The application of acat1 gene and acat1 gene or protein interferor and the products of the application interferor
A gene and product technology, applied in the direction of medical preparations containing active ingredients, gene therapy, biochemical equipment and methods, etc., can solve the problems of high cost and cumbersome diagnosis of kidney diseases
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Embodiment 1
[0078] The cultivation of embodiment 1 human kidney cancer Wilms cell (G-401)
[0079] cell culture
[0080] Cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO 2 In the cell culture incubator, the time to replace the cell culture medium is determined according to the state of the cells, and the cells are passaged in time.
[0081] cell inheritance
[0082] Pour out the original DMEM culture medium in the culture bottle, wash it twice with 1mL PBS, then add 0.5mL of 0.25% trypsin, cover the bottle cap and let it stand for about 1min, so that the trypsin can completely digest the adherent cells. Then add 4 mL of DMEM culture medium and blow the wall of the bottle to which the cells are attached to completely disperse the cells and suspend them in the culture medium. Adjust the passage ratio according to the test requirements, and put them into a new culture bottle to continue culturing.
Embodiment 2
[0083] The establishment of embodiment 2 IgA nephropathy model
[0084] (1) Selection of fatty acid concentration
[0085] Adjust the G-401 cell density to 5×10 4 cells / mL, inoculated in 96-well plate, 200 μL of cell suspension per well, replaced the culture medium after 6 hours, and incubated in the incubator until the cell confluence was 80%-90%.
[0086] Discard the culture solution in the wells of the culture plate, add normal DMEM culture solution to the control group, and add palmitic acid (palmitic acid, PA) culture solution with a concentration of 0, 0.25, 0.5, 1, 2 and 4 mmol / L respectively to the test group . Six replicates were set for each concentration, and 100 μL of normal DMEM medium was added to the control wells.
[0087] After culturing for 24 hours, add 10 μL of 0.5% MTT solution to each well, and add 100 μL DMSO after incubation for 4 hours. After the purple crystals are fully dissolved, set the wavelength to 490 nm for detection by a microplate reader, ...
Embodiment 3
[0095] Example 3 ACAT1 mRNA expression
[0096] PA stimulation induces the expression level of ACAT1 in the cell model of IgA nephropathy
[0097] Place G-401 cells in a 6-well plate and culture until the confluence reaches 80%-90%, add normal DMEM culture medium to the control group, and add an appropriate amount of PA stock solution to the other groups, so that the final concentration is 0.25mmol / L , 0.5mmol / L and 1mmol / L. After culturing in the incubator for 24 hours, the cells of each group were collected.
[0098] (1) Expression of ACAT1 mRNA level
[0099] The total RNA in G-401 cells was extracted by the TRIzol method in a dedicated console (DEPC water cleaning). Experimental consumables such as centrifuge tubes and pipette tips were soaked in DEPC water before extraction to remove RNase.
[0100] Extracting RNA from G-401 cells mainly includes the following steps:
[0101] a. Add 1mL Trizol to each well of the six-well plate, scrape off the cells with a cell scrap...
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