Application of adefovir dipivoxil in preparing antitumor drugs
A technology of adefovir dipivoxil and anti-tumor drugs, which is applied in the field of medicine, can solve the problem of the influence and effect of adefovir dipivoxil on the growth of cancer cells, and achieve the purpose of inhibiting the growth, division and proliferation of colon cancer cells , moderately priced effect
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Embodiment 1
[0029] Example 1 Study the anti-tumor mechanism of adefovir dipivoxil by superELISA technology
[0030] 1. Preparation of fusion protein KCTD12-his, CDK1-gst
[0031] (1) Expression and purification of KCTD12-his fusion protein
[0032] 1. Construction of the recombinant expression vector pET-KCTD12: According to the human KCTD12 protein gene sequence in the NCBI database (NCBI accession number: NM_138444.3), use Primer Premier 5.0 to design and synthesize primers encoding the KCTD12 gene, and add double restriction sites at both ends (The restriction site at the upstream end is BamH1, and the restriction site at the downstream end is EcoR1, both purchased from TAKARA Company), and the target gene band is amplified by PCR.
[0033] 2. The PCR product was detected by 1% agarose gel electrophoresis, and the target fragment was recovered and purified using a DNA recovery kit (Tiangen Biochemical Technology Co., Ltd., catalog number: DP214). Use endonucleases to double-digest th...
Embodiment 2
[0072] Example 2 Co-immunoprecipitation experiment
[0073] Co-immunoprecipitation experiment (Co-IP) is an important means to test the interaction of proteins in vivo. In order to further study and verify the anti-cancer mechanism and effect of adefovir dipivoxil, the present embodiment carries out co-immunoprecipitation experiment (Co-IP) on Adefovir dipivoxil. Fuvir dipivoxil for further study.
[0074] In advance, 4 groups of 1 million colon cancer HCT116 (purchased from ATCC; product number: CCL-247) or HT29 cells (purchased from ATCC, product number: HTB-38) were spread in cell culture dishes in each group, and DMSO was added respectively, Drug #1, Drug #2, Drug #3. DMSO was added according to the ratio of 1 μL: 1000 μL DMEM medium, drugs 1 to 3 were diluted with DMEM medium, and the final concentration was 10 μM; the treated cells were cultured at 37°C for 24 hours, and then the cells were washed twice with PBS. Add 1 mL of 0.25% trypsin to digest the cells to make th...
Embodiment 3
[0083] Embodiment 3 Adefovir dipivoxil on the influence of cancer cell division
[0084] 1. Spread the colon cancer cells HCT116 in a 6-well plate, with 500,000 cells per well. In the experimental group, 2 μL of 10 mM adefovir dipivoxil was added to 2 mL of complete culture solution to make the final concentration 10 μM; in the control group, 2 μL of DMSO was added to 2 mL of complete culture solution. Incubate at 37°C for 48 hours.
[0085] 2. The cells were digested and counted, and 500,000 cells were taken from each of the experimental group and the control group. Wash the cells with PBS, wash away the attached culture medium, and centrifuge at 300g for 5 minutes.
[0086] 3. Prepare a mixture of 600 μL PBS and FBS in advance, the preparation method is 540 μL PBS+60 μL FBS, mix well and pre-cool on ice. Take 300 μL of the mixture of PBS and FBS to resuspend the cells, then add 700 μL of pre-cooled 100% ethanol to each group, and rotate at 4°C overnight (16-18 hours) for ...
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