SiCCR5-carried engineered umbilical cord stem cell exosome, preparation method and application thereof in treatment of Alzheimer's disease

Abstract

The invention provides a siCCR5-carried engineered umbilical cord stem cell exosome, a preparation method and application of the siCCR5-carried engineered umbilical cord stem cell exosome in treatment of Alzheimer's disease, and belongs to the technical field of biological medicine. The invention provides a preparation method of a new drug for treating Alzheimer's disease, which comprises the following steps: preparing a lipid membrane, dissolving the lipid membrane, adding siCCR5 and umbilical cord mesenchymal stem cell exosome, and feeding siCCR5 into the umbilical cord mesenchymal stem cell exosome by using a cationic liposome extrusion method to obtain the engineering umbilical cord stem cell exosome carrying siCCR5. The engineered exosome prepared by the invention can repair brain tissues through tissue regeneration and regulate the brain microenvironment, and does not generate toxic and side effects on the organism. Moreover, the engineered exosome carries siCCR5 of a targeted specific gene, so that the targeted therapeutic property of the engineered exosome is higher than that of common hUCMSC-EVs, the anti-inflammatory effect can be achieved more efficiently, and the disease progress of the Alzheimer's disease is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an engineered umbilical cord stem cell exosome carrying siCCR5, a preparation method and application thereof in treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease is a neurodegenerative disease associated with neuronal damage and is the leading cause of dementia. Since aging leads to an imbalance in protein homeostasis, the incidence of Alzheimer's disease increases with age. In 2019, Alzheimer's disease was listed as the sixth leading cause of death in the United States. At the same time, COVID-19 (new coronavirus) has also had a significant impact on the mortality rate of Alzheimer's disease. It is estimated that by 2060, 13.8 million people in the United States will suffer from Alzheimer's disease, which will impose huge health care costs on families and society, which is not conducive to social progress.

[0003] Pathologically, Alzheimer's disease is manifested by amyloid plaques (Aβ) and neurofibrillary tangles (NFTs) formed by phosphorylation of tau protein; in addition, soluble amyloid β oligomers (AβO) can cause neuronal dysfunction, activate glial cells in the brain, and induce chronic neuroinflammation.

[0004] The Alzheimer's disease treatment drugs approved by the U.S. Food and Drug Administration (FDA) include memantine, donepezil, galantamine and rivastigmine. These drugs are cholinesterase inhibitors that can relieve patients' cognitive impairment by inhibiting the decomposition of acetylcholinesterase. However, due to the complex pathogenesis of Alzheimer's disease, there is currently no effective treatment or drug in the world that can fundamentally cure Alzheimer's disease, and can only relieve related symptoms. Therefore, new Alzheimer's disease treatment methods are urgently needed.

[0005] In recent years, mesenchymal stem cells have been regarded as a new treatment for Alzheimer's disease due to their characteristics of no immune rejection, anti-inflammatory and extensive differentiation, and have been used in clinical trials. Mesenchymal stem cells play a role in neurodegenerative diseases in three main ways: secreting growth factors for immune regulation, inhibiting neuroinflammation, and releasing exosomes by paracrine. Studies have shown that mesenchymal stem cells inhibit Alzheimer's disease mainly through paracrine exosomes, rather than the cells themselves. Exosomes contain substances such as proteins, lipids, DNA, mRNA and miRNA, which play an important role in cell communication, have no toxic side effects on the body, can repair brain tissue through tissue regeneration, regulate the brain microenvironment, and inhibit inflammation. Therefore, exosomes have broad prospects in the clinical application of treating Alzheimer's disease.

[0006] Chemokine CC subfamily receptor 5 (CCR5), a cell membrane protein belonging to the G protein-coupled receptor family, is an important receptor for the activation and mobilization of leukocytes, including monocytic lineage cells. CCR5 is expressed at a high level in microglia in the central nervous system. After CCR5 activation, different signal cascades will change the calcium flux and chemotaxis of cells, affect cell metabolic activities such as cell proliferation and apoptosis, promote the migration of activated cells, cause the release of proinflammatory factors, and stimulate downstream immune cells. Recent studies published in Cell have shown that CCR5 plays an important role in the regulation of learning and memory. Delayed expression of CCR5 in the CA1 region of mice can cause decreased neuronal excitability, close the time window of memory connection, and cause memory impairment in elderly mice. However, the combination of CCR5 and exosomes has not been used to treat Alzheimer's disease.

