Application of lobeline in preparing medicament for treating apoptosis of the nerve cell
A neuron and lobeline technology, applied in the field of biomedicine, can solve problems such as the central nervous system of lobeline that have not been reported
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Embodiment 1
[0058] Example 1 The effect of lobeline on the uptake of dopamine by D8 cells
[0059] 1. Establishment of in vitro screening model
[0060] Establishment of activity-screening cell lines targeting dopamine neurotransporter (DAT). By cloning the full-length coding sequence of rat DAT (GeneBank accession number: GI31000978) into the multiple cloning site of the pCDNA3 vector (Invitrogen, USA), transfection into Chinese hamster ovary cells (CHO) by electroporation, 48 hours Then cultured in 1640 medium containing G418. After 10 days, all the cells in the control group died, while many cell clones formed in the experimental group. After the clones were picked and cultured for one week, when the cells covered the bottom of the well, the medium was sucked off, and digested with trypsin as above. Cells in each well were seeded in corresponding wells of two 96-well plates. After confluency, one of the plates was used for isotopic flux measurements. The cells in the wells with hi...
Embodiment 2
[0064] Example 2 Lobeline inhibits the uptake of dopamine by rat striatal synaptosomes
[0065] Preparation of synaptosomes: Adult male SD rats were killed by decapitation and the striatum was isolated and rinsed with ice-cold 0.32M sucrose (dissolved in 1M phosphate buffer). Homogenize the solution with a glass homogenizer at 4°C for 10-12°C, centrifuge the homogenate at 1100g for 10 minutes at 4°C, and take the supernatant and centrifuge at 10,000g for 20 minutes at 4°C. The pellet was resuspended in artificial cerebrospinal fluid (103mM NaCl, 1mM CaCl2, 1mM KH2PO4, 25mM HEPES, 1mM MgCl2, 27mM NaHCO3, 5.4mM glucose, pH7.4, 1mM ascorbic acid, 0.01mM pargyline) for later use.
[0066] When experimenting with synaptosomes, different concentrations of lobeline hydrochloride and other compounds were first mixed with the same amount of synaptosomes for 5 minutes, then added with a final concentration of 0.1 μM tritium-labeled dopamine, placed at 37 ° C for 10 minutes, and passed t...
Embodiment 3
[0068] Example 3 The effect of lobeline on the reverse transport ability of DAT
[0069] D8 cells were cultured in a 48-well plate (Costar) until the plate was confluent (approximately 60,000 cells per well). Discard the culture medium, wash once with PBS, suck off the PBS solution, add 90ul HBS (10mM Hepes, 100mM NaCl, pH8.0) to each well, 10ul 3 H-DA (Amersham Pharmacia Biotech), 100 μM vitamin C and 100 μM parjiline, incubated at 25°C for 10 minutes; washed 3 times with PBS, added 90ul HBS to each well, 10ul drugs of different concentrations, incubated at 25°C for 20 minutes, pipetted The supernatant of each well was added to 1.2ml of scintillation fluid, and put into a liquid scintillation counter (Beckman LS 5000TA) to detect the content of the isotope (DMP value) to measure the effect of the drug on the transport activity of the dopamine transporter .
[0070] RESULTS: Lobeline did not significantly affect DAT-mediated dopamine reverse transport at the dose used to inh...
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