Pharmaceutical composition having antitumor collaborative synergistic effect
An anti-tumor drug and synergistic technology, which is applied in the direction of anti-tumor drugs, drug combinations, and medical preparations containing active ingredients, etc., can solve the problems of no related reports, no combination of gemcitabine fusion protein, etc., and achieve Obvious anti-tumor synergistic effect, synergistic anti-tumor effect, significant effect
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Embodiment 1
[0039] 《Example 1》Expression of MMP-2 in human colon cancer cells and human pancreatic cancer cells
[0040] Collect colon cancer cell lines HCT-15, HCT-116, HT-29 and pancreatic cancer cell lines BxPC-3, PANC-1, MIA PaCa-2 and other tumor cells in logarithmic growth phase in subculture, after cell lysate , use the BCA kit for protein quantification, each cell lysate sample is prepared according to the same loading amount (30μg in general), and then take an appropriate amount of 5× loading buffer to mix, denature in a boiling water bath for 5 minutes, add 10% SDS- Electrophoresis in PAGE gel. After electrophoresis, the membrane was transferred at a constant voltage of 80V, and the protein was transferred to the PVDF membrane. After blocking overnight with 5% milk, rinse the membrane with TBST 5 times, 5 minutes each time, and incubate overnight at 4°C with rabbit-derived anti-MMP-2 monoclonal antibody (diluted with 1×PBS at 1:1000). After washing with TBST, the membrane was ...
Embodiment 2
[0042] 《Example 2》Fusion protein dFv-LDP and doxycycline inhibit the proliferation of colon cancer HCT-15 cells
[0043]The HCT-15 tumor cells in the logarithmic growth phase were digested, the cells were counted, and spread on a 96-well plate. After 24 hours, different concentrations of drugs were added and the culture continued. After 48 hours, add MTT (blue tetrazolium) and incubate at 37°C for 4 hours, add DMSO (dimethyl sulfoxide) to dissolve the blue-purple particles, and measure the absorbance at 570 nm on a microplate reader. Subtract the background OD value (cell-free well) from the OD value of each test well, set up three parallel wells, and take the OD average value. The survival rate of cells is represented by T / C (%), T is the OD value of the administration group, and C is the OD value of the control group. Cell survival rate T / C (%)=(administration group OD-background OD) / (control group OD-background OD)×100%. Calculate the two-drug interaction coefficient CDI=...
Embodiment 3
[0045] 《Example 3》The fusion protein dFv-LDP and doxycycline inhibit the migration of tumor cells
[0046] Transwell assay technology is a device for simulating cell migration or invasion in vitro. Transfer the Transwell chamber to a new 24-well plate, add 100 μL and 600 μL of serum-free cell culture medium to the small chamber and the outer chamber, respectively, and place it in a 37°C incubator to pre-equilibrate for 1-2 hours. The subcultured tumor cells were digested to obtain cell pellets, and the cells were resuspended in serum-free medium and counted, and the cell density was adjusted to 1×10 6 individual / mL. Discard the balance solution in the small chamber and the outer chamber, take 100 μL of the adjusted cell suspension into the small chamber, and add the cell culture medium containing 20% serum to the outer chamber. The fusion protein dFv-LDP (100 μg / ml) and doxycycline (1 μg / ml) were added to each group respectively, mixed gently, and cultured in a cell cultur...
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