[0007] Therefore, how to transform exosomes so that they can serve as natural carriers to carry siCCR5, construct engineered exosomes that can edit genes, and realize cell and gene therapy is crucial to the treatment of Alzheimer's disease. Summary of the invention

[0008] The purpose of the present invention is to provide an engineered umbilical cord stem cell exosome carrying siCCR5, a preparation method and its application in the treatment of Alzheimer's disease, which can be used to treat Alzheimer's disease, realize cell and gene therapy, significantly improve the patient's memory ability, and enhance spatial memory ability.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a method for preparing engineered exosomes, comprising the following steps:

[0011] (1) Preparation of liposome membrane: DLin-MC3-DMA was dissolved in an organic solvent (chloroform: methanol = 9:1), and cholesterol and DOPC were further dissolved to prepare a liposome solution. The organic solvent was removed by rotary evaporation, and the solution was dried under an inert gas environment to obtain a lipid membrane.

[0012] (2) Preparation of engineered exosomes: The lipid membrane is hydrated with phosphate buffer, and siCCR5 and umbilical cord mesenchymal stem cell exosomes are added after ultrasonic treatment to obtain a mixed solution. The mixed solution is hydrated and filtered, squeezed and dialyzed under heat preservation conditions to obtain engineered exosomes.

[0013] Furthermore, in step (1), the organic solvent is a mixed solvent of chloroform:methanol in a volume ratio of 9:1; the molar ratio of DLin-MC3-DMA:cholesterol:DOPC is 25-34.5:35-65:1.5-40.

[0014] Furthermore, in step (2), the mass volume ratio of the lipid membrane to the PBS phosphate buffer during the hydration and dissolution is 100:3; and the mass ratio of the siCCR5: umbilical cord mesenchymal stem cell exosomes: lipid membrane is 2:1:50.

[0015] Furthermore, in step (2), the insulation temperature is 40-50°C.

[0016] Furthermore, in step (2), the siCCR5 can target CCR5 for knockdown, and the siCCR5 is composed of the sequence sense shown in SEQ ID NO.1 and the sequence antisense shown in SEQ ID NO.2.

[0017] Furthermore, in step (2), the extrusion step is:

[0018] The mixed solution was extruded for the first time through a filter with a pore size of 1.0 μm, then through a filter with a pore size of 0.1 μm for the second time, and finally through a filter with a pore size of 0.05 μm for the third time.

[0019] Furthermore, the filter used in the three extrusions is a polycarbonate filter, and the number of extrusions is 2 to 5 times.

[0020] Furthermore, in step (2), the dialysis step is:

[0021] The solution was dialyzed overnight at 4°C using a dialysis cassette with a pore size of 100 kDa, and the pH was adjusted to 7.2-7.4.

[0022] The present invention also provides an engineered exosome prepared by a method for preparing an engineered exosome.

[0023] The present invention also provides an application of engineered exosomes in the preparation of a drug for treating Alzheimer's disease.

[0024] The beneficial effects of the engineered exosomes described in the present invention compared with the prior art are:

[0025] The present invention utilizes lipids to transform exosomes, and uses a cationic liposome extrusion method to deliver siCCR5 into exosomes to obtain engineered umbilical cord stem cell exosomes carrying siCCR5. The engineered exosomes prepared by the present invention will not produce toxic side effects on the body, can repair brain tissue through tissue regeneration, regulate the brain microenvironment, inhibit inflammation, and carry siCCR5 directed to specific genes, which can have stronger targeted therapeutic properties than ordinary hUCMSC-Exos, more efficiently exert anti-inflammatory effects, improve the progression of Alzheimer's disease, realize cell and gene therapy, significantly improve the patient's memory ability, and improve spatial memory ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0027] Figure 1 This is a diagram showing the results of flow cytometry identification of cell surface marker proteins in Example 1;

[0028] Figure 2 This is a cell morphology picture of MSC-3P1 cultured on the second day in Example 1;

[0029] Figure 3 The results of flow cytometry analysis of surface markers of umbilical cord mesenchymal stem cells in Example 1;

