Application of IL-11 monoclonal antibody in inhabiting PI3K/Akt signal channel
A monoclonal antibody, 1. IL-11 technology, applied in the direction of antibodies, antineoplastic drugs, peptides, etc., can solve side effects and other problems, and achieve the effect of reducing toxic and side effects
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Embodiment 1I
[0017] Example 1 The effect of IL-11 on the proliferation of intestinal cancer cells
[0018] 1. Screening of IL-11 concentration
[0019] Dilute the digested HT-29 cell suspension of the 3rd to 5th generation by 10 4 / ml concentration inoculated in 12-well culture plate, 2ml per well, placed at 37°C, 5% CO 2 cultured in an incubator. After 12 hours of adherence, the culture medium was discarded, and 2 ml of DMEM complete medium with a final concentration of (2, 5, 8, 10 ug / ml) IL-11 was added, and a cell control without IL-11 and a repeated control were established at the same time. Continue culturing, add 80ul MTT to the cells at 6h, 12h, 24h, 36h, and 48h, add 150ul dimethyl sulfoxide after continuing to culture for 4h, shake for 10 minutes, and detect OD490.
[0020] 2. The effect of IL-11 on cell proliferation
[0021] cell suspension according to l0 4 / ml concentration inoculated in 12-well culture plate, 2ml per well, placed at 37°C, 5% CO 2 cultured in an incubat...
Embodiment 2I
[0026] The effect of embodiment 2IL-11 on PTEN, AKT, NF-κB in PI3K cell signaling pathway
[0027] Cell suspension by 5xl0 4 Inoculate in 12-well culture plate (with slide), 2ml per hole, at 37°C, 5% CO 2 cultured in an incubator. After 12 hours of attachment, the culture medium was discarded, and the experiment was divided into two groups. One group was added with 2ml of DMEM complete medium with a final concentration of (8ug / ml) IL-11. After 4h, a volume of 10ulIL-11 antibody was added to incubate for 4h. One group only added 2ml of DMEM complete medium with a final concentration of (8ug / ml) IL-11, and a signal inhibitor control group and a repeat group were set up at the same time. Continue to culture, collect cell supernatant from 4h, 6h, 8h, 12h, detect PTEN, AKT with ELISA kit;
Embodiment 3
[0028] Embodiment 3 detects the content of PTEN in the above-mentioned cell supernatant
[0029] 1. Method:
[0030] 1) Using an ELISA kit to detect the content of PTEN in the supernatant of the above cells.
[0031] 1. Adding samples: set blank wells, standard wells, and sample wells to be tested respectively. Add 100ul of sample diluent to the blank well, and add 100ul of standard substance or sample to be tested in the remaining wells respectively. Be careful not to have air bubbles. Cover or cover the target plate and react at 37°C for 120 minutes.
[0032] 2. Discard the liquid, spin dry without washing. Add 100ul of detection solution A working solution to each well (prepared by taking 1ul detection solution A plus 99ul detection diluent A, mix gently, and prepare within one hour before use), 37°C, 60 minutes.
[0033] 3. After incubating for 60 minutes, discard the liquid in the well, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, 350ul / well, and ...
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