WO2021114164A1 - Exosome, preparation method therefor and use thereof - Google Patents

Abstract

Provided is a method for preparing an exosome, which falls within the field of biotechnology. The method comprises the following steps: placing human astrocytes in a medium for culturing; performing circulatory stimulation on the human astrocytes via an ultrasonic apparatus; and continuing culturing the human astrocytes, which have been stimulated by means of ultrasound circulation, then collecting the cell culture supernatant, and performing centrifugal treatment on the cell culture supernatant to obtain the exosome. Also provided are the exosome obtained by means of the above-mentioned method and the use thereof. The above-mentioned preparation method has simple operations, and is low cost, and strongly controllable.

Description

Exosomes and preparation method and application thereof Technical field

The present invention relates to the field of biotechnology, in particular to an application of non-invasive ultrasound treatment of cells in the preparation of exosomes, exosomes and preparation methods and applications thereof.

Background technique

Alzheimer's disease (Alzheimer's disease, AD), also known as Alzheimer’s disease, is a degenerative disease of the central nervous system with an insidious onset and a chronic and progressive course. The disease is also the most common cause of dementia. It accounts for about 60-80% of the total number of dementias. Its main pathological features include senile plaques composed of β-amyloid (β-amyloid, Aβ) and neurofibrillary tangles (NFT) composed of hyperphosphorylated microtubule-associated protein tau. So far, the current treatment drugs for Alzheimer's disease can only relieve part of the symptoms at a specific stage of the disease's development, but cannot slow down or cure Alzheimer's disease. Therefore, the development of treatments for the cause of Alzheimer's disease is an important issue that needs to be solved urgently.

Exosomes are round membranous bodies wrapped in lipid bilayers. They are a type of extracellular vesicles that contain a variety of proteins, RNA, miRNA, growth factors and their receptors. Wait. Exosomes can spread between the central nervous system and peripheral circulation1. A variety of neurodegenerative diseases have abnormal accumulation and aggregation of protein2. The accumulation of excess substances in neuronal endosomes and lysosomes is an important factor that leads to nerve fragility in Alzheimer's disease. Extracellular vesicles (EVs) (exosomes and microvesicles) form an endogenous transport system that participates in the exchange of biomolecules (proteins and RNA) between cells. This makes EVs have great potential for drug delivery and regenerative medicine applications.

The current method for preparing exosomes has a complicated process, poor controllability and high cost.

Summary of the invention

The main technical problem solved by the present invention is to provide an exosomes with simple operation, low cost and strong controllability, and a preparation method and application thereof. In order to solve the above technical problems, a technical solution adopted by the present invention is:

A preparation method of exosomes includes the following steps:

Place human astrocytes in a culture medium for culture;

Use an ultrasound device to stimulate the human astrocytes in circulation;

The human astrocytes stimulated by the ultrasonic cycle were cultured and the cell culture supernatant was collected, and the cell culture supernatant was centrifuged to obtain exosomes.

In one of the embodiments, the cyclic stimulation conditions are: the probe frequency of the ultrasound device is 0.5-5MHz, the amplitude is 50-000mV, the pulse repetition frequency is 50-1000Hz, the duty cycle is 10%-70%, and the ultrasound device The power amplifier amplifies 5%-45%, circulates 20-200k times, the pulse interval is 2μs-2s, and the ultrasonic time is 1 minute to 10 minutes.

In one of the embodiments, the medium is high-sugar DMEM without exosomes.

In one of the embodiments, the human astrocytes stimulated by the ultrasound cycle are cultured for 24-72 hours and then the cell culture supernatant is collected.

In order to solve the above technical problems, a technical solution adopted by the present invention is to provide exosomes obtained by the above preparation method.

In one of the embodiments, the average diameter of the exosomes is about 50 nm to 150 nm.

In order to solve the above technical problems, a technical solution adopted by the present invention is to use the exosomes prepared by the above preparation method in the preparation of drugs for the prevention and/or treatment of Alzheimer's disease.

In order to solve the above technical problems, a technical solution adopted by the present invention is to use the exosomes prepared by the above preparation method to degrade β-amyloid protein.

The beneficial effect of the present invention is that compared with the prior art, the present invention uses medical ultrasound technology to make the central nerve cells and glial cells at the stimulation site different through different intensities, frequencies, pulse repetition frequencies, pulse widths, and durations. Biological effects. Astrocytes are the basis for the stability, defense and regeneration of the central nervous system. The morphology and function of astrocytes can be adjusted by environmental stimuli or drugs. The invention utilizes ultrasonic waves to stimulate human astrocytes, and the preparation method of the separated exosomes is simple in process, convenient in operation, strong in controllability and low in cost. The obtained exosomes can slow down or even reverse the toxicity of β-amyloid (Aβ) to nerve cells, reduce the deposition of Aβ plaques in the brain in mice, and can be used in new drugs for the treatment of Alzheimer's disease.