[0030] Figure 4 The results of osteogenic and adipogenic differentiation induction staining of umbilical cord mesenchymal stem cells in Example 1;

[0031] Figure 5 This is a flow chart of ultracentrifugation separation of exosomes in Example 1;

[0032] Figure 6 This is the electron microscopic image of hUCMSCs-Exos in Example 1;

[0033] Figure 7 This is a diagram showing the particle size analysis of hUCMSCs-Exos in Example 1;

[0034] Figure 8 The expression of hUCMSCs-Exos marker proteins in Example 1;

[0035] Fig. 9 For miRNA sequencing analysis in hUCMSCs-Exos in Example 1

[0036] Fig.10This is the engineering exosome technology route in Example 1;

[0037] Fig.11 This is a flow chart of exosome engineering by extrusion method in Example 1;

[0038] Fig.12 This is an electron micrograph of the engineered exosomes in Example 1;

[0039] Fig.13 The particle size analysis of engineered exosomes in Example 1;

[0040] Fig.14 hUCMSCs-Exos reduces the expression of CCR5 in microglia in Experimental Example 1, where a is a differential gene enrichment map; b is a gene expression heat map; c is a GO enrichment analysis-related pathway map; d is the changes in the expression of CCR5 at the protein and gene levels after BV2 was treated with hUCMSCs-Exos;

[0041] Fig.15 For the identification of transgenic mice in Experimental Example 1;

[0042] Fig.16 The results of the novel object recognition experiment and novel location recognition experiment of APP / PS1 mice in Experimental Example 1 are shown in FIG.

[0043] Fig.17 This is the result of the Morris water maze for APP / PS1 mice in Experiment 1, where a is the escape delay time of the mouse, b is the movement trajectory of the mouse, c is the number of times the mouse crosses, d is the time the mouse spends in the target quadrant, e is the total swimming distance of each group of mice, and f is the swimming speed of each group of mice. DETAILED DESCRIPTION

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0046] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0047] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0048] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0049] The present invention prepares an engineered exosome, which specifically includes the following steps:

[0050] (1) Preparation of liposome membrane: lipids are dissolved in an organic solvent, and cholesterol and DOPC are further dissolved to prepare a liposome solution; the organic solvent is removed by rotary evaporation, and the solution is dried under an inert gas environment to obtain a lipid membrane;

[0051] (2) Preparation of engineered exosomes: The lipid membrane is hydrated with phosphate buffer, and siCCR5 and umbilical cord mesenchymal stem cell exosomes are added after ultrasonic treatment to obtain a mixed solution. The mixed solution is hydrated and filtered, squeezed and dialyzed under heat preservation conditions to obtain engineered exosomes.

[0052] In the present invention, siCCR5 is a short nucleic acid chain designed for targeted knockdown of CCR5, comprising:

[0053] sense(SEQ ID NO.1):

[0054] GGUCAGUUCCGACCUAUAGTT;

[0055] antisense (SEQ ID NO. 2):

[0056] CUAUAGGUCGGAACUGACCTT.

[0057] Example 1

[0058] Embodiment 1 of the present invention provides a method for preparing engineered exosomes, which specifically comprises the following steps:

[0059] (1) Isolation and culture of umbilical cord mesenchymal stem cells

[0060] A. Isolation of primary umbilical cord mesenchymal stem cells:

[0061] Cut fresh healthy umbilical cord tissue into 2-3 cm small segments, peel off the Wharton's jelly, cut the Wharton's jelly into pieces, transfer it to a culture bottle, add an appropriate amount of culture medium, and place it in a CO2 incubator. Do not shake the culture bottle during this period to prevent the tissue blocks from falling off. When the crawling cells cover about 90% of the bottom area of ​​the cell culture bottle, the primary cells are passaged.

[0062] B. Subculture of umbilical cord mesenchymal stem cells

[0063] When the cells grow to 90%, discard the old culture medium in the culture bottle and add an appropriate amount of PBS to wash the cells. Aspirate the PBS, evenly add an appropriate amount of stem cell gentle digestion trypsin, and digest until the bottom of the culture bottle falls off, then add an equal amount of serum-free complete culture medium to terminate the digestion. Use a pipette to blow the bottom of the bottle, and transfer the cell suspension to a centrifuge tube, centrifuge at 1000rpm for 5 minutes at room temperature. Discard the supernatant, add an appropriate amount of complete culture medium to resuspend the cells and count the cells. Add an appropriate amount of complete culture medium to a clean culture bottle, inoculate the cells into the culture bottle, shake it thoroughly, and place it in a 37℃ CO2 incubator to complete the cell passaging.

