Cx43-mediated arteannuin B combined with cisplatin against lung cancer effect and related mechanism
An artemisinin and anti-lung cancer technology, applied in the field of medicine, can solve problems such as intercellular communication barriers and GJ dysfunction
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Embodiment 1
[0019] The effect of artemisinin B on gap junction
[0020] (1) Parachute assay
[0021] 1) Cell inoculation: Take the cells in the logarithmic growth phase, digest and resuspend them in medium for counting, and adjust the cell density to 1.5×10 5 Cell / mL is then inoculated in a 12-well plate, placed at 37℃, 5% CO 2 Incubate in an incubator for 48 hours for subsequent experiments.
[0022] 2) Preparation of loading solution: add 12.5 μL calcein-AM (1 mg / mL) to 487.5 μL serum-free medium by pipetting and mixing, so that the final concentration of calcein-AM is 25 μM.
[0023] 3) Preparation of "donor cells": select 1 well of cells as "donor cells", discard the supernatant, wash once with PBS, and add 500 μL of the above-mentioned prepared load solution. 12-well plate placed at 37℃, 5% CO 2 Continue to incubate for 30 min in the incubator. Aspirate and discard the loading solution, wash with PBS 3 times, 5 min each time. Trypsin digestion of the "donor cell" wells, centrifugation, res...
Embodiment 2
[0073] Study on the combined effect of artemisinin B and DDP in vitro
[0074] (1) MTT experiment
[0075] 1) Cell inoculation: discard the old culture medium in the culture flask, wash once with PBS, add trypsin digestion and centrifuge, centrifuge at 1000 rpm for 5 min, discard the supernatant, add complete medium to resuspend, and count on a hemocytometer. Adjust the cell concentration to 3×10 4 cell / mL for plating, add 100μL of cell suspension to each well of a 96-well plate, set a blank control group, and place it at 37℃, 5% CO 2 Cultivate in a cell incubator for 24 hours to make the cells grow adherently.
[0076] 2) Dosing treatment: After 24 hours, discard the supernatant, add the prepared liquids of different concentrations in the corresponding 96-well plate area, and add an equal volume of complete medium to the negative control group. The 96-well plate was placed in a cell incubator and incubated for 72 hours.
[0077] 3) Color reaction: Add 20μL / well of 5mg / mL MTT solutio...
Embodiment 3
[0091] Study on the combined effect of artemisinin B and DDP in vivo
[0092] 1) Establishment of tumor model: trypsinize A549 cells in logarithmic growth phase and collect them in a 15mL centrifuge tube; centrifuge at 1000rpm for 10min, discard the supernatant, and save the cell pellet; wash once with PBS, centrifuge at 1000rpm for 10min, and discard Clear, add PBS solution to resuspend, gently pipette to mix into a single cell suspension; use a hemocytometer to count, adjust the cell suspension concentration to 5×10 6 cell / mL; disinfect the right axilla of nude mice with 75% alcohol in the ultra-clean platform, and inject 0.2mL cell suspension per mouse subcutaneously. After the nude mice are inoculated with tumor cells, observe the tumor formation every day, weigh and measure the tumor volume every other day, when the tumor volume reaches 100mm 3 At that time, randomized administration.
[0093] 2) Animal grouping and administration:
[0094]
[0095] Observe the changes of the n...
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