| In this experiment, rabbit Intestinal Epithelial Cells were used as the research object and soybean agglutinin as experimental material to label the target sites of soybean agglutinin on the rabbit Intestinal Epithelial Cells, and study the different levels of soybean agglutinin on morphology, proliferation, differentiation and cell membrane integrity on rabbit intestinal epithelial cells, for further sduty of soybean anti-nutritional mechanisms provide a theoretical basis.1. Collagenase IV digestion was used to isolated and cultured the IEC of rabbit. The rabbit IECs were purified by the way of Phase difference digestion and adherence. And the IEC was identified by observing of morphology and detecting the cyto-keratin using the mouse anti-cyto-keratin monoclonal antibodies. The result shows that a small amount of adherent at 2-3d, proliferated at 6-8d, merged into pieces at 9-12d the IEC by digesting with collagenaseâ…£was well growth.95% for the purification of the IEC was obtained, cell junctions was tight, and the boundary was clear. The result of identification by detecting morphology and the cyto-keratin 8 was positive.2. Labeling the soybean agglutinin on primary cultured rabbit IEC with FITC fluorescent labeled soybean agglutinin In this experiment, soybean lectin binding to its target site was observed under the fluorescent microscope. The results showed that:soybean agglutinin labeled with FITC binding to rabbit intestinal epithelial cells, the large number of green fluorescence can be clearly seen under the fluorescence microscopy, cell boundary was clear. Accounting that a large number of target sites of soybean lectin were on the surface of rabbit intestinal epithelial cell.3. In this experiment, isolated and primary cultured of rabbit intestinal epithelial cell as a model to study the different levels of soybean agglutinin on the morphology, proliferation, differentiation and cell membrane integrity on rabbit intestinal epithelial cells. They were allocated to 6 groups, each group with 8 replicates,each replicate a hole. Each group inoculated soybean lectin concentrations were 0,0.25,0.50,1.00,3.00,5.00 mg/mL.Observed the morphology after dealing with the SBA and detected IEC proliferation with MTT colorimetric for 24h,48h,72h. and detected AKP activity,LDH activity in the culture medium and assayed the TEER value.The results showed that:With the increase of SBA concentration,morphological observation shows that, cell density was rising,but there were not any dead cells,which were observed in the culture medium. With the increase in SBA concentration and time, the IEC MTT values tended to increase, and increased significantly on 24h with 1 mg/mL SBA. AKP activity in the culture medium gradually decreased,5 mg/mL SBA treatment can significantly affect the degree of differentiation mature of rabbit IEC (P<0.01). With the increasing of concentration of SBA, permeability of the IEC increased, 5mg/mLSBA treatment, the TEER value of the cells were significantly lower than the other groups (P<0.01), LDH activity of cells was significantly higher than other groups (P<0.01).In summary, rabbit intestinal epithelial cells were isolated and purified successfully and found a large number of target sites of soybean lectin on the cell surface, and further experiment shows that as increased of the concentration of soybean agglutinin, proliferation of IEC can be enhanced for different leval in primary culture; but effected the degree of the differentiation mature of rabbits IEC; the permeability of the membrane on rabbits IEC was increased, epithelial barrier function was damaged.
|
PDF Full Text Request