腮腺细胞 z-bo 2020-10-15 245

　　1. Experimental method: 4-month-old experimental mini-pigs, conventional anesthesia, extract parotid alveolar tissue under aseptic conditions, remove glandular lobule envelope and other combined tissues, and then immerse in HamF12 medium (containing 100 U/ml penicillin, 0.1 mg/ml streptomycin). For small pieces smaller than 1 mm3, wash twice with Mg2 + Hank balanced salt solution without Ca2 +, and digest with HamF12 culture medium containing 2.5 mg/ml mixed collagenase for 1 hour at 37°C. Filter and place at room temperature for 10 minutes to contain 50 ml/L heat-inactivated fetal bovine serum, 10μg/ml reduced glutathione, 1 mmol/L putrescine, 5μg/ml transferase, 5μg/ml insulin Cell. Collect, add 10g/ml epidermal growth factor dexamethasone to HamF12 complete medium. The separated and digested cells were seeded on a 24-well Falcon culture plate covered with Matrigel at a concentration of 104 cells/ml. Inverted microscope, electron microscope, keratin antibody, α-amylase antibody immunohistochemical staining to observe the cells.

　　2. The experimental results obtained through phase contrast microscope observation: a few hours after seeding the separated parotid gland cells, single cells aggregated, and within 36 hours, the cells attached to the surface of the culture substrate, after which the single cells can be seen from the edge of the cell cluster It begins to extend, and most cells are seen within about 3 days. It grows in a single layer and adheres to the wall. The pixel size is relatively uniform, and most of them look round and closely aligned. On the 5th day, the cells can be seen growing and connecting into sheets, the central part of the cells is actively proliferating, and some cells are polygonal long spindles. Under these conditions, cells can survive for nearly 3 weeks. HE staining showed cell monolayer growth, most of which were round or round, and some cells were long spindles. All nuclei are stained and centered. The immunohistochemical staining results showed that the cultured cytokeratin monoclonal antibody staining was positive, the α-amylase polyclonal antibody staining was positive, and the vimentin monoclonal antibody staining was negative. The control composition of the fibrocytokeratin monoclonal antibody and the α-amylase polyclonal antibody staining were negative, and the vimentin monoclonal antibody staining was positive. Osmosis electron microscope observation: The cultured cells are round, centered or offset in the nucleus, moderately developed cytoplasmic mitochondria and thick and small vesicles, small Gorgi devices and many secretory particles with different electron densities. include. , Each secreted particle is surrounded by a complete unit membrane, the density within the particle is not uniform, and contains a large number of fine particles. There are sparse microvilli of different densities on the cell membrane surface. Some cells contain microfilaments in their cytoplasm and no secretory granules.

　　(1) Determination of protein secretion: After cells are cultured in a 24-well plate for 24 hours, the measured absorption value is 1.58±0.49, and the protein content is (3±0.93) mg/ml.

　　(2) Determination of α-amylase: The results showed that the content of α-amylase in the culture broth was the highest (343U) on the second day of culture. With the increase of in vitro culture time, the content of α-amylase decreased significantly. After 4 days of culture, the amylase content in the culture medium of the stimulated group was significantly higher than that of the unstimulated group (P\u003c0.05). On the 5th to 6th day of culture, the content of α-amylase in the culture broth decreased to 30% of the initial value, and on the 10th day of culture, the content of α-amylase in the culture broth fell below the initial value 8%.

　　(3) The role of isoproterenol: The long-term survival of parotid gland cells in vitro depends on the addition of secretions to the culture medium. The newly isolated acinar cells are morphologically similar to those in vivo. Without isopropyl group. After culturing in epinephrine medium for 2 days, the cells contracted, and the cell boundaries became blurred and died within one week. The addition of secreted isoproterenol can prevent the above changes. The parotid gland cells in the isoprenaline-added group and the isoprenaline-free group both expressed α-amylase, but the expression level was different, and the expression of the former was enhanced.

　　(4) The effect of different concentrations of serum on cell proliferation: When 50 ml/L of bovine fetal serum is added to HamF12 complete medium, parotid gland cells will attach to the wall faster and proliferate well. After adding 100 ml/L of bovine fetal serum, fibroblasts grew vigorously, and epithelial cells hardly grew. All cells grow slowly without serum.