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A kind of exosome containing mir-204 and its preparation method and application

A 1.mir-204, exosome technology, applied in the field of biomedicine and molecular biology, can solve the problems of restriction, immune response virus reactivation toxicity, difficulty in obtaining MSC cells, etc., achieve stable source, improve symptoms and signs, Resolving the effect of unstable transfection efficiency

Active Publication Date: 2022-02-18
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the outstanding problems currently exist include: difficulty in obtaining MSC cells; difficulty in culturing; unstable passage, etc., making it difficult to provide a stable source of exosomes, which restricts the progress of this achievement into clinical practice
However, viral vectors still pose a threat to the safety of the human genome, and a series of safety issues caused by possible immune responses, hidden dangers of integration into the genome, and the possibility of virus reactivation have been controversial.

Method used

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  • A kind of exosome containing mir-204 and its preparation method and application
  • A kind of exosome containing mir-204 and its preparation method and application
  • A kind of exosome containing mir-204 and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Preparation of exosomes loaded with miR-204

[0048] A kind of exosome loaded with miR-204, its preparation method comprises the following steps:

[0049](1) Synthesize the double-stranded oligonucleotide sequence of the miR-204 precursor, clone it into the pHBLV plasmid to construct the expression vector, and transfect it into 239T cells, collect the virus supernatant 72 hours after transfection, and filter it with a 0.45 μm filter , centrifuged at 72000×g for 120min to obtain concentrated lentivirus; transfect L929 cells with the prepared virus;

[0050] (2) Culture and expansion and drug screening with puromycin, and the L929 cell line with stable and high expression of miR-204 was screened out by GFP fluorescence flow cytometry.

[0051] (3) Collect the cell culture medium obtained in step (2), and perform differential centrifugation and extraction of exosomes by ultrahigh-speed centrifugation to obtain L929-exo loaded with miR-204.

[0052] The experime...

Embodiment 2

[0053] Example 2 Preparation of mouse dry eye model and intervention with exosomes

[0054] (1) The 6-8 week old C57 mice were selected, and the slit lamp examination showed no abnormalities, and no abnormalities were found in the tear secretion and corneal fluorescein staining. Divided into 2 groups by random grouping method, 15 rats in each group;

[0055] (2) Two groups of mice were given eyedrops in both eyes respectively, in which group 1 was given 0.2% benzalkonium chloride + 0.2ug / uL L929-exo loaded with miR-204 (prepared in Example 1), and group 2 was given 0.2% benzalkonium chloride + 0.2ug / uLMSC-exo, 5ul / eye each time, for 7 consecutive days.

Embodiment 3

[0056] Example 3 dry eye index detection

[0057] After 7 days of eye drops treatment, on the 8th day, the two groups of mice were intraperitoneally injected with 4.3% chloral hydrate, the injection dose was 10ul / g, to anesthetize the mice, and the measurement of tear secretion after dry eye induction, the anterior segment Photographs, tear film break-up time and corneal epithelial fluorescein staining scores.

[0058] (1) Detection of tear secretion: Place phenol red cotton thread in the middle and outer 1 / 3 of the conjunctival sac of the lower eyelid of the mouse with ophthalmic microtweezers, take out the reading after 1 min, measure and record the red part of the phenol red cotton thread in millimeter units. Each eye was tested twice according to the above method, and the final result was the average of the two measurement results.

[0059] (2) Tear film breakup time: drop 1 μl of 1% liquid sodium fluorescein into the conjunctival sac of mice, and after 3 times of assiste...

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Abstract

The present invention provides an exosome loaded with miR-204, which can significantly improve the symptoms and signs of dry eye disease. By highly expressing miR-204 in L929 cells, the present invention obtains exosomes loaded with miR-204 that are safe, high-yield, and stable in source, which solves many limitations such as difficult cultivation and poor stability of MSC-derived exosomes in practical applications, At the same time, it solves the problems of unstable transfection efficiency of liposome transfection, large differences between different cells, high toxicity of liposome transfection reagents, short action time, and poor stability, and promotes clinical transformation.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to an exosome containing miR-204 and its preparation method and application. Background technique [0002] Dry eye, also known as keratoconjunctival sicca syndrome (keratoconjunctivitis sicca), refers to the abnormality of tear quality or quantity caused by any reason, or abnormal dynamics leading to a decrease in tear film stability, accompanied by eye discomfort and (or) A general term for various diseases characterized by ocular surface tissue damage. There are many causes of dry eye and it affects a wide range of people. At present, the treatment effect is limited, which greatly affects the life and work of patients and brings a heavy burden to the society. [0003] Exosomes (exosomes) are a type of nano-scale membranous vesicles secreted by cells that carry cytoplasmic components, which contain various biologically active substances such as...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775C12N15/867C12N5/10A61K31/7105A61K48/00A61P27/02
CPCC12N5/0662C12N15/86C12N5/0656C12N15/1138A61K31/7105A61P27/02C12N2510/00C12N2740/15043C12N2310/141
Inventor 柳夏林何嫦周恬
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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