Application of cdk6 small molecule inhibitors in reducing the resistance of liver cancer cells to antineoplastic drugs or radiotherapy
A technology of small molecule inhibitors and liver cancer cells, which is applied in the field of medicine, can solve problems such as enhanced CDK6 activity, and achieve the effect of improving therapeutic effect and reducing tolerance
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Embodiment 1
[0023] Example 1 Western blotting method to detect the tolerance of liver cancer cells to 5FU and CIS
[0024] It is believed that the culture method of serum-free medium enhances the drug resistance of chemotherapy drugs 5FU and CIS of liver cancer cells by immunoblotting method, which is positively correlated with the expression of CDK6;
[0025] Spheroid culture method (serum-free medium culture method): Hepatoma cells were inoculated in a low-adhesion 6-well culture plate, and treated with EGF (20ng / ml), bFGF (20ng / ml), B27, LIF (leukemia inhibitory factor, 10ng / ml), 2mmol / L L-glutamine and 40U / ml heparin in DMEM-F12 medium (SFM), cultivated in a 37°C, 5% CO2 incubator for 7 to 10 days.
[0026] Adhesive culture method (serum-free medium culture method): Inoculate liver cancer cells in low-adhesion 6-well culture plates, culture in 37°C, 5% CO2 incubators with DMEM medium containing 10% FBS.
[0027] CCK8 detects the toxic effect of cells on chemotherapeutic drugs 5FU and...
Embodiment 2
[0030] Example 2 CDK6 expression in liver cancer tissues at different advanced stages
[0031] Immunohistochemical analysis of CDK6 expression in liver cancer tissues at different advanced stages showed that CDK6 expression may be positively correlated with liver cancer progression, indicating that CDK6 immunohistochemical method can be used as an indicator of drug resistance in liver cancer. If the expression of CDK6 gene is high in patients with liver cancer, it may indicate that the chemotherapy effect is not good, and targeted drugs CDK4 / CDK6 inhibitors can be used to enhance the therapeutic effect.
[0032] Immunohistochemistry: Paraffin-embedded sections of 30 cases of liver cancer tissues of different advanced stages and 10 cases of normal liver tissues were used for tissue chip construction. Each sample on the chip has a diameter of 0.6mm and a spacing of 0.1mm. After the tissue chip was dewaxed and hydrated by gradient, endogenous peroxidase was blocked with 0.3% hyd...
Embodiment 3
[0034] Example 3 High expression of CDK6 promotes resistance to chemotherapy drugs 5FU and CIS in liver cancer cells
[0035] 3.1 Construction of liver cancer cell lines with high expression of CDK6 gene: including packaging and preparation of virus with high expression of CDK6 gene and screening of liver cancer cell lines with high expression of CDK6 gene.
[0036] 3.2 Packaging and preparation of CDK6 gene high-expression lentivirus: construct the related lentivirus expression vector (pLVX-Puro-CDK6), put 293T packaging cells in a 60mm culture dish before transfection, and inoculate 1-2×10 6 , Transfection can only be carried out when the cell adherent growth density reaches about 80%. The recombinant lentiviral vector, pMD-G, and pPax2 vectors were co-transfected into 293T packaging cells by liposome-mediated method, the plasmid ratio was 8:5:3, and the total amount was 6 μg. Place the transfected 293T cells in 5% CO 2 , 37 ° C incubator culture. After 48 hours and 72 ho...
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