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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

Active Publication Date: 2022-05-13
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there is no report on the application of spherical microRNA in the treatment of gastric cancer

Method used

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  • A kind of spherical microrna and its preparation method and application
  • A kind of spherical microrna and its preparation method and application
  • A kind of spherical microrna and its preparation method and application

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Experimental program
Comparison scheme
Effect test

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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Abstract

The present invention relates to the technical field of biomedicine, and specifically provides a spherical microRNA and its preparation method and application. The spherical microRNA is constructed by dissolving potassium cyanide after coupling non-coding single-stranded nucleotides with gold nanoparticles; The non-coding single-stranded nucleotide contains two stem-loop structures, and the 5' end of the antisense strand contains a sulfhydryl modification, which can be combined with gold nanoparticles through a gold-sulfur bond. The prepared spherical microRNA has an inhibitory effect on gastric cancer cells, and can be applied to the treatment of esophageal cancer, pancreatic cancer, colon cancer, liver cancer, breast cancer, endometrial cancer and other tumors.

Description

technical field [0001] The invention relates to the technical field of biomedicine, specifically provides a spherical microRNA and its preparation method and application, and further relates to a nano-gold miR-193b microsphere and its preparation method and application. Background technique [0002] Gastric cancer is one of the leading causes of cancer-related deaths worldwide. Most of the cases come from Asia. It is the second most common malignant tumor in China and poses a huge threat to people's lives and health. Early diagnosis and treatment play an important role in early gastric cancer, which can have a longer quality of life and survival period. However, patients with advanced stage often metastasize and spread. Even after surgery and chemotherapy, radiotherapy, the prognosis is still poor. However, the routine examination of gastroscopy in our country is not popularized, and the discovery of gastric cancer is already in the advanced stage, which brings great diffic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/69A61K31/7088A61K47/52A61P35/00A61P35/04C07H1/00C07H21/02C12N15/113
CPCA61K47/6923A61K31/7088A61P35/00A61P35/04C12N15/1137C07H21/02C07H1/00C12N2310/531C12N2310/353C12Y304/21073
Inventor 周海燕胡永斌王宽松胡忠良肖德胜周建华
Owner CENT SOUTH UNIV
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