CN112402456A

CN112402456A - Application of induced extracellular vesicle derived from mesenchymal stem cell in treating Alzheimer's disease

Info

Publication number: CN112402456A
Application number: CN202010641070.0A
Authority: CN
Inventors: 赵陆; 寇晓星; 施松涛
Original assignee: Medical Micro Cell Biotechnology Guangzhou Co ltd
Current assignee: Medical Micro Cell Biotechnology Guangzhou Co ltd
Priority date: 2020-07-06
Filing date: 2020-07-06
Publication date: 2021-02-26

Abstract

The invention provides application of induced extracellular vesicles in a product for treating Alzheimer's disease. The invention discovers that the following advantages are achieved when the induced extracellular vesicles are used for treating the Alzheimer's disease: the yield of IEVs is richer, and a single MSC can yield 300-1000 IEVs; the preparation process of IEVs is simple, short in time consumption, low in requirements on reagents and equipment and good in treatment effect. Therefore, the extracellular vesicle therapy based on IEVs derived from MSCs has good application prospect. The present invention finds that IEVs as physiological vesicles can reach the cranium through the blood brain barrier; and can be combined with the intracerebral metabolite beta amyloid protein and directly discharged out of the body through the skull by a non-classical way to reach the surface layer of the hair, thereby playing a therapeutic role.

Description

Application of induced extracellular vesicle derived from mesenchymal stem cell in treating Alzheimer's disease

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to application of an inducing extracellular vesicle derived from mesenchymal stem cells in treating Alzheimer's disease.

Background

Alzheimer's disease is the most common neurodegenerative disease, the most prominent manifestation of the disease is cognitive impairment, and 50% -75% of dementia is caused by alzheimer's disease. Alzheimer's disease was reported in the us in 2018 as the sixth disease of all-cause death in 2014 and 2015, and was the first disease to change in ten-cause death at a 15.7% rate of increase. For 30 years, biomedical researchers have been working on finding therapeutic strategies for alzheimer's disease, but there are no drugs that are effective in ameliorating the symptoms. It has been shown that extracellular exosomes (exosomes) can improve symptoms by carrying or clearing amyloid beta, a metabolite in the brain, but clinical studies have not been successful.

Apoptosis is programmed cell death characterized by marked cell contraction, chromatin condensation and cell membrane blebbing. Apoptosis produces a large number of apoptotic bodies, containing a variety of cellular components. Staurosporine (STS) is capable of inducing apoptosis of Mesenchymal Stem Cells (MSCs) in vitro, during which a large number of extracellular vesicles are produced. Previous studies have focused primarily on apoptosis itself, and the concomitant production of extracellular vesicles lacks systemic and intensive research. Previous studies by the inventors/subject groups of the present application confirmed that extracellular vesicles derived from STS in vitro have an important regulatory effect on MSCs homeostasis, and can significantly improve the bone destruction in osteoporotic mice. There are no reports of treatment of alzheimer's disease by extracellular vesicles.

Disclosure of Invention

The invention aims to provide application of induced extracellular vesicles in a product for treating Alzheimer's disease.

In order to achieve the purpose, the invention adopts the technical scheme that:

the use of induced extracellular vesicles in products for the treatment of alzheimer's disease.

Extracellular vesicles-this term is a term created by the International Society for Extracellular Vesicles (ISEV), and vesicles can be classified according to their biosynthesis or release pathway: exosomes (exosomes) having a diameter of 30-150nm, originating from the endocytic pathway, and having a density of about 1.11-1.19g mL "1; microparticles/microvesicles (microparticles/microviscles) are released directly from the plasma membrane, with a diameter of about 100-; apoptotic bodies (bleb) are about 50nm-2 μm in diameter and arise from apoptosis; tumor vesicles (large oncospheres) about 1-10 μm in diameter, produced by release from tumor cells; and various other EV subgroups. Due to the differences in the size of the different EV subgroups and their contained biomolecules, several groups have now begun to characterize the composition of the EV subgroups. Recent papers claim successful subpopulation classification of extracellular vesicles based on general surface proteomic analysis or transcriptional profiling of individual EV populations. Different extracellular vesicle subsets can be roughly separated by various means such as differential centrifugation, filtration, immunoaffinity, chromatography, flow cytometry sorting and density gradient centrifugation for selecting different density regions, but the methods can not be completely purified to specific subsets, and the separated subsets are usually enriched in a certain subset and simultaneously carry other extracellular vesicle subsets.

Preferably, the induced extracellular vesicles are mesenchymal stem cell-derived induced extracellular vesicles.

Preferably, the mesenchymal stem cells comprise oral maxillofacial tissue, bone marrow, adipose, umbilical cord-derived mesenchymal stem cells.

