牙髓干细胞 z-bo 2020-10-15 321

　　1. For the experimental method, please choose experimental miniature pigs that are 4 to 6 months old. Under anesthesia, the incisors of the mini pork milk were removed, and the spinal cord was incised under aseptic conditions. Cut the pulp tissue into small pieces of about 1 mm3 and digest them with enzymes. Cultivate in high-sugar DMEM medium. (1) Selection of monoclonal cells: Collect the third-generation cells, seed them in a 24-well plate at a cell density of 50 to 100 cells per milliliter, culture for 10 days, and select clones with more than 50 cells. I will. Use the filter paper method to transfer to a new 24-well plate, transfer the cells to a 6-well plate after fusion, and expand the culture to 2 x 105. (2) Identification by immunofluorescence staining: take 15 monoclonal cells and inoculate In a 24-well plate, 2×104 cells per well, high-sugar DMEM medium, 5% CO 2, 37°C. Add 4% for 24 hours. Fix with 0.5 ml paraformaldehyde, add 0.5 ml 0.3% Triton 100 to each well at room temperature, leave it at room temperature for 5 minutes, then add 10% blocked normal goat serum and 0.5 ml at room temperature, and keep it for 60 minutes. Discard the blocking solution and add 1:200 mouse antibody to each well. 0.2 ml human Nestin antibody, 1:600 mouse anti-human β-tubulin III antibody, 1:500 mouse anti-Butavimentin antibody, 1:200 mouse anti-human STRO-1 antibody, overnight at 4°C. Aspirate the primary antibody, discard it, and rinse 3 times with PBS. Add 0.2 ml of secondary antibody to each well, incubate at 37°C for 30 minutes, then observe and take pictures with a fluorescence microscope. (3) Induction differentiation experiment group: select monoclonal cells that are positive for STRO-1 expression, induction group A: monoclonal origin cell group; induction B group: mixed cell origin group; control group A: monoclonal origin cell group; control Group B: Mixed cell source group.

　　2. Culture results (1) Morphological characteristics of dental gum cells in miniature pig deciduous teeth: When observed with an inverted microscope, the cell morphology is diverse, spindle cells and polygonal cells grow in sheets, and the cell bodies are full and cytoplasmic. It is uniform and the core is round. The core is clear. Nerve-like cells grow radially like grass, with small cell bodies and transparent cytoplasm. As the cells pass, the nerve-like cells disappear mainly in the form of spindle cells or polygonal cells.

　　(2) The morphological characteristics and immunofluorescence staining identification results of the deciduous teeth of the monoclonal miniature pigs: The deciduous teeth of the miniature pigs were inoculated at a low density, and the colony formation rate was 2.74%. The cloned colony cells had polygonal immunity among the 15 monoclonal cells stained with fluorescence Six of the perivascular surface markers and bone marrow stromal precursor cell markers STRO-1 were positive, and the positive rate was about 76.4%. Under the microscope, it was observed that the cells with strong positive expression of STRO-1 were polygonal cells, and some spindle cells were observed to express weakly positive STRO-1. Vimentin was a typical feature of mesenchymal-derived cells. The test results showed that all Vimentin was positive in 15 monoclonal cells. All 15 twigs and deciduous gingival cells express neuron cell specific antibody Nestin, and the positive rate is about 69.7%.

　　(3) Mineralization induction results: Miniature pig deciduous dentin cells were cultured in vitro for 4 weeks, and Vonkossa staining was performed under the induction of mineralization solution. All six monoclonal origin induction groups A, the black positive reaction area is obvious and dense, alkali The expression of sex phosphatase is clear, showing positive areas of red staining. There is no significant difference between inducer B and inducer A. Control groups A and B are scattered in the mineralization points, and the red staining area is very small. The result of the alkaline phosphatase quantitative test is that there are significant differences in the alkaline phosphatase activity between the induction group A and the induction group B, between the control group A and the control group B, and between the induction group A and the induction group B There is no significant difference in alkaline phosphatase activity.

　　(4) Fat induction results: After induction of IBMX, indomethacin and hydrocortisone, some cells in groups A and B were induced to float. The dental pulp stem cells of three-week-old miniature pig deciduous teeth can be induced into adipocytes. Fat droplets have a clear refractive index under a phase contrast microscope. With the extension of the induction time, the lipid droplets in the cytoplasm gradually increase, aggregate, swell and are placed in the grape cluster. Group A induced by 6 kinds of monoclonal sources has 4 wells, which are stained with oil red and orange-red positive particles can be seen in the cells. The positive rate induced by fat is 4 times lower. According to calculations, 20 holes under an optical microscope Approximately 5 cells with lipid droplets appeared in the visual field. %. In the induction B group of 6 mixed sources, there were almost no positive oil red stains in 2 wells. The control group had no orange-red positive particles, and oil-red staining was negative.

　　(5) The result of neuronal cell induction: 6 kinds of monoclonal derivation induced. Group A has two holes. The morphology of the cells changes drastically. The original large and flat cell body shrinks, the edges of the cells become irregular, and there are many small cells. Finger-like protrusions. Two weeks after induction, the cells undergo significant morphological changes, and the cell bodies further shrink to form round, irregular cones and triangles. Some cells have multiple protrusions and branches, and the ends are similar to nerve cells. End. After induction, the result of immunofluorescence staining was positive for β-tubulin-III antibody and negative for STRO-1 antibody. According to the results of cell morphology analysis and immunofluorescence staining, the deciduous dental pulp stem cells were differentiated into nerve cells. No obvious nerve cells appeared in the induced group B and the control groups A and B.