[0064] C. Identification of umbilical cord mesenchymal stem cells

[0065] 1) Morphological identification

[0066] The cell morphology was observed under a microscope. Figure 2 shown.

[0067] Figure 2 The cells showed fibroblast-like growth.

[0068] 2) Marker identification

[0069] The expression of cell surface markers CD44, CD73, CD90, CD105, CD34, and CD45 was measured by flow cytometry, and IgG isotype control was used as a negative control. The results are shown in Figure 1 shown.

[0070] Figure 3 The results showed that the expression rates of mesenchymal stem cell positive markers CD44, CD73, CD90, and CD105 were all >95%, and the expression rates of mesenchymal stem cell negative markers CD34 and CD45 were less than 1.0%. The identification results met the standards, indicating that the cells were mesenchymal stem cells.

[0071] 3) Identification of osteogenic and adipogenic differentiation of stem cells

[0072] MSCs have differentiation potential, that is, they can be directed to differentiate into bone and fat cells under specific induction conditions. The differentiation ability after adipogenic and osteogenic induction in vitro can be identified by specific staining.

[0073] Osteogenic differentiation and identification steps:

[0074] Inoculation of mesenchymal stem cells: Take cells in the logarithmic growth phase and add 2×10 4 cells / cm 2 The cells were inoculated into a six-well plate at a density of 100 μg / mL and cultured at 37°C and 5% CO2 until the confluence was 60-70%. The supernatant was discarded and the differentiation induction medium was added. The osteogenic induction medium was DMEM complete medium (DMEM + 10% FBS + 1% P / S + 200 μM sodium ascorbate + 100 μM β-glycerophosphate + 0.1 μM dexamethasone), and the adipogenic differentiation medium was (DMEM + 10% FBS + 1% P / S + 1 μM dexamethasone + 0.5 mM 3-isobutyl-1-methylxanthine + 200 μM indomethacin + 10 μg / mL insulin). Cell differentiation induction: The osteogenic / adipogenic induction medium was replaced every 2-3 days, and the cells were cultured at 37°C and 5% CO2 for about 14-21 days, and the cell morphology was observed. The time to terminate the cell induction was determined according to the cell condition, and staining was performed for identification. Cell fixation: aspirate the culture medium and wash once with an appropriate amount of 1×PBS, discard it and take 2mL of 4% neutral formaldehyde solution to cover the bottom of the culture vessel, fix at room temperature for 30min, discard the fixative and wash twice with PBS.

[0075] Osteogenic differentiation Alizarin red staining: let stand at room temperature before use, add 1mL 1% Alizarin red staining solution (pH4.2) to each well and stain at 37℃ for 30min, remove the Alizarin red staining solution, wash twice with PBS, and add appropriate amount of PBS to prevent the cells from drying. Adipogenic differentiation Oil Red O staining: use ddH2O and Oil Red O stock solution to prepare Oil Red O working solution (Oil Red O stock solution: ddH2O = 3:2), filter with a filter membrane after preparation, and prepare it before use. Add 1mL of working solution to the cleaned induction wells, let stand at 37℃ for staining for 30min, remove the Oil Red O working solution, wash twice with PBS, and add appropriate amount of PBS to prevent the cells from drying.

[0076] Induction evaluation: Observe the osteogenic and adipogenic staining effects under a microscope, and perform image acquisition and induction evaluation. When the induction is successful, the calcium nodules will appear red or orange after combining with the alizarin red dye, the bone nodules formed after osteogenic differentiation will be stained purple, and the fat droplets after adipogenic differentiation will be stained red. Figure 4 shown.

[0077] (2) Isolation of umbilical cord mesenchymal stem cell exosomes (hUCMSCs-EVs)

[0078] A. Expand and culture the umbilical cord mesenchymal stem cells obtained in step (1) according to Figure 5 In the centrifugation step shown, the subcultured cells were centrifuged at 300 g for 5 min, the cells were removed and then centrifuged at 2000 g for 20 min, the supernatant was filtered through a 0.22 μm filter membrane, then dissolved in PBS and centrifuged at 120,000 g for 120 min to obtain exosomes hUCMSCs-EVs.

