牙周膜细胞培养 z-bo 2020-10-15 329

　　1. Experimental method: 6-month-old mini-pigs weighing about 25 kg will be anesthetized regularly. Pull out the eight anterior teeth (4 dogs and 4 incisions) of the upper and lower jaws of the miniature pig under aseptic conditions, and immediately put them into a sterile packaging bottle containing D-Hank balanced salt solution. Contains double antibodies. Shake gently for 5-10 minutes to wash away blood stains on the surface of the tooth roots, and then immediately send it to the laboratory under an ice bath. In an ultra-clean workbench, use a needle holder to fix the root surface of the tooth neck upward, and use a 20 ml sterile syringe to suck sterile saline to clean the root surface. Rinse repeatedly. A sharp subgingival curette scrapes off one third of the periodontal ligament at the root, and cuts the tissue into small pieces of about 1 mm3, each with a distance of about 5 mm, and evenly place them in a 25 cm2 disposable sterile culture In the bottle. Vaccination. under. Invert the culture flask, and inject 3 ml of high-glycemic DMEM culture medium into the flask and cell culture box (5% CO2, 37°C), which contains 200 ml/L fetal bovine serum, 100 U/ml penicillin, 100 g/L Streptomycin. Incubate (100% humidity). After attaching the small tissue fragments for 2-4 hours, slowly turn the culture flask over and lay it flat so that the liquid slowly covers the small tissue fragments and starts the culture. Within one week after the inoculation, the tissue block is not firmly attached. Liquid exchange and observation should be avoided to prevent the tissue block from being impacted and floating. After that, change the culture medium every three days, add 3-4ml culture medium each time, carefully observe the cell growth, and swim.

　　2, experimental results

　　(1) The success rate of periodontal ligament cell culture in miniature pigs: Among the 4 miniature pigs and 4 dogs sampled, the periodontal ligament of 4 incisors obtained by tissue block culture method was normal. Was cultivated. The success rate of cell culture is 50%, the success rate of incisor culture is 100%, and the success rate of canine teeth is 0.

　　(2) Cell proliferation and morphological performance: From the 7th day to the 14th day of the primary culture, observe the cells swimming around the tissue mass with an inverted microscope. The cells expand around the tissue mass, and single cell clusters without tissue mass can also be seen. The cells are spindle-shaped, star-shaped or polygonal, with small protrusions, abundant cell bodies, clear cytoplasm, and round or oval nuclei. The units are arranged radially and spirally. In the primary culture of about 30 days, when the cells were fused, the cells reached 80% to 90% of the bottom of the flask and passed 1:2. The periodontal ligament cells of miniature pigs grow rapidly after passage and can be passaged again after 4-6 days. Different cell types have different sensitivity to trypsin. The spindle-shaped fibroblast-like cells can be digested in 3-5 minutes without changing their shape. Epithelial-like cells are less sensitive to trypsin and grow in clusters. Periodontal ligament fibroblasts can be separated from epithelioid cells by cell passage.

　　(3) Immunochemical identification results: The third generation of miniature porcine periodontal ligament cells were observed by immunohistochemical staining under an optical microscope. The cells stained positive for anti-vimentin. The positive particles are evenly distributed in the cytoplasm and the nucleus is transparent. Does not stain. Anti-keratin staining was negative, indicating that the periodontal ligament cells of miniature pigs were derived from mesenchymal tissue.

　　(4) Cell proliferation curve: The proliferation curve of periodontal ligament cells of miniature pigs is S-shaped, with a clear retention period, exponential growth period and stationary period. Within 24 hours after inoculation, the number of cells decreased due to trypsin digestion, and then gradually increased. Cell proliferation accelerated from day 3 and entered the exponential proliferation phase. When the number of cells reached a peak on day 8, the cells grew on a Petri dish, and did not divide due to contact inhibition, and proliferation slowed to a plateau.