Neuroprotective composition, preparation process thereof and medical uses thereof
A technology of neuroprotection and composition, which is applied in the direction of biochemical equipment and methods, drug combination, nervous system diseases, etc., and can solve problems such as inefficiency
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Embodiment 1
[0052] Example 1: Isolation and culture of dental pulp stem cells (DPSC)
[0053] The upper teeth were harvested intact from 3-week-old male Wistar rats, and then the tissue was extracted using a syringe needle and transferred to a 25 cm 2 flask (Corning, Inc., New York, USA). The pulp was washed twice with phosphate buffered saline and treated with type II collagenase (50-100 U / ml) for 1 hour. The tissue was then centrifuged at 200 rpm for 4 minutes to obtain cell pellets, which were subsequently washed twice with phosphate buffered saline. Supplemented with 10% FBS ( The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) nutrient mixture F12 (DMEM / F12; Life Technologies, Karlsruhe, Germany) in Grand Island, New York, USA) for several days until the cell number reached more than 1 × 10 6 indivual. Cells were trypsinized to detach and washed twice with phosphate-buffered saline, then resuspended in 0.5 ml phosphate-buffered saline. Deplete the cell suspen...
Embodiment 2
[0054] Example 2: Preparation of biologically active media fractions
[0055] Conditioned medium was produced as follows: to 150 cm 2 80% confluent MSCs of 2-5 passages in cell culture flasks (Corning, Inc. New York, USA) were added with 20 mL / bottle of serum-free DMEM / F12 (Life Technologies, Karlsruhe, Germany), and then cultivated for 72 hours. The DMEM / F12 was supplemented with 10 ml of Hank's balanced salt solution (HBSS; Life Technologies, Carlsbad, CA, USA) and 300 μl of penicillin-streptomycin (1%). For the in vitro and in vivo experiments described below, a Tangential Flow Filtration (TFF) system (Millipore Co., St. Charles, MO, USA) equipped with a 5 kDa cut-off membrane (Millipore Co., Billerica, MA, USA) was used Conditioned medium was further concentrated according to the manufacturer's instructions, thereby obtaining medium fractions with nominal molecular weights equal to and less than 5 kDa.
Embodiment 3
[0056] Embodiment 3: rat SAH model
[0057]Male Wistar rats (250 to 300 g) were used in this example. All surgical procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and animal experiments were approved by the local Animal Care and Use Committee. The anesthetic was 2.5% isoflurane with 70% nitrous oxide and 27.5% oxygen. Administration was administered through a tracheostomy tube to ensure deep sedation, which was confirmed by the absence of pain reflexes in the hindlimb and forelimb as well as absence of the corneal reflex. Maintain normal, effortless breathing throughout the procedure. Animals were placed on a heat blanket, temperature was monitored with a rectal probe, and body temperature was maintained at 37°C. Monitor blood pressure and maintain it at 100-120mmHg.
[0058] Animals were randomly divided into three groups, namely: SAH group, in which 0.3 ml of fresh autologous blood was inj...
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