Construction method and application of cell strain for expressing NTNG1 gene
A cell line and gene technology, applied in the direction of genetically modified cells, applications, cells modified by introducing foreign genetic material, etc.
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Embodiment 1
[0059] 1. qRT-PCR detection of NTNG1 expression
[0060] Fresh tissue specimens obtained during surgery were cooled in liquid nitrogen and stored in a -80°C freezer. After all the liver cancer tissues and paracancerous tissues were cut up, the total RNA was extracted with Trizol (TaKaRa, A-79061) reagent, and the total RNA was reverse-transcribed into cDNA according to the instructions of the kit. Reaction conditions: 95°C pre-denaturation, enzyme activation (30s), 95°C denaturation (5s), 60°C (30s), 40 cycles. With βactin as internal reference, with 2 -ΔΔCt The relative expression level of target gene NTNG1 mRNA was calculated by method. The upstream primer of NTNG1 is 5′-CTTGGAAGGAGTATCCCAAGC-3′, the downstream primer is 5′-TGTGGCATAATACTGATAGGGCT-3′; the upstream primer of β-actin is 5′-CACCATTGGCAATGAGCGGTTC-3′, and the downstream primer is 5′-AGGTCTTTGCGGATGTCCACGT-3′. The experiment was repeated three times. Conclusions such as figure 1 a.
[0061] Establishment of...
Embodiment 2
[0106] CCK-8 assay (cytotoxicity detection)
[0107] The specific steps of the CCK-8 experiment were performed according to the manufacturer's standard protocol. Cells were diluted in serum-free medium and seeded in 96-well cell culture plates at a density of 2000 cells / well. The plate was incubated at 37°C in 5% CO 2 incubator. To measure the growth rate of the cells, replace 100 μL of the spent medium with an equal volume of fresh medium containing 10% CCK-8. Cells were then further incubated at 37°C for 3 hours. Finally, a microplate reader (5082Grodig, Tecan, Austria) was used to measure the value of cell absorbance at 450 nm. The experiment was repeated 3 times and the results were recorded.
[0108] 1. Plate colony formation experiment
[0109] Cells in the logarithmic growth phase were taken from the cells in good growth state, digested with trypsin, resuspended with medium, inoculated into six-well plates with 500 cells per well, and cultured normally for 1520 da...
Embodiment 3
[0118] Flow Cytometry
[0119] Collect the HepG2 and SMMC7721 cells of each group transfected for 48 hours, digest with EDTA-free trypsin, wash twice with PBS, add 500 μL of binding buffer to resuspend the cells, and then add 5 μL of annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), protected from light, incubated at room temperature for 15 min, and then detected the apoptosis rate of cells in each group on a flow cytometer (BD, USA). Then the cells were collected and fixed with 70% ethanol at 4°C for 2 hours, treated with PI (50 μg / mL) and RNaseA I (100 μg / mL), incubated at 37°C in the dark for 30 minutes, and placed in a flow cytometer The changes of DNA content in cells were analyzed in each period. Experiments were repeated three times.
[0120] Conclusion: NTNG1 inhibits apoptosis and regulates cell cycle progression in human hepatocellular carcinoma cells
[0121] The uncontrolled proliferation of tumor cells caused by apoptosis and cycle regulati...
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