miRNA regulation of platelet gelsolin and its application in screening antiplatelet drugs
An anti-platelet, drug candidate technology, applied in the direction of drug combination, microbial determination/inspection, medical preparations containing active ingredients, etc., can solve problems such as inability to express the same genus and prolonged bleeding time
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0024] Example 1: Culture and Induced Maturation of Human MEG-01 Megakaryocytes
[0025] 1. Experimental grouping: 1.MEG-01 group: MEG-01 cells were cultured with RMPI-1640+10%FBS+4mmol / L L-glutamine culture medium; 2.MEG-01 / TPA group: MEG-01 cells were cultured with The culture solution of RMPI-1640+10%FBS+4mmol / L L-glutamine was kept at 37℃, 5%CO 2 Conditioned suspension culture, the initial concentration of each well of 6-well plate was 5×10 5 cell. The final concentration of TPA was 10nM to induce the differentiation of MEG-01 cells for 3 days. Cell images (200×) were collected with an inverted Olympus microscope.
[0026] 2. FACS surface staining
[0027] Aspirate 500 μL of MEG-01 cells induced for 3 days and the cells of the blank control group, first wash the two groups of cells with PBS (staining solution) containing 5% serum for 1-2 times, centrifuge at 500 g for 5 min, and adjust the cell concentration to 1×10 6 / mL. Add 2 μL anti-human Fc blocking antibody pe...
Embodiment 2
[0032] Example 2: Research on the expression levels of miRNA and Gelsolin protein in MEG-01 and MEG-01 / TPA cells
[0033] 1. Detection of miRNA expression level
[0034] Take 1×10 7 Quantities of MEGA-01 and MEGA-01 / TPA cells in the exponential growth phase were used to extract the RNA of these two cells by Trizol.
[0035] For the reverse transcription of miRNA, each miRNA needs to use its specific reverse transcription primer, as shown in Table 1. The reverse transcription method is as follows: Take a certain amount of RNA (0.1ng-5μg), add 1μL oligo miRNA-specific reverse primer to an RNase free tube, and add a certain amount of DEPC water to make the final volume 12μL. Place the tube in warm water at 65°C for 5 minutes. After the incubation period, quickly place the tubes on ice. Add 4μL 5×reaction buffer, 2μL 10mM dNTP mix, 1μL RNase inhibitor and 1μL reverse transcriptase to the tube, mix gently, place in a 42℃ water bath for 1 hour, then place in a 70℃ water bath in...
Embodiment 3
[0060] Example 3 Verification that miR-124 directly regulates the expression of Gelsolin
[0061] 1. Construction of 3'-UTR-containing vectors and mutant 3'-UTR-containing vectors
[0062] (1) Enzyme cut empty plasmid
[0063] 2 μg pmirGLO empty plasmid was double digested with PmeI and XbaI, the enzyme digestion system is as follows:
[0064] Table 4 enzyme digestion reaction system
[0065]
[0066]
[0067] Digest overnight at 37°C, then add 4 μL of 5M NaCl and 100 μL of ice-free ethanol to the EP tube after digestion, and mix well. Place in -80°C refrigerator for 30 minutes, centrifuge at 12,000 rpm for 15 minutes, quickly wash twice with 75% ice-free ethanol, and blow dry. Finally with 10 μL ddH 2 O dissolved.
[0068] (2) Synthesize the target DNA fragment
[0069] The following fragments were synthesized (sequences in bold in italics are miRNA binding sequences or binding mutation sequences, and the underlined sequences are NotI enzyme-cut recognition sequen...
PUM
Login to view more Abstract
Description
Claims
Application Information
Login to view more - R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap