Application of Kir2.1 as target spot to preparation of reagent or medicine for treating, preventing or diagnosing medulloblastoma
A medulloblastoma and drug technology, applied in anti-tumor drugs, drug combinations, biological tests, etc., can solve problems such as growth retardation, endocrine dysfunction, cognitive function decline, etc.
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Embodiment 1
[0023] Through immunohistochemistry and data analysis, the results were as follows figure 1 shown.
[0024] Immunohistochemical staining
[0025] 1. Slicing: After all paraffin specimens are uniformly numbered, put them in a -20°C refrigerator for pre-freezing, and then perform the steps of slicing, spreading and air-drying. The thickness of the slices was all 3 μm.
[0026] 2. Dewaxing: Place the paraffin sections on an iron shelf, bake in an oven at 60°C for 45 minutes, and then quickly soak them in xylene for 15 minutes. Put it in another vat of xylene and soak for 15 minutes. Then soak in 100% alcohol for 10 minutes, 95% alcohol for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, tap water for 5 minutes, and PBS for 5 minutes.
[0027]3. Antigen retrieval: Rinse with PBS 3 times, 5 min each time. Put the slices in a pressure cooker filled with restoration solution, heat to 100°C, cover the lid, and apply high pressure for 2min30s. Then rinse the cool...
Embodiment 2
[0057] Invasion migration and Western Blot experiments were carried out, and the results were as follows figure 2 It can be seen.
[0058] Medulloblast culture
[0059] The medulloblasts used in this experiment were all cultured in DMED medium containing 10% FBS, and kept in the cell incubator CO 2 Conditions of content 5%, temperature 37°C, humidity 95%. When the cell growth confluence reached 80%-90%, it was digested and subcultured with trypsin cell digestion solution, and both MB428 and MB913 cells were subcultured in 1 passage and 3 passages. Half of each cell was cryopreserved when it was transferred to the second passage, and the cells from the third to fifth passage were used for experiments.
[0060] Establishment of a medulloblast cell line stably silencing Kir2.1
[0061] 1. Kir2.1-shRNA interference lentivirus and nonsense sequence control lentivirus provided by Santa Cruze Company.
[0062] 2. For medulloblasts in the logarithmic growth phase, after washing ...
Embodiment 3
[0114] Co-immunoprecipitation experiments were carried out, and the results were as follows image 3 shown.
[0115] co-immunoprecipitation
[0116] The co-immunoprecipitation experiment used the Co-IP kit of Thermo Company, and the specific operation steps were as follows:
[0117] 1. Antibody and resin cross-linking
[0118] (1) Mix the cross-linked resin gently, pipette 80 μL into the chromatography column, centrifuge at 1000×g for 1 min, and wash twice with Couplingbuffer.
[0119] (2) Mix 10 μg of primary antibody or corresponding IgG, 10 μL of sodium cyanoborohydride and an appropriate volume of Coupling buffer to form 200 μL of antibody cross-linking mixture, seal the bottom of the chromatography column, and add the mixture to the chromatography column, 4 Rotational crosslinking at ℃ for 2-4h:
[0120] 2. Cell collection, lysis and pre-clearing
[0121] (1) When the medulla overexpressing Kir2.1 cells are cultured until the confluence reaches 80%, the medium is rem...
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