[0079] B. Exosome identification

[0080] 1) Electron microscopy observation

[0081] The isolated exosomes were observed using electron microscopy. Figure 6 shown.

[0082] Figure 6 It was shown that hUCMSCs-EVs presented a typical pie-shaped double-layer membrane structure.

[0083] 2) Exosome particle size

[0084] The particle size of the separated exosomes was detected, and the results were as follows Figure 7 shown.

[0085] Figure 7 The results of five repeated measurements of the nanoparticle size showed that the diameter of the obtained exosomes was distributed around 100 nm, which was consistent with the specified particle size range of exosomes.

[0086] 3) Exosome marker protein detection results by Western Blotting

[0087] TSG101, CD63, and ALIX proteins were selected as marker proteins for exosomes. TSG101 is a protein associated with the ESCRT complex. CD63 protein is a member of the four-transmembrane protein family, and they are directly involved in the sorting of ESCRT-dependent and ESCRT-independent extracellular vesicle contents. ALIX protein is directly involved in the process of cleaving and detaching from the plasma membrane to form an independent membrane structure during the formation of membrane vesicles. Therefore, the expression of the above proteins can be used to identify whether it is an exosome. The results are as follows Figure 8 shown.

[0088] Figure 8 It was shown that the extracted exosomes successfully expressed three marker proteins.

[0089] 4) High-throughput sequencing results of miRNA in hUCMSCs-EVs

[0090] As a key core component in exosomes, the present invention performs high-throughput sequencing on the types and contents of miRNA. The results are as follows Fig. 9 shown.

[0091] Fig. 9 The results show that the higher content of miR-21 and miR-146 are related to various signaling pathways, regulating the expression of downstream target genes and affecting cell metabolism. Currently, a large number of studies have focused on miRNAs, using them as targets to study various metabolic-related diseases.

[0092] (3) Construction of engineered exosomes

[0093] according to Fig.10 , 11 The process shown is to prepare engineered exosomes, and the specific preparation method is as follows:

[0094] A. Preparation of liposome solution: Lipids were dissolved in a chloroform / methanol mixture (9 / 1; v / v) and prepared into a liposome solution with a concentration of 4 nmol and a total volume of 1 mL at a molar ratio of 25:35:40 (DLin-MC3-DMA:DOPC:cholesterol).

[0095] Among them, DLin-MC3-DMA was purchased from MCE Biotechnology Company; DOPC was purchased from McLean Reagent Co., Ltd.; and cholesterol was purchased from Sima Biotechnology Co., Ltd.

[0096] B. Preparation of liposome film by rotary evaporator: Add the prepared liposome solution into a 5 mL round-bottom flask, evaporate the organic solvent in situ by vacuum evaporation at 45°C, and dry the obtained lipid film under nitrogen flow for about 20 minutes.

[0097] C. Hydration extrusion: Hydrate the lipid membrane with phosphate buffer (the reagents and the configuration ratios used are as follows: Figure 2 As shown), ultrasonic for 5 min; add a mixture of siCCR5 (Suzhou Jima Gene Co., Ltd.) and umbilical cord mesenchymal stem cell exosomes at a mass ratio of 2:1, and then fully hydrate and dissolve at a ratio of siCCR5, exosomes and total lipid membrane of 2:1:50. The suspension was maintained at 45°C and then extruded 5 times through a polycarbonate filter with a pore size of 1.0 μm, then extruded 5 times through a polycarbonate filter of 0.1 μm, and finally extruded 5 times through a polycarbonate filter of 0.05 μm using a hand extruder. Subsequently, the liposomes were dialyzed overnight at 4°C using a dialysis cassette with a pore size of 100 kDa (Slide-A-Lyzer G210K, 22.5-30 μM regenerated cellulose membrane) to change the pH to 7.4 and remove unencapsulated siRNA to obtain engineered exosomes.

[0098] (4) Detection of engineered exosomes

[0099] A. Electron microscopy was performed on engineered exosomes. The results were as follows Fig.12 shown.

