Use of berberine in the preparation of hedgehong signaling pathway inhibitors
A signaling pathway, berberine technology, applied in the field of berberine preparation of Hedgehong signaling pathway inhibitors, can solve problems such as tumors that have not yet been seen, and achieve the effects of being beneficial to clinical application, inhibiting tumor growth, and low price
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Embodiment 1
[0024] Example 1: The specific inhibitory effect of BBR on Hedgehog signaling pathway in vitro
[0025] 1. The inhibitory effect of BBR on the Hedgehog signaling pathway in vitro:
[0026] 1): Gli-luciferase dual luciferase reporter gene test
[0027] NIH3T3 (American Type Culture Collection) cells were treated with Dulbecco's modified Eagle's medium (DMEM) (GIBCO) containing 10% (v / v) newborn bovine serum (GIBCO), penicillin / streptomycin (Hyclone), 37°C, 5% CO2 Constant temperature culture. Inoculate 3×10 4 cells in a 48-well plate. After 24 hours, Gli-dependent firefly luciferase reporter and TK-Renillaluciferase reporter vectors were transfected with transfection reagent lipo2000 (Invitrogen). 48 hours after transfection, the cells were treated with medium containing 0.5% newborn bovine serum (GIBCO), SAG (100 nM) or Shh-N conditioned medium (ShhN-CM) and BBR prepared in different concentrations. The reaction was terminated after 36 h. Luciferase activity was detected...
Embodiment 2
[0042] Example 2: In vivo anti-tumor effect experiment of BBR on Hh-dependent medulloblastoma
[0043] 1. BBR inhibits the expression of Gli1 mRNA in medulloblastoma cells
[0044] Select a well-grown Ptch + / - P53 - / - Tumor cells (MB cells), Neurobasal-A Medium (GIBCO), B-27 Supplement (GIBCO), Sodium Pyruvate (GIBCO), L-Glutamine, isolated from spontaneously grown medulloblastoma tissue of C57B / L mice (GIBCO), penicillin / streptomycin (Hyclone), 37 ° C, 5% CO2 constant temperature culture, inoculated 5 × 10 6 After 24 hours, the cells were added with different concentrations of BBR, and the total RNA was extracted with Trizol after 24 hours. The endogenous gene GUSB was used as a reference, and the reverse transcription kit ( RT Master Mix, Takara) reverse transcription, with Premix Ex Taq TM (Tli RNaseH Plus) (Takara) was quantified in real time according to the instructions, using the iCycler iQsystem (Bio-Rad) real-time fluorescent quantitative PCR instrument to quan...
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