Hepatocellular carcinoma sorafenib-resistant cell strain and construction method thereof
A technology of human liver cancer cell lines and drug-resistant cells, applied in the field of biomedicine, can solve the problems of lack of liver cancer cell lines and drug resistance of patients, and achieve the effect of high scientific research and production application value.
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Embodiment 1
[0032] Example 1: Validation of induction and establishment of LM3 / SORA cells of drug-resistant liver cancer
[0033] ① After resuscitating LM3 cells of human liver cancer cell line, use DMEM culture medium containing 10% fetal bovine serum at 37℃, 5% CO 2 Cultivate in an incubator. The LM3 cells in the logarithmic growth phase were seeded in a 24-well plate. In order to find the maximum drug-resistant concentration of cells that can tolerate sorafenib (the concentration at which cells can survive and grow slowly), add sorafenib to each culture well to the final concentration of 0.5μmol / L and 1μmol / L, respectively , 2μmol / L, 5μmol / L, 10μmol / L, 15μmol / L, 20μmol / L, passage or change in time.
[0034] After each passage or fluid change, Sorafenib was added to the original concentration and treated continuously for 3 months. The maximum drug resistance concentration at which cells can survive and grow slowly after the addition of sorafenib is the screening concentration.
[0035] ② Th...
Embodiment 2
[0037] Example 2: Morphological observation of LM3 / SORA cells of the drug-resistant liver cancer strain
[0038] (1) Observe the morphology of living cells with an inverted phase-contrast microscope: Take LM3 control cells and LM3 / SORA resistant cells in logarithmic growth phase, wash with saline and change the solution, and observe the morphology of living cells under an inverted microscope (×200 times). Through the light microscope, it can be observed that the LM3 cells are basically the same size, polypseudopod-like polygons, and some are round (such as figure 2 a); and LM3 / SORA cells are of different sizes, round and polygonal, and the polygonal shapes are different (such as figure 2 b).
Embodiment 3
[0039] Example 3: Determination of the growth curve of LM3 / SORA cells of the drug-resistant liver cancer strain
[0040] Take LM3 and LM3 / SORA cells in logarithmic growth phase, digest with 0.5% trypsin and count them, inoculate into a 96-well plate at a density of 5000 cells per well and set up 3 multiple wells, add 200μL medium to each well , Placed at 37℃, 5% CO 2 Incubate in an incubator for 0h, 24h, 48h, 72h, 96h; aspirate the medium at each time point, add 100μL of CCK8 working solution to each well, incubate in the incubator for 1 hour, put it in the microplate reader and measure at 450nm wavelength , Read the OD value of each well and calculate the average value, draw the growth curve with time as the abscissa and the average OD value as the ordinate.
[0041] After continuously testing the growth status of the two cells, it is found that the drug-resistant cell line LM3 / SORA has a faster proliferation rate than the normal tumor cell line LM3, and the curve is still upward ...
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