[0100] Fig.12 The hybrid particles were shown to be quasi-circular with a typical "cup mouth" structure, and the size was mainly between 30-150nm. The identification results were consistent with the morphological identification range of hybrid particles. Electron microscopy images proved the effective hybridization of liposome exosome siCCR5.

[0101] B. Engineered exosome size

[0102] Since the hybrid particles are hybridized from exosomes, siRNA and lipid particles, and the sizes of these three particles are different, in order to detect their purity, the present invention conducted nanoparticle size analysis on the hybridized samples. The results are as follows: Fig.13 shown.

[0103] Fig.13 It shows that the distribution of engineered exosome particles is relatively uniform, and the particle size is in the range of 90-100nm, which is consistent with the particle size identification.

[0104] Example 2

[0105] Example 2 of the present invention provides a method for preparing engineered exosomes, which differs from Example 1 in that the ratio of exosomes to total lipids in Example 2 is 1:25.

[0106] Test Example 1

[0107] Experimental Example 1 of the present invention detected the therapeutic effect of the engineered exosomes prepared in Example 1 on Alzheimer's disease, and the specific method was as follows:

[0108] (1) Establishment and testing of cell models

[0109] Since Alzheimer's disease is closely related to microglia, when starch plaques are deposited in the brain, microglia will trigger an inflammatory response, release a variety of inflammatory factors, and migrate to the plaques to engulf and clear them, causing the body's immune response. Therefore, the present invention uses LPS to establish an inflammatory model of microglia.

[0110] BV2 microglia were cultured at 1×10 5The cells were subcultured at a density of 100 / well (six-well plate) and divided into three groups: control normal group, LPS inflammation model group, and EVs exosome treatment group. After 12 hours of cell attachment, 1μg / ml LPS was added to the LPS inflammation model group and EVs exosome group for 24h. After 24h, the culture medium of the EVs group was replaced with basal culture medium, and 20μg / ml hUCMSC-EVs were added for 24h. The results are shown in the figure. Fig.14 .

[0111] The present invention uses qPCR to detect the expression of inflammatory factors. Fig.14 The results showed that after 24 hours of treatment with 1 μg / ml LPS, the expression of inflammatory factors increased significantly. After exosome treatment, the expression of inflammatory factors decreased significantly, indicating that exosome treatment inhibited the inflammatory response of microglia. At the same time, after hUCMSC-EVs treatment, the migration ability of microglia was promoted, which facilitated the movement of microglia to the starch deposition site for removal.

[0112] The present invention found through sequencing that after exosome treatment, the expression of CCR5 at the gene and protein levels in microglia cells was significantly reduced ( Fig.14 ,bd), proving that umbilical cord stem cell exosomes are associated with CCR5, and that CCR5 is also closely related to the inflammatory response of microglia ( Fig.14 -a). Therefore, the present invention intends to use umbilical cord stem cell exosomes as carriers and carry siCCR5 for the treatment of Alzheimer's disease.

[0113] (2) Establishment and testing of mouse model

[0114] (1) Animal grouping

[0115] The experimental animals used were APP / PS1 transgenic mice, which have five familial AD mutant genes, including three APP mutant genes and two PS1 mutant genes. The mice have behavioral and pathological characteristics of human AD, with amyloid protein deposits and over-activated glial cells in the hippocampus and cortical tissues of the brain. Since APP / PS1 transgenic mice are heterozygous, we mated male APP / PS1 transgenic mice with female C57BL / 6 wild-type mice. Theoretically, the probability of obtaining APP / PS1 transgenic mice is one in four, so it is necessary to identify the genotype of the offspring produced by mating. In this study, the PCR method was used to detect the genotype of the DNA of the tails of one-month-old mice using primers for the APP and PSEN genes. The positive bands are transgenic model mice, see. Fig.15 .

[0116] As shown in Table 1, the experiment was divided into four groups: normal wild-type mice, AD disease group + PBS group, AD disease + umbilical cord stem cell exosome group, and AD disease + siCCR5 engineered umbilical cord stem cell exosome group, with six mice in each group. Exosomes were injected into the mice via nasal administration at a dose of 50 mg / kg, once a week, and continuously injected for one month. The difference before and after exosome injection was observed.

