Application of magi-1 missense mutation (p.w845r) in renal podocyte function
A technology of podocytes and cells, which is applied in the field of constructing the apoptosis model of renal podocytes with MAGI-1 mutation, can solve the problems such as the lack of reports of renal podocyte apoptosis models, and achieve the reduction of cell adhesion ability, good cell model conditions, The effect of increasing the rate of apoptosis
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Embodiment 1
[0033]Example 1, MAGI-1 experimental model affects kidney foot cell function after knock down
[0034]First, design MAGI1 siRNA
[0035]
[0036]Second, the experiment group
[0037]A, normal group: MPC5 cells.
[0038]B, blank interference group: MPC5 cell + scrambled siRNA group.
[0039]C, MAGI1 interference group: MPC5 cell + MAGI1 siRNA1 group.
[0040]D, MAGI1 interference group: MPC5 cell + MAGI1 siRNA2 group.
[0041]E, MAGI1 interference group: MPC5 cell + MAGI1 siRNA3 group.
[0042]F, MAGI1 interference group: MPC5 cell + MAGI1 siRNA4 group.
[0043]Third, cell culture
[0044]MPC5 cells are first placed at 33 ° C, 5% CO2Proliferation culture is carried out in the incubator; when the MPC5 cell fusion is 50% -70%, the medium is transferred to 37 ° C, and placed at 37 ° C, 5% CO2Differentiation in the incubator was subsequent and cultured, and subsequent experiments were carried out after synchronization.
[0045]Fourth, cell transfection
[0046]1. Take a good MPC5 cell with good growth, with 5 × 105 / Well inoc...
Embodiment 2
[0058]Example 2, QRT-PCR detection of experimental effects of renal foot cell function
[0059]First, sample processing
[0060]1. Collect MPC5 cells to add 1 ml Trizol to the EP tube and shock 5 min.
[0061]2, add chloroform (200 μL) oscillating until emulsified, qi 4 min; 12000 g from 4 ° C for 15 min; lysis liquid is divided into three layers, and the colorless supernatant into a new EP tube.
[0062]3, isopropyl alcohol precipitation: add an equal volume of isopropanol to the upper cleaner, mix well, deposit 10 min at room temperature; 12000 g 4 ° C for 10 min, and a precipitate in the bottom of the tube.
[0063]4, ethanol cleaning: discard the supernatant, adding 70% ethanol 1 ml, oscillating to the precipitation block floating, 12000 g of 4 ° C is centrifuged for 5 min, and ethanol is discarded.
[0064]5, precipitate dissolution: Dry 2-5 min at room temperature, adding an appropriate amount of RNA dissolved solution (such as 20 μl) to dissolve precipitation, and then dissolve 30 min after pu...
Embodiment 3
[0075]Example 3, Western blot testing of kidney foot cell functional effects
[0076]First, protein cleavage solution formulation
[0077]Add PMSF, Cocktail, Triton-100 and Navo in the base lysate3.
[0078]Second, the sample preparation
[0079]1, discard the culture medium, add 1 ml of PBS (0.01M pH 7.2-7.3) per well to add 1 ml of 4 ° C, wash the cell, discard the lotion, repeat the above operation twice, then place the cellular hole plate in ice. on.
[0080]2, each cell plus 150 μl of the configured lysate, cleaves 30 min on ice.
[0081]3. After the cleavage is finished, the cell fragments were collected, and 4 ° C was centrifuged for 15 min.
[0082]4. Determination of protein concentrations: The kit is determined by Biyun Tian BCA protein concentration.
[0083]5. Adjust the protein concentration based on the concentration measurement results to ensure that different groups of protein concentrations are consistent and stored at -80 ° C.
[0084]Third, Western blot test
[0085]1. Preparation of electroph...
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