A kind of pharmaceutical composition and its application
A composition and drug technology, applied in the direction of drug combination, drug delivery, pharmaceutical formulation, etc., can solve the problems of lack of space and tissue specificity, neuron degeneration, death, etc., and achieve the effect of long duration of drug action and low toxicity
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Embodiment 1
[0060] (1) constructing an adeno-associated virus carrying a chemical genetic gene;
[0061] (1') Plasmid construction: construct the chemical genetic gene, red fluorescent gene and nerve cell-specific promoter on the pAAV plasmid to obtain pAAV-syn DIO-hM4Di-mCherry, the schematic diagram is as follows figure 1 shown;
[0062] (2') Transfection of cells: the plasmid obtained in step (1') and the viral packaging plasmid pHelper and pAAV-RC9 of the adeno-associated virus were co-transfected into 293FT cells, and the concentration of the transfection reagent was 5-8 mg / L per 200,000 cells of liposomes;
[0063] (3') Harvesting virus: change the medium 24 hours after transfection, the culture medium after the medium change is DMEM containing 5mM sodium pyruvate, perform ultracentrifugation to rupture the membrane 16 hours after the fluid change, the centrifugal speed is 5000g, and suspend the membrane after the membrane rupture The liquid was passed through a 20% sucrose filter...
Embodiment 2
[0068] (1) constructing an adeno-associated virus carrying a chemical genetic gene;
[0069] (1') Plasmid construction: the chemical genetic gene, red fluorescent gene and nerve cell-specific promoter were constructed on the pAAV plasmid to obtain pAAV-syn DIO-hM4Di-mCherry;
[0070] (2') Transfection of cells: the plasmid obtained in step (1') and the viral packaging plasmid pHelper and pAAV-RC9 of the adeno-associated virus were co-transfected into 293FT cells, and the concentration of the transfection reagent was 5 mg / L of lipid per 200,000 cells. plastid;
[0071] (3') Harvesting virus: change the medium 20 hours after transfection, the culture medium after the change is DMEM containing 4mM sodium pyruvate, and perform ultracentrifugation to rupture the membrane 12 hours after the fluid change, the centrifugal speed is 4000g, and suspend the membrane after the membrane is ruptured. The liquid was passed through a 20% sucrose filter column, and the supernatant was collecte...
Embodiment 3
[0076] (1) constructing an adeno-associated virus carrying a chemical genetic gene;
[0077] (1') Plasmid construction: the chemical genetic gene, red fluorescent gene and nerve cell-specific promoter were constructed on the pAAV plasmid to obtain pAAV-syn DIO-hM4Di-mCherry;
[0078](2') Transfection of cells: the plasmid obtained in step (1') and the viral packaging plasmid pHelper and pAAV-RC9 of the adeno-associated virus were co-transfected into 293FT cells, and the concentration of the transfection reagent was 8 mg / L of lipid per 200,000 cells. plastid;
[0079] (3') Harvesting virus: change the medium 30 hours after transfection, the culture medium after the medium change is DMEM containing 8mM sodium pyruvate, perform ultracentrifugation to rupture the membrane 20 hours after the fluid change, the centrifugal speed is 6000g, and suspend the membrane after the membrane rupture The solution was passed through a 20% sucrose filter column, and the supernatant was collected...
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