Effect Of Different Dietary Copper Sources And Levels On Copper Transport In Chicken Intestinal Epithelium Cells

This paper based on primary culture of chicken intestine epithelium cells, and studied the effects of dietary copper sources and levels on copper transport in chicken intestine epithelium cells used copper sulfate and copper methionine as test material. This paper consist of two experiments:first, primary isolated culture and purification in chicken intestine epithelium cells; second, the effect of dierary copper sources and levels on chicken intestine epithelium cells.Experiment one:Firstly we mechanically shear ground the tissues, then isolated culture chicken intestine epithelium cells by collagenase Ⅳ digestion, and finally purified the chicken intestine epithelium cells in differential digestion and differential adhesion method. Morphology observation and immunohistochemical method identified the purified cells. These results suggested that cells obtained by collagenaseIVdigestion are growing in good condition. We could get adherent cells in inverted microscope after3-4d, then cells propagated obviously at7-9d, at last the cells merged at12-15d. The concentration of purified cells is over90%, and results of morphology observation and identification of cytokeratin-18monoclonal antibody showed that purified chicken intestine epithelium cells presented masccline.Experiment two:Isolated culture chicken intestine epithelium cells were used in this experiment,and it consists of two groups:one is copper sulfate, the other is copper methionine.Add0,15.6,31.2,46.8,62.4,78μmol/L Cu in culture medium with10%serum. Morphology observation and cells MTT colorimetric technique were performed to study the effect of different concentration of copper sources on IEC propagation, and the effect of different concentration of copper sources on IEC membrane permeability was detected in accordance to the changes of LDH activity in supernate. The effect of different concentration of copper sources on IEC cell mature differentiation was analyzed by the changes of AKP activity in supernate, and the amounts of copper in subcellular organelle were examined to discuss copper transport in vovo.The results showed that copper sulfate can efficiently promote cell multiplication in copper sulfate group, and its MTTOD570value is significantly higher than control group(P<0.05).The effect of experimental group added46.8μmol/L Cu on propagation of chicken intestine epithelium cells is the strongest at48h. Copper methionine can also efficiently enhance cell multiplication, but its strongest effect is generated at36h.The difference of cell multiplication activity among each concentration level is extremely significant(P<0.01),as well as that of each time, and the difference of interaction of concentration and time is extremely significant(P<0.01).As the increasing of concentration and prolongating of time, the value of cell AKP decreased.The effect of groups with78μmol/L copper sulfate and copper methionine on chicken IEC mature differentiation is extremely significant(P<0.01).As the increasing of copper concentration, the membrane permeability of chicken IEC rised gradually.The LDH activity of group added78μ mol/L copper sulfate and copper methionine is significantly higher than other groups(P<0.01).The amount of copper in nucleolus of groups added78μ mol/L and62.4μ mol/L copper sulfate is extremely higher than other copper sulfate groups(P<0.01), the amount of copper in mitochondria of groups added46.8μ mol/L copper sulfate is extremely higher than other copper sulfate groups(P<0.01),this is particularly true of groups added78μ mol/L copper sulfate in lysosomes and groups added78μ mol/L and62.4μ mol/L copper sulfate in cytolymph.The amount of copper in nucleolus in groups added78μ mol/L and62.4μ mol/L copper methionine is significantly higher than other groups(P<0.01), amount of copper in mitochondria in groups added46.8μ mol/L copper methionine is significangly higher than that of other groups, and the amount of copper in lysosome in groups added78μ mol/L copper methionine is extremely higher than that of other groups.The difference of copper amount in lysosome between groups added15.6μ mol/L and46.8μ mol/L is not significant(P>0.05),but the amount of copper in cytolymph of goups added78μ mol/L copper methionine is significantly higher than that of other groups.In conclusion, this experiment managed to isolated culture and purify chicken intestine epithelium cells, and copper in different concentration can promote propagation of chicken intestine epithelium cells and influence the mature differentiation of chicken intestine epithelium cells in different degree. The copper can also enhance membranaceous permeability of chicken intestine epithelium cells. Copper in intestine epithelium cells is distributed in each subcellular organelle, especially in cytolymph.