The use and method of the compound of fasudil and the pharmaceutical composition thereof
A technology of fasudil and its compounds, applied in drug combination, drug delivery, medical preparations containing active ingredients, etc., can solve the problem of not finding adult neural stem cells, and achieve the effect of protecting nerve function
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Embodiment 1
[0050] Example 1: Fasudil promotes the proliferation of brain neural stem cells in vitro
[0051] 1. Experimental cells and their preparation
[0052] Pregnant Kunming (KM) mice (purchased from Shanghai Slack Experimental Animal Co., Ltd.) at 17-18 days of pregnancy were killed by dislocation of the neck, and the skin was disinfected with 75% alcohol. The fetal mice were stripped under aseptic conditions and placed in ice-cold DMEM medium (purchased from GIBCO, USA). Carefully separate fetal mouse brain tissue, peel off meninges and blood vessels, remove cerebellum, brainstem and basal ganglia, take out hippocampal tissue, and place it in ice-cold DMEM by blowing gently to form a single-cell suspension. The cell suspension was collected and centrifuged at 1000 rpm for 5 minutes at 4°C. The supernatant was discarded and resuspended with freshly prepared neuron culture medium (purchased from GIBCO, USA). Count the cells and calculate the density of the cells in the cell sus...
Embodiment 2
[0063] Example 2: Fasudil promotes brain neural stem cell proliferation test in vivo
[0064] 1. Test animals
[0065] KM mice (purchased from Shanghai Slack Experimental Animal Co., Ltd.). After the animals arrived in the laboratory, they were fed with clean grade, free to eat and drink, and rested for 5-7 days.
[0066] 2. Mouse model of hypoxia
[0067] Divide 32 KM mice into 4 groups, 8 in each group, and hypoxia in hypoxic system (8% oxygen) for 2 hours, 4 hours, 6 hours, 8 hours. A hypoxia meter was placed in the hypoxia box to dynamically detect the oxygen concentration in the hypoxia box to ensure the controllability of the degree of hypoxia in the mice. The box can accommodate 30-40 mice, and 32 mice can be placed in one time to ensure the comparability of hypoxic conditions. After 72 hours of reoxygenation, the brain was perfused with normal saline, and the brain was quickly frozen in liquid nitrogen and stored at -80°C. After immunoblotting and immunohistochem...
Embodiment 3
[0080] Embodiment 3: the protective effect of fasudil on neurons
[0081] 1. Test cells
[0082] The test cells of this example were prepared by the method described in the test cells and their preparation in Example 1.
[0083] 2. Glucose and hypoxia injury model
[0084] The glucose and hypoxia injury model of this example was prepared by the method described in the glucose and hypoxia injury model in Example 1.
[0085] 3. Administration
[0086] According to the pre-experiment comparing different concentrations of fasudil in Example 1, an intermediate concentration, namely 10 μg / ml, was selected in this example. All the other methods are exactly the same as the administration method of Example 1.
[0087] 4. Controlled trials
[0088] After the test model was determined, the same volume of PBS was added as the control of fasudil under the same experimental conditions.
[0089] 5. Cell LDH Release Rate Determination
[0090] In order to determine the damage of neur...
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