Application of Loureirin B in preparation of Kv1.3 channel blocker
A channel blocker, kv1.3 technology, applied in the field of medicine, can solve the problems that no reports of dragon's blood B treating autoimmune diseases have been seen, and the lack of dragon's blood B can achieve the effect of clear ingredients and controllable quality
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Embodiment 1
[0022] Example 1 The patch clamp experiment technique was used to detect the inhibitory effect of dragon blood B on mammalian Kv1.3 channel function.
[0023] Jurkat E6-1T cells (ATCC TIB152) and HEK293T cells (ATCC ACS4500) were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 100 units / mL penicillin, and 100 μg / mL streptomycin, respectively. ) and DMEM medium (Life Technologies, Grand Island, NY, USA), the cells were placed at 37°C and 5% CO 2 cultured in an incubator. The cDNA encoding the mKv1.3 channel protein in the vector pSP64 (gifted by Professor Stephan Grissmer, University of Ulm, Ulm, Germany) was subcloned into pIRES2-EGFP (Clontech, Inc., Mountain View, CA, USA) vector. The constructed clones were analyzed by DNA sequencing to confirm the correctness of their nucleotide sequences. The constructed vector was transfected into HEK293T cells with Lipofectamine 2000 (Invitrogen), and used for electrophysiological exper...
Embodiment 2
[0025] Example 2 Using calcium imaging technology to detect the effect of dragon blood B on free Ca in Jurkat T cells 2+ Inhibitory effect of concentration
[0026] The efflux of intracellular potassium ions caused by the opening of Kv1.3 channel can lead to the repolarization of T cell membrane potential, and the repolarization of T cell membrane potential is to promote the extracellular Ca of Jurkat T cells. 2+ The main electrical potential energy of the influx, thereby increasing the free Ca in Jurkat T cells 2+ concentration.
[0027] sarcoplasmic reticulum Ca 2+ - The ATPase inhibitor Cyclopiazonic acid (CPA) empties the calcium ions in the cellular calcium pool, and then uses Ca 2+ The indicator dye Fura-2 determines the effect of ascarin B on intracellular Ca in Jurkat T cells 2+ The effect of concentration changes.
[0028] When there is no Ca in the extracellular fluid 2+ 10 μM CPA induced Ca in Jurkat T cells 2+ The increase in concentration is small. When 2m...
Embodiment 3
[0030] Example 3 ELISA was used to detect the inhibition of the release of the inflammatory factor IL-2 in Jurktat cells by dragon blood B.
[0031] The opening of the Kv1.3 channel on the Jurkat T cell membrane causes the repolarization of the cell membrane potential, which is the main potential energy to maintain the continuous influx of extracellular calcium ions. Blocking the Kv1.3 channel current on the Jurkat T cell membrane will correspondingly prevent the influx of extracellular calcium ions, thereby reducing the concentration of intracellular free calcium ions. The cytokine IL-2 secreted by Jurkat T cells is affected by the intracellular free calcium concentration, and a decrease in the intracellular free calcium concentration will attenuate the release of the inflammatory cytokine IL-2.
[0032] 10 μg / mL phytohemagglutinin (PHA) was added to the cell culture medium to activate Jurkat T cells for 24 hours, and the concentration of inflammatory cytokine IL-2 released b...
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