Effects Of Glycinin On The Mechanical Barrier Function Of Porcine Intestinal Epithelial Cells

In this study, the effects of purified Glycinin on porcine intestinal epithelial cells activity, permeability, the distribution and the genes expression of the intercellular tight junction proteins were investigated in vitro using porcine intestinal epithelial cells as an experimental model.Experiment1:Isolation and culture of porcine intestinal epithelial cells was done in this experiment. The tissue pieces culture method and collagenase IV digestion method were applied to isolate the porcine intestinal epithelial cells. The porcine intestinal epithelial cells were purified by the method of phase difference digestion and adherence. The porcine intestinal epithelial cells were identified using the cytokeratin from the mouse anticytokeratin monoclonal antibodies in the purified and stable subculture cells. The results indicated that a large amount of highly active porcine intestinal epithelial cells were obtained by the tissue culture and collagenase IV digestion method. Cells were adherent for majority and emigrated for minority at1-2d, and obviously proliferated at5-7d, and merged into pieces at9-10d, then presented "paving stone shape" at17-20d. After the morphological observation and immunohistochemical evaluation, the positive cells rate can reach95%.Experiment2:The effects of Glycinin of different levels on the activity and permeability of the porcine intestinal epithelial cells were investigated in this experiment. The porcine intestinal epithelial cells were treated with the five different concentrations (0-4.0mg/mL) of Glycinin to compare the change of the epithelial activity and permeability. The epithelial activity was determined by MTT after incubating24h. The LDH activity in the culture medium and the TEER were detected to study the epithelial permeability. The results showed that the MTT values of porcine intestinal epithelial cells tended to decrease with the increasing Glycinin. Glycinin can reduce the epithelial activity, and inhibit their proliferation. Along with the increase of Glycinin concentration, LDH activity in the culture medium gradually grew up, and the TEER value of the cells gradually went down. When the concentration of Glycinin is over2mg/mL, the TEER values of the cells were significantly lower than the control group (P<0.01). Seen from two results, the Glycinin can increase the permeability of the porcine intestinal epithelial cells.Experiment3:The distribution and gene expression of tight junction protein ZO-1and Occludin in porcine intestinal epithelial cells treated with Glycinin were developed in this experiment. Porcine intestinal epithelial cells were treated with0,0.5,1.0,2.0or4.0mg/mL Glycinin and each treatment with3replicates. After incubating24h, the distribution of tight junction proteins in porcine intestinal epithelial cells was determined using cell immunofluorescence test. The gene expression of ZO-1and Occludin were measured by the real-time quantitative FQ-PCR. The immunofluorescence data showed that the tight junction proteins ZO-1and Occludin decreased with the increasing Glycinin, but the change of ZO-1was not obvious. When the concentration reached2mg/mL, immunofluorescent staining weakened significantly and appeared to rupture. With the increase of Glycinin concentration, the relative expression of ZO-1and Occludin mRNA in porcine intestinal epithelial cells declined. In the group of Glycinin concentration higher than2mg/mL, tight junction protein ZO-1and Occludin mRNA relative expression were significantly lower than the control group(P<0.01). The above results indicated that Glycinin can lead to the damage of the tight junction proteins of ZO-1and Occludin in porcine intestinal epithelial cells with dose-dependence.