Description of the drawings

In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the specific embodiments or the description of the prior art. Obviously, the appendix in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work.

Fig. 1 is a transmission electron micrograph of astrocyte exosomes without ultrasound stimulation according to an embodiment;

Figure 2 is a schematic diagram of astrocyte exosomes stimulated by ultrasound;

FIG. 3 is a schematic diagram of a nanoparticle tracking analysis (NTA) result of astrocyte exosomes without ultrasound stimulation according to an embodiment;

4 is a schematic diagram of the results of nanoparticle tracking analysis (NTA) of ultrasound-stimulated astrocyte exosomes according to an embodiment;

Fig. 5 is a proteomic heatmap of astrocyte exosomes that have not undergone ultrasound stimulation and ultrasound-stimulated astrocyte exosomes according to an embodiment;

Fig. 6 is a schematic diagram of an embodiment of the result of the ultrasound-stimulated exosomes cck-8 secreted by astrocytes;

Fig. 7 is a schematic diagram showing the in vivo therapeutic effect of exosomes secreted by ultrasound-stimulated astrocytes on APP/PSI transgenic Alzheimer’s disease mice according to an embodiment, wherein the diagram in A is stained with Thioflavin S The method detects the deposition of Aβ plaque, the green is the plaque, and the blue is the nucleus. In the figure B, the β1-42 antibody immunofluorescence staining detects the distribution of Aβ1-42 in the brain, the green is Aβ, and the blue is the nucleus;

Other diagram explanation: HA-EXO means astrocyte exosomes, US-HA-EXO means ultrasound-stimulated astrocyte exosomes.

Detailed ways

The embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If no specific conditions are indicated in the examples, it shall be carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer.

A preparation method of exosomes includes the following steps:

S110, placing the human astrocytes in a culture medium for culture.

Specifically, the medium is high-sugar DMEM without exosomes. In one embodiment, human astrocytes (HA) cells are cultured in a cell culture flask, and exosomes-free high-sugar DMEM medium is added, and the cells are cultured in a 37° incubator. After 24 hours Stimulate the cell culture dish with an ultrasound device. The total time lasts 5 minutes. After the stimulation, the cell morphology was observed under a microscope, and the cell morphology was good, no difference from unstimulated cells.

S120. Use an ultrasound device to stimulate the human astrocytes in circulation;

Specifically, in one embodiment, the cyclic stimulation conditions are: the probe frequency of the ultrasound device is 0.5-5MHz, the amplitude is 50-000mV, the pulse repetition frequency is 50-1000Hz, the duty cycle is 10%-70%, and the ultrasound device The power amplifier amplifies 5%-45%, circulates 20-200k times, the pulse interval is 2μs-2s, and the ultrasonic time is 1 minute to 10 minutes. Circulating ultrasound can independently adjust multiple ultrasound parameters, which is helpful for the diversification of ultrasound parameters and the precise stimulation of cells.

S130: Continue culturing the human astrocytes stimulated by the ultrasonic cycle and collect the cell culture supernatant, and centrifuge the cell culture supernatant to obtain exosomes.

Exosomes prepared by the above preparation method.

In one of the embodiments, the average diameter of the exosomes is about 50 nm to 150 nm, and exhibits a typical exosomes particle size distribution.

The application of the exosomes prepared by the above preparation method in the preparation of drugs for the prevention and/or treatment of Alzheimer's disease.

The application of the exosomes prepared by the above preparation method for the degradation of β-amyloid.

Example

Culture human astrocytes (HA) cells in a cell culture flask, add exosomes-free high-sugar DMEM medium, and wait for the cells to be cultured in a 37° incubator. After 24 hours, use an ultrasound device to culture the cells Dish for stimulation. The total time lasts 5 minutes. After being stimulated by ultrasound, HA cells were placed in a 37° incubator for continued cultivation. After 24 hours, 48 hours, and 72 hours, the cell culture supernatant was collected. The cell culture supernatant was subjected to a series of centrifugation treatments, 400g for 15 minutes, 2000g for 30 minutes, 10000g for 60 minutes, and 100,000g for 90 minutes, and the resulting pellet was exosomes. Refer to Figure 1 and Figure 2. Figure 1 is a transmission electron microscope image of astrocyte exosomes that have not been stimulated by ultrasound; Figure 2 is a schematic diagram of astrocyte exosomes stimulated by ultrasound.

