The Influence Of The Extracted Soybean Glycinin And β-conglycinin On The Function And Molecular Mechanism Of Mice Intestinal-epithelial Cells In Primary Cultivation

By using the isolated and purified soybean as subjects and by using techniques of mouse intestinal epithelial cells in vitro techniques,the paper studies the purified glycinin andβ-conglycinin on intestinal epithelial cells in mice physiological functions and the impact on biochemical parameters, and explores the cellular and molecular level of antigen protein on the digestion and absorption-related gene expression.Two experiments were conducted.Part 1:Glycinin andβ—conglycinin was isolated and purified by means of the characteristics 7S globulin and 11S globulin of different dissolubility at PH6.1 to PH 6.6(optimization pH6.2 to 6.4).The first was to prefract two kinds of globulin, then, salt out with sulfate ammonium and gel filtration, as much as possible to remove constituents of non- antigen protein to obtain glycinin andβ—conglycinin with relative higher purity coefficient.The results indicate:Soybean powder being extracted for 2 hours through 0.03mMTris-HCl, ratio of dosage liquor:1 to 20, room temperature, PH8.0, crude 11S with 71.53 percents protein and crude 7S with 60.73 percents protein are separated. Salted out with sulfate ammonium and gel filtration, 11S with 84.2 percents protein and 7S with 79.5 percents protein are obtained..Part 2:The experiment studies the effect of different level purified glycinin andβ—conglycinin(0,1mg/ml,5mg/ml,10 mg/ml) on integrity and function of mice intestinal epithelial in vitro.The results indicate:The amounts of cells in the group of glycinin andβ—conglycinin decrease gradually.The cell MTT absorption at 570 nm wavelength (MTT OD570) is very significantly affected by glycinin andβ-conglycinin concentration (P< 0.01). Glycinin andβ—conglycinin is positively correlated significantly with LDH, AKP, GOT, GPT, interleukin-2, interleukin-6, interleukin-8 and NO (P<0.05), glycinin andβ—conglycinin is positively correlated significantly with protein content and retention, GOT of the cell, but does not affect NH3 (P>0.05)Cellular and molecular level of the experimental results show that, P-conglycinin, soybean globulin decrease PEPT1 and DMT1 gene expression.β-conglycinin significantly reduces the PEPT1 gene expression (P<0.05), high-dose P-conglycinin are affected more siglificantly. The effects of P-conglycinin on DMT1 gene expression are significantly greater than the impact on soybean globulin.In this paper, experimental studies have shown that soybean meal antigen protein significantly affects cell nutritional physiological functions. The role ofβ-conglycinin is significantly larger than glycinin.β-conglycinin inhibits digestive tract epithelial cells with the major nutrients (protein) digestion and absorption and the related genes (PEPT1) expression is significantly stronger than that in genes (DMT1). Soybean antigen protein of the cellular and molecular level caused by the substance of change is caused by the integrity of intestinal epithelial cells, changes in physiological functions,and cell physiology and biochemistry parameters changed significantly, resulting in the reduction of cell proliferation, or even causing intestinal inflammatory cytokines (IL-2, IL-6, IL-8) level of the main reasons for significant changes.