The invention also provides a product, the active ingredient of which comprises the inducing extracellular vesicles as described above, for use in the treatment of alzheimer's disease.

Preferably, the product has any one or more of the following uses:

(1) increasing the number of neurons in the CA1 area;

(2) reducing beta-amyloid deposition;

(3) inhibiting inflammatory responses in the alzheimer's brain;

(4) increasing the expression of beta-amyloid in the head skin;

(5) clearing beta-amyloid in neurons.

Preferably, the product is a medicament.

Preferably, the medicament further comprises a pharmaceutically acceptable carrier.

Preferably, the pharmaceutically acceptable carrier includes an antioxidant and a stabilizer.

The invention has the beneficial effects that: the invention provides application of inducing extracellular vesicles in a product for treating Alzheimer's disease, and develops a new method for treating Alzheimer's disease. Compared with exosomes, Induced Extracellular Vesicles (IEVs) are more advantageous, mainly represented by: the yield of IEVs is richer, and a single MSC can yield 300-1000 IEVs; the preparation process of IEVs is simple, short in time consumption, low in requirements on reagents and equipment and good in treatment effect. Therefore, the extracellular vesicle therapy based on IEVs derived from MSCs has good application prospect. The present invention finds that IEVs can reach the cranium through the blood brain barrier; and can be combined with the intracerebral metabolite beta amyloid protein and directly discharged out of the body through the skull by a non-classical way to reach the surface layer of the hair, thereby playing a therapeutic role.

Drawings

FIG. 1 is a flow chart of the specific operation of inducing collection of bone marrow mesenchymal stem cell-derived IEVs.

FIG. 2 is an electron micrograph of bone marrow mesenchymal stem cell-derived IEVs as observed by transmission electron microscopy.

Fig. 3 is a particle diameter distribution graph of bone marrow mesenchymal stem cell-derived IEVs as shown by nanoparticle tracking analysis.

FIG. 4 is a graph showing the results of analyzing surface membrane proteins of bone marrow mesenchymal stem cell-derived IEVs by flow cytometry.

FIGS. 5A-D are content analyses of bone marrow mesenchymal stem cell-derived IEVs: FIG. 5A shows the results of quantitative analysis of proteomics of MSCs, MSCs-Exosomes and MSCs-IEVs by DIA quantification technique; FIG. 5B is a heat map of specific highly expressed proteins screened for IEVs; FIG. 5C is the results of GO enrichment analysis of differential proteins by IEVs expressing Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntexin4 molecules; FIG. 5D is the result of Western Blot verifying that MSCs, MSCs-Exosomes, MSCs-IEVs express Annexin V, Flotillin-1, Cathererin 11, Integrin alpha 5 and Syntexin 4.

FIG. 6 is a graph of the effect of bone marrow mesenchymal stem cell-derived IEVs on Alzheimer's disease brain tissue architecture: figure 6A analyzes changes in structure of hippocampal dentate gyrus (SGZ) region of alzheimer's disease model (AD) with and without IEVs treatment by HE staining comparison. FIG. 6B shows the change in neuron width in CA1 region. Indicating that the hippocampal size and number of neurons in the CA1 region were restored in AD mice after treatment with the IEVs as compared to those before treatment.

Figure 7 is a graph of the effect of bone marrow mesenchymal stem cell-derived IEVs on alzheimer's disease brain inflammation and Α β: FIG. 7A compares the reduction in microglial expression in the DG region of the Alzheimer's disease model (AD) following treatment with IEVs by immunofluorescence staining. Figure 7B is an immunofluorescent stain demonstrating a reduction in hippocampal Α β plaques in the alzheimer disease model (AD) following IEVs treatment. Indicating that administration of the IEVs treatment significantly ameliorates inflammation and Α β plaque (β -amyloid) deposition in the alzheimer's brain.

FIG. 8 shows the tail vein injection of green fluorescence labeled IEVs to mice, and the expression of the IEVs in brain tissue was examined at 1 day, 3 days, and 7 days, respectively, indicating that the IEVs can enter the brain tissue through the blood brain barrier.

FIG. 9 shows the intracerebral medullary canal injection (CM) of IEVs, which were examined for intracranial efflux at 1 day and 6 days, respectively. Indicating that it may be expelled transcranially from the cranium to the skin surface.

FIG. 10 shows the expression of parietal skin A β after tail vein IEVs treatment. FIG. 10A is the expression of head skin metabolite A β (400 ×); fig. 10B is a statistical result of the expression of the head skin metabolite a β. Indicating that the reduction in the expression of a β in the parietal skin of alzheimer's mice can be restored after the administration of the IEVs.

Fig. 11 is a graph of clearance of Α β by bone marrow mesenchymal stem cell-derived IEVs. FIG. 11A is a graph of Ass binding to the IEVs; fig. 11B is a graph of the clearance of Α β within neurons by IEVs. In vitro bone marrow mesenchymal stem cell-derived IEVs are described to bind to A β and are capable of clearing A β from neurons.

