A kind of spherical microrna and its preparation method and application
A mir-193b, spherical technology, applied in the field of biomedicine, can solve the problems that there are no reports on the application of spherical microRNA gastric cancer, and achieve good pharmaceutical properties, significantly inhibit growth, and good stability
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Embodiment 1
[0057] The preparation method of spherical microRNA, the steps are as follows:
[0058] 1) Preparation of gold nanoparticles: Add 98 mL of water and 2 mL of 50 mM HAuCl into a three-neck round bottom flask 4 Aqueous solution, heated at 150 ° C and condensed to reflux. Then quickly add 1 mL of sodium citrate aqueous solution with a concentration of 388 mM, continue to reflux for 20 min, stop heating, stir and cool to room temperature, and centrifuge at 16000 rpm for 10 min to collect gold nanoparticles stabilized by sodium citrate;
[0059] 2) Design and chemically synthesize single-stranded miR-193b nucleotide sequence: AACUGGCCCUCAAAGUCCCGCU.
[0060] 3) miR-193 nucleotide pretreatment: Take 8 nmol of miR-193b nucleotide, dissolve in 889 μL ultrapure water, aliquot and store in a -80°C refrigerator in the dark; when using, take an appropriate amount in an enzyme-free tube , add TCEP solution (final concentration 1 mM), then incubate at room temperature for 1 h;
[0061] 4) C...
Embodiment 2
[0065] Detection of spherical microRNAs directly entering gastric cancer cells and upregulating miR-193b:
[0066] 1) Cell culture: RAW264.7 cells were cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO 2 and cultured in a humidified cell culture incubator. When the cells cover about 80-90% of the bottom of the bottle, discard the old culture medium, wash once with PBS, add 5ml of fresh complete medium, blow and scrape the cells along the edge of the culture bottle repeatedly to form a uniform single-cell suspension. solution, the cells were seeded in 6-well plates, the seeding density was about 40%~50%, about 1.2×10 6 cells / well, and the experimental treatment was carried out 1 day after seeding.
[0067] 2) Treatment of each group: set free microRNA group and spherical microRNA group, add free microRNA or spherical microRNA to the 1640 culture medium containing 10% fetal bovine serum so that the final concentration is 50nM. On the day of the exper...
Embodiment 3
[0071] Example 3 Expression levels of uPA mRNA in gastric cancer cells after different treatments.
[0072] 1) Cell culture: gastric cancer cell BGC-823 cells were inoculated in cell culture flasks supplemented with complete medium (DMEM medium with 10% fetal bovine serum), and incubated at 37°C and 5% CO 2 and cultured in a humidified cell culture incubator. When the cells cover about 80-90% of the bottom of the bottle, discard the old culture medium, wash once with PBS, add 0.25% trypsin to digest, and observe under an inverted microscope. It is found that the cells become round and shrink, and the gap increases, discard and digest solution, add 5ml of fresh medium containing complete medium to stop the digestion, gently pipet the adherent cells repeatedly along the edge of the culture flask to form a uniform single-cell suspension, and inoculate the cells in a 6-well plate at a seeding density of about 40% to 50%. , about 1.2×10 6 cells / well.
[0073] 2) Treatment of eac...
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