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Objective: To establish IEC cell lines by separation Ji Chang epithelial cells (IEC) and primary cultured enzyme digestion of cell proliferation, promoting cell proliferation optimal serum concentrations of CO 2 concentration and temperature. Then the IEC cell experimental model studies carrying copper silicate nanoparticles (CSN) morphology of chicken IEC, proliferation, differentiation and cell migration after injury affect. Method: 1. Different digestive enzymes in vitro separation chicken IEC and training. 2.IEC growth, proliferation detected by MTT assay and cell counting. Inverted microscope, transmission electron microscopy observation IEC growth, morphology. 4.IEC identified with alkaline phosphatase, and H-E staining. Results: 1. 300U/mL Ⅺ collagenase and 0.1mg/mL Ⅰ neutral protease at 37 ℃ digestion 40min, a large number of cells can be collected by centrifugation. Observed under an inverted microscope, the mucosal suspension by the crypt-like epithelial cell clusters, cells were ball or pebble-like, clear boundary, abundant cytoplasm, nuclear round or pebble-like nuclear chromatin sparse empty bright nucleolus 1 to 2. 2 in DMEM medium containing 2.5 to 5.0% fetal calf serum, 39 ° C, 5 to 7.5% CO 2 culture, after a certain time, under a transmission electron microscope, can be observed to the cell having typical intestinal epithelial crypt cell characteristics, IEC surface with a large number of microvilli, cell tight junctions between cells in the short term, desmosomes adherent cells are rich in mitochondria and endoplasmic reticulum. Adherent IEC 1 ~ 2d, 6 ~ 7d proliferated significantly, 10 to 12d merged into slices, the cells were polygonal monolayer. 3 The test alkaline phosphatase as markers Immunoperoxidase, the results show that the separation of the cell culture to day 7, (91.164 ± 6.97)% cells were blue-black reaction. 4 compared with the control group, add 30μg/mL 50μg/mL CSN after cell morphology did not change, in the first 10 days, the cell surface of microvilli than that without the CSN more apparent; add CSN wells in cell proliferation more evident, the cells of the hole in the first 10 days have been completely merged into the sheet, without adding holes in the first 10 days, the cells there is not completely convergence zone; CSN is the number of migrated cells in the unit area can be significantly increased (P LT; 0.05) can promote damage cells quickly repair. Conclusion: 1. Determine Ⅺ with 300U/mL collagenase, and 0.1mg/mL Ⅰ neutral protease digestion method for better cell separation methods, including 2.5% of 5? S is more appropriate serum concentration, 39 ℃ and 5% to 7.5% CO 2 is a better culture conditions. Chicken IEC microscope and histochemical identification, the cultured cells to IEC. Add 2 in primary cultured chicken IEC 30μg/mL, 50μg/mL CSN, can promote the IEC proliferation, differentiation, and repair of damaged cells.
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