The results of Nanoparticle Tracking Analysis (NTA) combined with Figures 3 and 4 are as follows. The results show that the average diameter of exosomes is about 133.1±1.2nm for US-HA-EXO, and about 132.3± for HA-EXO 1.5nm.

Combined with the iTRAQ quantitative proteomics results of exosomes in Figure 5, it can be seen that the exosomes secreted by astrocytes after ultrasound stimulation are compared with the exosomes secreted by the non-ultrasound group. The exosomes are related to the proteasome function. The increase in protein content of exosomes helps to degrade abnormally folded proteins.

After Aβ1-42 (10μmol/L) was added to SH-SY5Y cells for 48 hours, fresh serum-free medium was replaced, and US-HA-EXO was added to incubate for 24 hours. CCK-8 measured the proliferation activity of SH-SY5Y cells. The results are shown in Figure 6, showing that exosomes secreted by ultrasound-stimulated astrocytes can reverse the toxicity of Aβ1-42 to SH-SY5Y cells.

To verify the in vivo therapeutic effect of US-HA-EXO on APP/PSI transgenic Alzheimer's mice. After injection of US-HA-EXO into the tail vein, 1 week, 2 weeks, and 3 weeks later, mouse brain tissues were taken, fixed, dehydrated, and Aβ plaque deposition was detected by Thioflavin S staining method, and immunofluorescence staining with Aβ1-42 antibody The distribution of Aβ1-42 in the brain was detected, and the results showed that the US-HA-EXO treatment group had a significant reduction in Aβ plaques and a decrease in the distribution of Aβ1-42. Refer to Figure 7. In Figure 7 A, the thioflavin S staining method is used to detect the deposition of Aβ plaques, the green is the plaque, and the blue is the cell nucleus. In B, Aβ1-42 antibody immunofluorescence staining was used to detect the distribution of Aβ1-42 in the brain. The green is Aβ and the blue is the nucleus.

Compared with the prior art, the present invention uses medical ultrasound technology to produce different biological effects of central nerve cells and glial cells at the stimulation site through different intensities, frequencies, pulse repetition frequencies, pulse widths, and durations. Astrocytes are the basis for the stability, defense and regeneration of the central nervous system. The morphology and function of astrocytes can be adjusted by environmental stimuli or drugs. The invention utilizes ultrasonic waves to stimulate human astrocytes, and the preparation method of the separated exosomes is simple in process, convenient in operation, strong in controllability and low in cost. The obtained exosomes can slow down or even reverse the toxicity of β-amyloid (Aβ) to nerve cells, reduce the deposition of Aβ plaques in the brain in mice, and can be used in new drugs for the treatment of Alzheimer's disease.

The above are only implementations of the present invention, and do not limit the scope of the present invention. Any equivalent structure or equivalent process transformation made using the content of the description and drawings of the present invention, or directly or indirectly applied to other related technical fields, The same reasoning is included in the scope of patent protection of the present invention.

Claims (8)

A preparation method of exosomes, which is characterized in that it comprises the following steps: Place human astrocytes in a culture medium for culture; Use an ultrasound device to stimulate the human astrocytes in circulation; The human astrocytes stimulated by the ultrasound cycle are cultured continuously, and the cell culture supernatant is collected, and the cell culture supernatant is centrifuged to obtain exosomes. The method for preparing exosomes according to claim 1, wherein the cyclic stimulation conditions are: the probe frequency of the ultrasound device is 0.5-5MHz, the amplitude is 50-000mV, the pulse repetition frequency is 50-1000Hz, and the working The period is 10%-70%, the power amplifier of the ultrasonic device is amplified by 5%-45%, the cycle is 20-200k times, the pulse interval is 2μs-2s, and the ultrasonic time is 1 minute-10 minutes. The method for preparing exosomes according to claim 1, wherein the medium is high-sugar DMEM without exosomes. The method for preparing exosomes according to claim 1, wherein the human astrocytes stimulated by the ultrasound cycle are cultured for 24-72 hours and then the cell culture supernatant is collected. Exosomes prepared by the preparation method of any one of claims 1-4. The exosomes of claim 5, wherein the average diameter of the exosomes is about 50 nm to 150 nm. The preparation method according to any one of claims 1 to 4 or the use of the exosomes according to claim 5 or 6 in the preparation of drugs for the prevention and/or treatment of Alzheimer's disease. The preparation method according to any one of claims 1 to 4 or the use of the exosomes according to claim 5 or 6 in the degradation of β-amyloid protein.

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