Nano gold mir-885-5p conjugate and its preparation method and application
A technology of conjugates and gold nanoparticles, applied in the field of liver cancer, to achieve significant growth, good stability, and good pharmacological properties
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Embodiment 1
[0045] Example 1 The preparation and identification of nano-gold particles with a particle size of about 13nm
[0046] 1) Preparation of aqua regia: prepare according to the volume ratio of concentrated hydrochloric acid and concentrated nitric acid as 1:3, that is, measure 90ml of concentrated hydrochloric acid with a measuring cylinder and put it in a 500ml clean beaker, then measure 30ml of concentrated nitric acid, and slowly move along the glass rod under stirring Add it into concentrated hydrochloric acid, stir it evenly, and it turns yellowish brown (nitrosyl chloride is produced, and has a chlorine smell, all preparation and use must be carried out in a fume hood), seal the mouth of the beaker with a parafilm, and mark it as king with a marker pen Water back up.
[0047] 2) 10ml 25mM HAuCl 4 Solution preparation: weigh 0.0995g of HAuCl 4 .3H 2 Dissolve O in 10ml of ultrapure water, mix well, wrap in foil paper and store in dark at 4°C for later use.
[0048] 3) 100...
Embodiment 2
[0063] Example 2 The coupling method of nano-gold and miR-885-5p, the steps are as follows: 1) Preparation of 0.1% DEPC: Take 0.446ml of 1.12g / ml DEPC into 500ml of ultrapure water, place it in an ultrasonic cleaner for ultrasonic treatment for 2 hours until there are no oil droplets;
[0064] 2) Preparation of 10mMPBS: Accurately weigh NaCl8g, KCl0.2g, KH2PO40.2g, Na2HPO4.12H2O2.9g with an analytical balance, dissolve in 100ml enzyme-free sterile water, dissolve fully after ultrasonication, and then use enzyme-free sterile water to determine Capacity up to 1L;
[0065] 3) 0.18MPBS preparation: according to the PBS preparation plan, first prepare 0.2M, pH8.0 PBS with enzyme-free sterile water, and then dilute to 0.18M according to the proportion;
[0066] 4) Design and chemically synthesize miR-885-5pmimics, and connect Cy5 fluorescent dye to the 5' end of its sense strand, and connect six-carbon alkylthiol to the 5' end of its complementary strand. The sequence is as follows...
Embodiment 3
[0078] Example 3: Detection of miR-885-5p-AuNPs directly entering liver cancer cells and significantly upregulating miR-885-5p:
[0079] 1) Cell culture: Liver cancer cell lines Huh7, Hep3B, and HepG2 cells were placed in Dulbecco’s modified Eagle medium (DMEM) medium with 10% fetal bovine serum, 37°C, 5% CO 2and cultured in a cell incubator with saturated humidity; when the cells grow to about 90% confluent, discard the old culture medium in the culture bottle and wash once with sterilized PBS. Add 0.25% trypsin digestion solution for digestion, and lay the culture bottle flat so that the digestion solution covers the bottom of the bottle. Observe under an inverted microscope and find that the cytoplasm of the cells has retracted and the gap has increased, immediately discard the digestion solution, add 6-8ml of fresh DMEM culture solution containing 10% fetal bovine serum to stop the digestion, and repeat along the edge of the culture bottle Pipette adherent cells to form a...
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