[0117] Table 1 In vivo experimental groups and treatments

[0118] Grouping number Reagents dose Injection frequency time WT 5 Normal saline 15μL Twice a week 4 weeks AD 5 Normal saline 15μL Twice a week 4 weeks AD-Exos 5 hUCMSC-Exos 30μg(15μL) Twice a week 4 weeks AD-siCCR5Exos 5 hUCMSC-siCCR5Exos 30μg(15μL) Twice a week 4 weeks

[0119] (2) New object recognition experiment

[0120] 1) Before testing, eliminate any unfamiliarity with the mice and stroke them every day to avoid irritation during handling.

[0121] 2) During the adaptation period, mice were allowed to move freely in the experimental apparatus (without objects) for 10 min.

[0122] 3) During the familiarization period, two identical objects (A and B, ensuring that the objects have no odor and are fixed and cannot be moved) are placed in the apparatus, with the objects approximately 10 cm away from the two side walls (to provide experimental animals with space to explore).

[0123] 4) Place the mouse in the device with its back facing the object at an equal distance from the object, and use a camera and software to record the time the mouse spends exploring each object within 5 minutes (exploration of the object is counted as touching the object with the mouth or nose and getting close to the object within 2-3 cm).

[0124] 5) After 24 hours, conduct the test by replacing one of the two identical objects with a different object (AC) and placing the mouse into the device with its back facing the object at an equal distance from the object. Use a camera and software to record the time the mouse spends exploring each object within 5 minutes.

[0125] (3) New location recognition experiment

[0126] In the training of the new object recognition experiment, two identical objects were placed on the same side. In the new position recognition experiment, the training period remained unchanged. In the test phase, the two objects were placed in diagonal positions. The mice were placed in the device with their backs facing the objects at equal distances from the objects. The camera equipment and software were used to record the exploration time of the mice on the objects in each position within 5 minutes. The results are as follows: Fig.16 shown.

[0127] Fig.16The results showed that in the experiments of new object recognition and new location recognition, the AD model mice spent less time exploring new objects (new locations), which was basically the same as the time exploring old objects (old locations). However, the Exos and siCCR5 Exos groups significantly increased the proportion of exploration time for new objects (new locations), and the effect of siCCR5 Exos was more significant, indicating that stem cell exosomes can significantly improve the memory ability of mice, and the siCCR5 engineered exosomes have a better memory improvement effect.

[0128] (4) Morris water maze test

[0129] A. Train the mice first:

[0130] 1) In water maze training, the platform should be located in the center of the pool, 1 cm from the water surface, so that the animals are aware of its presence. The water temperature should be 26°C.

[0131] 2) Each animal will undergo three consecutive trials. The animal will be placed on the platform for 20 seconds.

[0132] 3) The water maze has 4 quadrants. Place the animal in one of the quadrants. Place the animal in the water by supporting it with your hands and gently placing it tail first. Do not place the animal head first to avoid causing stress.

[0133] 4) Allow the animal to swim to find the platform for no more than 60 seconds. At first, the animal may swim at the edge of the pool, looking for an exit. Eventually, the animal will learn to find the platform and climb up.

[0134] 5) Once the animal reaches the platform, stop the timer and record the time. If it cannot find the platform within 60 seconds, record it as one minute. If the animal cannot reach the platform, do not pick it up. You can use a glass or plastic rod to guide the animal to the platform and let the animal stay on the platform for 15 seconds. Repeat the same procedure for the other two quadrants in different directions for each trial.

[0135] 6) After the animal has completed all three trials, dry it with a towel. Repeat the three-trial training process for all animals in sequence. Keep the same direction for all animals and record their times.

[0136] B. After the animals have been trained for five days, they are ready for the water maze test.

[0137] 1) Before the start of the water maze experiment, fill the pool with tap water and heat it, and set the platform 1 cm below the water surface. Use skim dry milk, or 125 ml of non-toxic white temporary paint, or food coloring to make the water opaque.

[0138] 2) Lighting and water temperature should be the same as during training.

[0139] 3) The animal is analyzed until it reaches the platform and the time taken is recorded. If the animal does not reach the platform within 60 seconds, the handler will guide it to the platform, just as in training. The animal is allowed to be on the platform for 10 seconds, then it is wiped dry and returned to the holding cage.

[0140] 4) Perform 4 trials for each animal, placing them from different quadrants, with the animal handler returning to the same designated location during each trial. The order of testing should be: first trial for all animals, second trial for all animals, third trial for all animals, and so on. The interval between trials should be at least 2 minutes.

