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Nicotinic acetylcholine receptor targeted circDYM over-expressed exosome, and preparation method and application thereof

A nicotine acetylcholine and targeted technology, which is applied in the field of medicine, can solve the problems of slow onset of depression drugs, complex conditions, and many adverse reactions, and achieve the effect of reducing depression-like behaviors

Pending Publication Date: 2020-06-19
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the onset of depression drugs is slow, and there are many adverse reactions. In addition, the causes of depression are diverse and the condition is complicated. Some patients with depression still do not respond to the treatment drugs, and even develop drug resistance.

Method used

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  • Nicotinic acetylcholine receptor targeted circDYM over-expressed exosome, and preparation method and application thereof
  • Nicotinic acetylcholine receptor targeted circDYM over-expressed exosome, and preparation method and application thereof
  • Nicotinic acetylcholine receptor targeted circDYM over-expressed exosome, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of nicotinic acetylcholine receptor-targeted overexpressed circDYM exosomes

[0034] A method for preparing nicotinic acetylcholine receptor-targeted overexpressed circDYM exosomes, comprising the following steps:

[0035] (1) HEK-293T cells were cultured using fetal bovine serum and DMEM high-glucose medium;

[0036] (2) Adjust HEK-293T cells to 1*10^5-1*10^6, transfect circDYM-GFP-LVs lentivirus according to the infection ratio of 3MOL, and transfect for 24 hours;

[0037] (3) Replace the fresh medium, add 1-10ug plasmid for every 1*10^5-1*10^6 cells, and transfect for 24 hours;

[0038] (4) After 48 hours of transfection, replace the fresh medium, continue to cultivate for 48 hours, and then collect the cell supernatant;

[0039] (5) Purify the obtained exosomes by gradient centrifugation: centrifuge at 300g for 5 minutes to remove floating cells; centrifuge at 3000g for 30 minutes to remove dead cells; centrifuge at 10000g for 60 minutes to r...

Embodiment 2

[0043] Example 2 Electron microscope identification method of overexpressed circDYM exosomes targeting nicotinic acetylcholine receptors

[0044] Prepare 20 ul of overexpressed circDYM exosomes targeting nicotinic acetylcholine receptors, mix thoroughly and drop onto a sample-loading copper grid with a diameter of 2 mm, and let stand at room temperature for 5 minutes. Blot the residual liquid on the edge of the copper grid with filter paper, then place the copper grid upside down on a 30g / L phosphotungstic acid (pH6.8) droplet, and perform negative staining at room temperature for 5 minutes. The copper mesh was dried under incandescent lamp, observed and photographed by transmission electron microscope, the separation and identification results of the overexpressed circDYM exosomes targeting nicotinic acetylcholine receptors are as follows: figure 1 As shown, the overexpressed circDYM exosomes targeting nicotinic acetylcholine receptors are disc-shaped vesicles with a diameter...

Embodiment 3

[0045] Example 3 Identification method of surface marker protein of overexpressed circDYM exosomes targeting nicotinic acetylcholine receptors

[0046]Prepare 15% SDS-PAGE electrophoresis gel, fully lyse the nicotinic acetylcholine receptor-targeted overexpressed circDYM exosomes and target control exosomes, add 1 / 4 volume of 5×SDS loading buffer, Boil for 5 minutes, load 200 μg of the total amount of protein, transfer the protein to PVDF membrane by electrotransfer (350mA, 120min), block with TBS / T containing 50g / L skimmed milk at room temperature for 1h, and mix with HA / Lamp2b / CD63 / TSG101 respectively / GM130 antibody (1:500) react overnight at 4°C, wash the membrane with TBS / 0.5%Tween 20 for 3 times the next day, incubate with HRP-labeled secondary antibody at 37°C for 1h), wash the membrane with TBS / 0.5%Tween20 for 3 After several times, premixed HRP chemiluminescence substrate was added and detected by chemiluminescence gel imaging system. figure 2 The results of Western...

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Abstract

The invention discloses a preparation method for a nicotinic acetylcholine receptor targeted circDYM over-expressed exosome. The preparation method comprises the following steps: culturing HEK 293T cells by using a DMEM high-sugar culture medium; transfecting the HEK 293T cells with a pcDNA GNSM-3-RVG-10-Lamp2b-HA plasmid and a circDYM-GFP-LVs slow virus; collecting a cell supernatant, and sequentially carrying out centrifuging to remove floating cells, dead cells and cell debris through a gradient centrifugation method; and re-suspending an obtained precipitate so as to obtain the nicotinic acetylcholine receptor targeted circDYM over-expressed neural stem cell exosome. The nicotinic acetylcholine receptor targeted circDYM over-expressed exosome prepared by using the preparation method disclosed by the invention can effectively target the brain through a blood brain barrier, significantly reduces depression-like behaviors of a depression model mouse, has an anti-depression effect andis expected to be developed into a potential drug for treating depression.

Description

technical field [0001] The invention belongs to medical technology, and specifically relates to a nicotinic acetylcholine receptor-targeted exosome overexpressing circDYM, a preparation method and application thereof. Background technique [0002] Depression is a common affective disorder syndrome characterized by low mood. The main clinical symptoms of depression include low mood, slow thinking, decreased willpower, cognitive impairment and physical symptoms. Depression has the characteristics of high recurrence rate, high disability rate and high suicide rate, which brings a heavy burden to patients, families and society, and has become a serious social problem. [0003] For a long time, there has been a lack of ideal drugs for the treatment of depression in clinic. The traditional tricyclic and tetracyclic antidepressants and monoamine oxidase inhibitors have been significantly reduced due to serious adverse reactions, and have been withdrawn from the scope of clinical ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N15/113A61K31/7105A61P25/24
CPCC12N5/0623C12N15/86C12N15/113A61K31/7105A61P25/24C12N2510/02C12N2740/15043
Inventor 姚红红俞晓毓沈灵居敏姿叶清清唐天慈王宇
Owner SOUTHEAST UNIV
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