A kind of composition with antitumor effect and application thereof
A technology of anti-tumor effects and anti-tumor drugs, applied in the field of compositions with anti-tumor effects
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Embodiment 1
[0043] Determination of mitochondrial membrane potential
[0044] Cell Treatment: Lymphoma cells (Raji) were treated with 1 × 10 6 The density of cells / well was seeded into 12-well plates, and after overnight culture, the experimental group was stimulated with 10 mM metformin solution for 18 h. Cells were collected and stained with JC-1 (Biyuntian, C2006), and mitochondrial membrane potential was detected by flow cytometry.
[0045] The experimental results are as figure 1 The analysis showed that the mitochondrial membrane potential was detected by flow cytometer JC-1. JC-1 was an ideal fluorescent probe for the detection of mitochondrial membrane potential, which could detect cells, tissues or purified mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form aggregates (JC-1 red(aggregates)), which manifests as red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggr...
Embodiment 2
[0048] Assays for mitophagy
[0049] Cell treatment: cervical cancer cell lines (Hela cells) stably expressing mt-mKeima protein were treated at 2 × 10 5 Cells / well were seeded in 12-well plates, and after overnight culture, 10 mM metformin solution was added to continue treatment for 18 h, cells were collected, and the red and green fluorescence intensity of mt-mKemia protein was detected by flow cytometry.
[0050] The experimental results are as figure 2 , where mt-mKeima is a mitochondrial-localized protein that is sensitive to pH, mt-mKemia protein emits green fluorescence when it is in the neutral environment of mitochondria (mt-mKeima at neutral pH), and when mt-mKemia is at the end of mitophagy, mt-mKemia The protein is in an acidic environment (mt-mKemia at acidic pH) as the mitochondria enters the lysosome, and the protein turns to emit red fluorescence. Therefore, by detecting the changes of red and green fluorescence of mt-mKeima protein, the situation of mitoch...
Embodiment 3
[0054] Detection of the effect of Metformin (Met) and its composition on tumor cell apoptosis by flow cytometry
[0055] 1. Cell Processing
[0056] 1) Lymphoma cells (Raji) or cervical cancer cell line Hela were treated with 1 × 10 6 cells / well and 2 x 10 5 The density of cells / well was seeded in 12-well plates;
[0057] 2) After overnight incubation, add metformin solution (Met, 50μm-30mM) or composition (metformin / cyclosporine A solution, 10mM / 5μM; metformin / Mdivi-1 solution, 10mM / 10μM; or metformin / chloroquine solution, 10mM / 50μM;) After culturing for 36-48h, cell death was detected.
[0058]Among them, in the metformin / cyclosporine A composition solution, the added concentrations of the two are 10 mM and 5 μM, respectively; in the metformin / Mdivi-1 composition solution, the added concentrations of the two are 10 mM and 10 μM, respectively; The metformin / chloroquine composition In the solution, the addition concentrations of the two were 10 mM and 50 μM, respectively....
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