Detailed Description

In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.

Example 1

This example provides a method for preparing an induced extracellular vesicle, comprising the steps of:

(1) extraction of IEVs:

culturing MSCs (oral and maxillofacial tissues, bone marrow, fat or umbilical cord derived MSCs) in vitro, inducing cells by adopting various methods such as staurosporine, paclitaxel or ultraviolet irradiation, taking staurosporine as an example for inducing and extracting IEVs, flushing 2 times by PBS when 80% -90% of MSCs cells cultured in vitro are confluent, adding a serum-free culture medium containing 500nM staurosporine, incubating for 16-24h at 37 ℃, collecting cell supernatant, centrifuging for 10 min at 800g at 4 ℃, collecting supernatant, centrifuging for 10 min at 2000g at 4 ℃, collecting supernatant for 30 min at 2000 ℃, collecting supernatant again, and centrifuging for 30 min at 16000g at 4 ℃, wherein the obtained precipitate is the IEVs. 1ml PBS was resuspended and washed, and the pellet was centrifuged again at 16000g for 30 minutes at 4 ℃ to obtain washed IEVs. In use, an appropriate amount of buffer such as PBS is taken to resuspend IEVs.

(2) Detection of IEVs:

performing quantitative analysis on the extracted IEVs by adopting a flow cytometry technology; the diameter of most vesicles is below 1um and about 200nm when observed by a Transmission Electron Microscope (TEM).

The Nanoparticle Tracking Analysis (NTA) results were consistent with those observed by transmission electron microscopy, with IEVs particles below 1um averaging 244nm in diameter, as shown in FIG. 2 (transmission electron microscopy) and FIG. 3 (nanoparticle tracking analysis).

Analysis of surface membrane proteins of IEVs using flow cytometry showed that, in fig. 4, MSCs-derived IEVs were able to express similar surface proteins as MSCs, i.e. CD29, CD44, CD73, CD166 positive, CD34, CD45 negative. Meanwhile, IEVs are able to express the ubiquitous surface proteins CD9, CD63, CD81 and C1q of extracellular vesicles. In addition, a large amount of Phosphatidylserine (PS) is distributed on the membrane surface of IEVs.

Proteomic quantitative analysis of MSCs, MSCs-exoms (exosomes), MSCs-IEVs was done using protein DIA quantification technique. The result shows that 170 proteins are specifically and highly expressed in IEVs, and combined with the GO enrichment analysis result of differential proteins, western blot verification is carried out, and MSCs-IEVs are found to be capable of specifically and highly expressing Annexin V, Flotillin-1, Cadherin 11, Integrin alpha 5 and Syntexin 4. The above 5 proteins are expected to be characteristic protein markers for distinguishing MSCs-derived IEVs from exosomes, as shown in fig. 5.

Example 2 animal experiments

Using the APP/PS1 transgenic Alzheimer's disease mouse animal model, about 200 μ L of a PBS suspension of MSC-IEVs (IEVs about 8X 10) was injected via the tail vein⁸One), once a week, four consecutive times, animals were sacrificed one month after the end of all injections and samples were collected. In the trace-related experiments on IEVs, the extracted and collected IEVs were labeled by membrane staining with PKH67, and then tail vein injection was performed, and samples were collected after the corresponding days, and the experimental results are shown in FIGS. 6-11.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The use of induced extracellular vesicles in products for the treatment of alzheimer's disease.

2. The use of claim 1, wherein the inducing extracellular vesicles are mesenchymal stem cell-derived inducing extracellular vesicles.

3. The use of claim 2, wherein the mesenchymal stem cells comprise oral maxillofacial tissue, bone marrow, adipose, umbilical cord-derived mesenchymal stem cells.

4. The use according to any one of claims 1 to 3, wherein the inducing extracellular vesicles are mesenchymal stem cell-derived inducing extracellular vesicles.

5. A product comprising the induced extracellular vesicles according to any one of claims 1 to 4 as an active ingredient for use in the treatment of Alzheimer's disease.

6. The product of claim 4, wherein the product has any one or more of the following uses:

(1) increasing the number of neurons in the CA1 area;

(2) reducing beta-amyloid deposition;

(3) inhibiting inflammatory responses in the alzheimer's brain;

(4) increasing the expression of beta-amyloid in the head skin;

(5) clearing beta-amyloid in neurons.

7. The product of claim 6 or 7, wherein the product is a medicament.

8. The product of claim 8, wherein the medicament further comprises a pharmaceutically acceptable carrier.

9. The product of claim 9, wherein the pharmaceutically acceptable carrier comprises an antioxidant and a stabilizer.