Modulation Of Biological Function Of Intestinal Epithelial Cell By Bifidobacterial Secretion Adhesin In Vitro

Bifidobacteria, the predominant bacteria in the human intestinal microflora, are considered to be microorganisms with a great influence on human health. There have been many studies demonstrating that strains of bifidobacteria have anti-infectious properties against enteropathogenic bacteria. However, there existed a few flaws when live bifidobacteria agents was used in gut barrier dysfunction: live bifidobacteria agents were difficult to produce and preserve, and also difficult to breed sufficiently due to lack of local gas and growth substrate. In recent years, it was reported the occurrence of bifidobacteria adhering to the human intestinal cells by a mechanism of adhesion which involves a proteinaceous component. It was observed that SBT2928 produced a proteinaceous factor, binding inhibitory factor (BIF), which prevented the binding of the ETEC to GAl in vitro, and the binding of ETEC to the human intestinal epithelial cell line HCT-8 was reduced by BIF treatment in a dose-dependent manner. These results showed there was adhesin component in bifidobacteria. In 1999, we extracted and purified a protein with a molecular weight of 16 KDa from spent culture supernatant of bifidobacterium adolescentis 1027 (B. ado 1027), but it's physiological functions was not studied. In the present study, our aims were: (1) To produce mass bifidobacterial secretion adhesin by chemistry chromatography, and observe the role of adhesion of human bifidobacterial strain to cultured human intestinal epithelial cells mediated by it in vitro. (2) To evaluate7competitive inhibition of adherence of enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli and Clostridium difficile to intestinal epithelial cells by adhesin in vitro. (3) To study the effect of adhesin on proliferation, apoptosis and morphology of intestinal epithelial cells. (4) To detect the effect of adhesin on expression of NF- K B activity, which could regulate expression of cytokines, and hope to investigate the mechanism of adhesin in molecular levels.Isolation and purification of bifidobacterial secretion adhesin and its role on adhesion of human bifidobacterial strain to cultured human intestinal epithelial cells The adhesin of bifidobacterium was isolated and purified by ammonium sulfate deposition, Superdex 75 gel filtration and Q-Sepharose FF ion exchange chromatography, and the adhesin was analyzed by SDS-PAGE. A protein with a molecular weight of 16 KDa was obtained. Adhesion test was used to study the adhesion of human bifidobacterial strain to cultured human intestinal epithelial cells. Results showed that the adhesion was time and dose-dependent, and adhesiveness achieved saturation when adhesin in the concentration of 30ug/ml, and lasting 3 hours. The adhesion of bifidobacteria to Lovo cell was no significantly difference between pretreatment of adhesin and spent culture supernatant (18.28 + 6.55 and 19.90 ?.07, respectively. P>0.05), but the adhesion was significantly stronger than that of by the pretreatment of PBS(8.50?.30, P.01). The adhesion of bifidobacteria to intestinal epithelial cell was linear. These results suggested a 16KDa protein was purified and identified as the adhesin of bifidobacterium. The binding of human bifidobacterial strains to human intestinal epithelial cells mediated by the adhesin appeared to be specific, time and dose-dependent.Competitive inhibition of adherence of enteropathogens to intestinal epithelial cells by bifidobacterial secretion adhesin The number of adhesion of ETEC, EPEC and Clostridium difficile to Lovo cells was counted and the results were analyzed by comparison after 30 min of incubation indifferent concentration of adhesin. Results showed the adhesin at thegconcentration of 10 ug/ml, 20 ug/ml and 30 ug/ml except at 1 ug/ml and 5 ug/ml could significantly inhibit the adhesion of ETEC, EPEC and clostridium difficile to intestinal epithelial cell line Lovo. Moreover, we observed that the ability of inhibition was enhanced with increase in the concentration of adhesin. Inhibition of...