Method for preparing human retina epithelial cell (ARPE) toxic model by utilizing amyloid protein A beta 25-35 and application of human retina epithelial cell (ARPE) toxic model
An amyloid and epithelial cell technology, applied in biochemical equipment and methods, animal cells, nervous system cells, etc., can solve the problems of inactivity, cumbersome preparation of oligomers, and inability to obtain ARPE cytotoxicity models. Easy-to-master, technically easy-to-use effects
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Embodiment 1
[0037] Example 1: Preparation and detection of Aβ25-35 oligomer-induced ARPE cytotoxicity model
[0038] Use 0.25% trypsin-EDTA digestion solution to digest APRE cells in the proliferating phase, add 1ml digestion solution to a 10cm cell culture dish, digest for 2-5 minutes until the cells are in the shape of quicksand, add 1-2ml PRMI1640 complete medium (composition: 89% DMEM / F12 medium + 10% FBS + 1% double antibody) gently pipetting to single cells. After centrifuging at 1000rpm for 5 minutes, remove the supernatant, re-add PRMI1640 complete medium to make a single cell suspension, and inoculate into a 96-well cell culture plate, adjust the final cell seeding density to 15%, and the volume of each well is 100 μl.
[0039] After the inoculated cells were completely attached to the wall for 12 hours, the medium was discarded, and the culture medium containing different concentrations of Aβ25-35 oligomers (0 μmol / L, 0.3 μmol / L, 1.0 μmol / L, 5.0 μmol / L, 20 μmol / L, 60 μmol / L L) ...
Embodiment 2
[0041] Example 2: Detection of cell cycle-related genes in the ARPE cytotoxicity model induced by Aβ25-35 oligomers
[0042] ARPE cells were inoculated on 6cm cell culture dishes respectively, and Aβ25-35 oligomers were added to induce ARPE cytotoxicity model according to the treatment of cell inoculation and optimal concentration conditions (60 μmol / L) in Example 1, without adding Aβ25-35 oligomers Somatic cells were used as controls.
[0043] After Aβ25-35 oligomers treated ARPE cells for 48 hours, both cells were collected and total RNA was extracted (3 wells of cells in each group were repeated), and after reverse transcription, Q-PCR technology was used to analyze the relevant cell cycle in the cells. The specific gene CCNEmRNA expression, specific primer information is shown in Table 1. The ANOVA program in the SASV8 statistical software was used to test the difference of data by T-test, and the results were expressed as mean ± standard error, with P<0.05 as the standar...
Embodiment 3
[0047] Example 3: Activity detection of apoptosis-related key proteins in the ARPE cytotoxicity model induced by Aβ25-35 oligomers
[0048] ARPE cells were inoculated on 6cm cell culture dishes respectively, and Aβ25-35 oligomers were added to induce ARPE cytotoxicity model according to the treatment of cell inoculation and optimal concentration conditions (60 μmol / L) in Example 1, without adding Aβ25-35 oligomers Somatic cells were used as controls.
[0049] After Aβ25-35 oligomers treated ARPE cells for 48 hours, the two kinds of cells were collected and the total cell protein was extracted (3 wells of each group were repeated), and Western blotting technology and specific CASP3 antibody were used to analyze the protein prepared by this method. Changes in the activity of the key control protein CASP3 of apoptosis in an Aβ25-35 oligomer-induced ARPE cytotoxicity model.
[0050] The detection found that compared with the control cells without addition, the activity of CASP3 w...
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