[0141] 5) The underwater platform was removed for testing. In 60-second intervals, each mouse swam in the pool to explore the location of the platform before. The exploration trajectory of each mouse during training and testing was recorded using the water maze software for subsequent analysis.

[0142] 6) When all exploratory trials were completed, the animals were dried and the pool was drained.

[0143] Results Fig.17 .

[0144] In the mouse water maze experiment, Fig.17 Figure a shows that on the third day of training, the WT group's escape latency was significantly shortened and they were able to find the platform quickly. On the fourth day of training, the Exos group's escape latency was shortened; on the fifth day, the effect of the siCCR5-Exos group was more obvious. Fig.17 In the test trajectory in middle b, the number of times APP / PS1 crossed the platform within 60 s and the time in the target quadrant were significantly lower than those of WT. Both indicators increased in the Exos group, and were more significant in the siCCR5-Exos group. Fig.17 Zhongehe Fig.17 The results in middle f showed that there was no significant difference in swimming speed and total swimming distance among the mice in each group, indicating that the differences between mice were not affected by swimming ability. The above results show that exosomes can effectively improve the spatial memory ability of mice, and the effect of engineered exosomes siCCR5-Exos is better than that of ordinary exosomes.

[0145] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing engineered umbilical cord stem cell exosomes carrying siCCR5, characterized in that: The steps include: (1) Preparation of liposome membrane: Dissolve DLin-MC3-DMA in an organic solvent, and then dissolve cholesterol and DOPC to prepare a liposome solution; remove the organic solvent by rotary evaporation, and dry under an inert gas environment to obtain a lipid membrane; (2) Preparation of engineered exosomes: Use PBS to dissolve the lipid membrane, add siCCR5 and umbilical cord mesenchymal stem cell exosomes after ultrasonic treatment to obtain a mixed solution, and continue filtering, squeezing and dialyzing under insulation conditions to obtain engineered exosomes.

2. The method for preparing engineered umbilical cord stem cell exosomes carrying siCCR5 according to claim 1, characterized in that: In step (1), the organic solvent is a mixed solvent of chloroform and methanol in a volume ratio of 9:1; the molar ratio of DLin-MC3-DMA: cholesterol: DOPC is 25-34.5:35-65:1.5-40.

3. The method for preparing engineered umbilical cord stem cell exosomes carrying siCCR5 according to claim 1, characterized in that: In step (2), the mass ratio of the lipid membrane to PBS during the hydration and dissolution is 100:3; the mass ratio of siCCR5: umbilical cord mesenchymal stem cell exosomes: lipid membrane is 2:1:

50.

4. The method for preparing engineered umbilical cord stem cell exosomes carrying siCCR5 according to claim 1, characterized in that: In step (2), the insulation temperature is 40-50°C.

5. The method for preparing engineered umbilical cord stem cell exosomes carrying siCCR5 according to claim 1, characterized in that: In step (2), the siCCR5 can target CCR5 for knockdown, and the siCCR5 is composed of the sequence sense shown in SEQ ID NO.1 and the sequence antisense shown in SEQ ID NO.

2.

6. The method for preparing engineered umbilical cord stem cell exosomes carrying siCCR5 according to claim 1, characterized in that: In step (2), the filtering and extruding steps are: The mixed solution was extruded through a filter with a pore size of 1.0 μm for the first time, then through a filter with a pore size of 0.1 μm for the second time, and finally through a filter with a pore size of 0.05 μm for the third time.

7. The method for preparing engineered umbilical cord stem cell exosomes carrying siCCR5 according to claim 6, characterized in that: The filter used for the three-stage extrusion is a polycarbonate filter, and the number of extrusions is 2 to 5 times.

8. The method for preparing engineered umbilical cord stem cell exosomes carrying siCCR5 according to claim 1, characterized in that: In step (2), the dialysis step is: Dialyze overnight at 4°C using a 100 kDa dialysis cassette and adjust the pH to 7.2-7.

4.

9. An engineered exosome prepared by the method for preparing engineered umbilical cord stem cell exosomes carrying siCCR5 according to any one of claims 1 to 8.

10. Use of the engineered exosomes according to claim 9 as a drug for treating Alzheimer's disease.

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