Cell strain capable of realizing long-term passage after mouse embryo fibroblasts are subjected to gamma ray irradiation, and construction method of cell strain
A technology of fibroblasts and gamma rays, applied in the field of biomedicine, can solve the problems of not being able to better reveal the cause of changes in the pathological mechanism of cell irradiation, the method of cell model construction has not been studied in depth, and the value of cells is limited. The effect of high scientific research and production application value
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Embodiment 1
[0025] Example 1: Establishment and verification of stable long-term passage mouse embryonic fibroblast (NIH3T3) cells after irradiation
[0026] Take mouse embryonic fibroblast (NIH3T3) cells and culture them with DMEM medium containing 10% newborn bovine serum (NCBS) and 1% penicillin-streptomycin dual antibiotic solution, at 37°C, 5%CO 2 Cultivate in an incubator; when the degree of cell mixing reaches 90%, use 1ml of trypsin containing EDTA (the mass concentration of EDTA is 0.25%) to digest, after digesting for 5min, use 2ml of culture medium to stop the digestion of cells in the culture flask, and put the culture flask After the 3ml liquid in the medium was repeatedly pipetted evenly, it was transferred into a 15ml centrifuge tube and placed at a Co60γ-ray source for irradiation, with a total irradiation dose of 10Gy. After the irradiated cells were taken back to the cell room, they were laid flat in the culture flask and continued to be cultured. After the cells reache...
Embodiment 2
[0027] Example 2: Morphological observation of long-term passage mouse embryonic fibroblast (NIH3T3) cells after irradiation
[0028] Observe the morphology of live cells with an inverted phase-contrast microscope: take mouse embryonic fibroblast (NIH3T3) control cells and mouse embryonic fibroblast (NIH3T3) irradiated cells in the logarithmic growth phase respectively, wash and change the liquid with normal saline, and place them under the inverted microscope (×200 times) to observe the living cell morphology of the 1st passage and the 3rd passage cells. The cell morphology of the first generation of cells did not change significantly after gamma ray irradiation. The density gradually decreased, the boundary was blurred, irregular vesicles appeared in the cytoplasm of some cells, and the cell membrane became transparent and bright, showing a pre-apoptotic shape ( figure 2 ).
Embodiment 3
[0029] Embodiment 3: Determination of the proliferation curve of mouse embryonic fibroblast (NIH3T3) cells that can be passed on for a long time
[0030] Normal control cells and irradiated mouse embryonic fibroblasts were digested with trypsin and then added a suitable new medium to resuspend the cells, according to 5×10 3 The concentration of cells / ml was inoculated into a 96-well plate, and each of the control group, the first generation and the third generation of the irradiation group had 5 controls to ensure that the cells were evenly dispersed in each culture well, and placed in the incubator overnight , to be detected after it adheres to the wall. The method is to add 10 μl of CCK-8 solution to each well, incubate in a 37°C incubator for 1 hour, detect the absorbance OD value at a wavelength of 476 nm, and measure continuously for 5-7 days according to the actual growth of the cells. SPSS 19.0 software system was used for statistical analysis, and P﹤0.05 was considere...
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