P<0.05P<0.01), which was dose-dependent.(3) Lactobacillus casei EPS could promote T lymphocytes of IEL, LPL, PPLand MLNL secrete IL-17for the in vitro induced by ConA; Lactobacillus casei EPS could promote T lymphocytes of MLNL in secreting TNF-α at200μg/mL(P<0.01), depress T lymphocytes of IEL in secreting TNF-α (P<0.01); comparing with the inhibition ofγδ T cell group, could confirmed IEL γδ T lymphocytes is the major cell of secreting IL-17and TNF-α, and EPS can inhibit the in vitro induced by ConA of IEL γδ T lymphocytes secretion of TNF-α (P<0.01), the promotion of IEL γδ T lymphocytes secretion of IL-17(P<0.01).(4) Gavage EPS could improve the CD3+γδ T lymphocytes content in the IEL, LPL, and PPL percentage, from15.4%,6.8%,0.3%to17.6%,10.9%,0.9%; improve the CD8+γδ T lymphocytes percentage content in the IEL and LPL, from15.4%,3.7%to23.4%,14.2%; comparing with the inhibition ofγδ T cell group, confirmed that γδ T lymphocytes involved in intestinal IL-17and TNF-α mRNA expression, and EPS could act on the intestinal γδ T lymphocytes and thus increase the mRNA expression of IL-17; confirmed that γδ T lymphocytes participate in the peripheral blood of IL-17and IFN-γ secretion, and EPS could promote γδ T lymphocytes secrete IL-17and IFN-γ.In summary, the study could draw the following conclusions:Lactobacillus casei EPS could influence the proliferation of T lymphocytes in the mouse small intestine gut-associated lymphoid tissue and the secretion fo TNF-α IL-17cytokine in vitro induced by ConA. Especially from both vitro and vivo, EPS could regulate the number and major cytokine secretion of γδ T lymohocyte subset. The results werre helpful for clarifying the Lactobacillus casei EPS on the regulation of intestinal immune system and identified effect of EPS from Lactobacillus casei on theγδ T lymohocytes of gut-associated tissues in mouse small intestine..