| Part One Effect of External Bifidobacterium on the Production of DC in the Intestine of Mice Objective:The first part explores the effect of the external bifidobacterium on the production of dendritic cells (DCs) in the intestine of mice. Methods:Thirty-six four-week-old to five-week-old BALB/C mice were randomly divided into four groups each having 9 mice. The four groups of mice were administrated orally respectively with live bifidobacteria, heat-killed bifidobacteria (1×109CFU/ml), spent culture supernatant (SCS) or saline normal, once daily 0.5ml per mouse. After seven consecutive days of oral administration, the mice were killed on the 9th day, and their intestinal samples were used to evaluate the production of intestinal DCs by the immunohistochemical method. Results:Under the microscope, DCs were present in the intestinal lamina propria of mice, the cells had large irregular protuberance and had tight contact with surrounding cells; the sizes of DCs were varied and the morphologies were irregular; and the nucleuses were irregular and eccentric. Counting showed that the bifidobacterium could improve the number of DCs in the small intestine of mice. There was significant difference between the control group and the live bifidobacterium group, the heat-killed bifidobacterium group and SCS group(p<0.05). The heat-killed bifidobacterium group was compared with the live bifidobacterium group and the SCS group, a conclusion was drawn that there was obvious difference(p<0.05). Conclusions:External bifidobacterium can improve the number of DCs in small intestine of mice. The effect of live bifidobacterium is the most obvious. The research concludes that the bifidobacterium and its state of being alive have important effect on the differentiation and development of DC. DC is important for bifidobacterium to exert its function of immunity. Part Two Effect of Bifidobacterium on the Maturation and Function of DC derived from Monocyte in vitro Objective:The second part studies whether the bifidobacterium play a part in the functioning of the dendritic cell (DC) stimulating the proliferation of T lymphocyte, and having effect on the generation of cytokine secreted by the DC, which is derived from the normal adult peripheral blood monocyte in vitro. Methods:Peripheral blood mononuclear cells (PBMCs) were separated from normal adult peripheral blood by Ficoll. They could obtain monocytes after plastic-adherent culture. When the monocyte was induced only by the heat-killed bifidobacterium, to see whether it could change cell morphology and become DC by phase-contrast microscope morphological observation. On the other hand, normal adult immature DCs were induced by monocyte cultured in granuloeyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4).Immature DCs were exposed to various dosage heat-killed bifidobacterium (100ug/ml,10ug/ml) as experimental groups on the 7th day, while the control group was exposed without heat-killed bifidobacterrium, LPS at 1ug/ml was added to some cultures as positive control. The morphology of DC was observed by phase-contrast microscope and electron microscope. On the 9th day of the culture, the immune function of DC detected through the stimulation of the proliferation of allogeneic T lymphocytes was examined by MTT assay. At the same time, the level of IL-12 in DC culture supernatants and the level of IFN-γ in DC and T lymphocyte co-cultured supernatants were analyzed by ELISA. It was testified whether the bifidobacterium hadeffect on DC. Results:(1) Monocyte could not been changed cell morphology only induced by the heat-killed bifidobacterium; (2) The characteristics of phase-contrast microscope and electron microscope indicated that immature DCs stimulated by the heat-killed bifidobacterium had typical morphology, which was similar to the morphology of positive control mature DCs. The mixed leukocyte reaction (MLR) test showed that by the stimulation of the heat-killed bifidobacterium, the induced DCs could remarkably stimulate the proliferation of... |
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