An exosome mixture for treating Alzheimer's disease and its preparation method

Abstract

This application relates to the technical field of Alzheimer's disease treatment drugs, specifically disclosing an exosome mixture for treating Alzheimer's disease and its preparation method. The exosome mixture comprises Exo-miR-431 and Exo-miR-140-5p-in; the exosome content in the exosome mixture is (1.5-10)×10⁻⁶. 11 The exosome mixture of this application can be used for the treatment of Alzheimer's disease, and has the advantage of significantly improving Alzheimer's disease.

Description

Technical Field

[0001] This application relates to the technical field of drugs for treating Alzheimer's disease, and more specifically, it relates to an exosome mixture for treating Alzheimer's disease and a method for preparing the same. Background Technology

[0002] Alzheimer's disease (AD), commonly known as senile dementia, is characterized by senile plaques, neurofibrillary tangles, and the loss of cholinergic neurons in the basal ganglia of Minert, leading to hippocampal atrophy and gradual loss of perception (including cognition, personality, thoughts, behavior, and even smell and taste). The main pathological feature of this disease is the gradual accumulation and plaque formation of β-amyloid (Aβ) protein inside and outside brain neurons, blocking normal communication between neurons. The pathological mechanisms of Alzheimer's disease include: ① The amyloid hypothesis: M2 microglia can engulf Aβ plaques, but an excessive number of Aβ plaques leads to the exhaustion and gradual loss of M2 microglia function, causing neurotoxicity and resulting in neuronal death and neurodegeneration. ② The Tau protein hypothesis: Tau protein is one of the microtubule-associated proteins and can regulate the stability of microtubule assemblies. However, in Alzheimer's patients, Tau protein is highly phosphorylated or mutated, forming neurofibrillary tangles at nerve cells or linear tangles at dendrites and axons. ③ Inflammation hypothesis: Reactive glial proliferation and neuroinflammation are hallmarks of Alzheimer's disease. Overactivation of M1 microglia releases cytokines such as IL-1β and TNF-α, damaging the dendrites and spikes of neurons, leading to the gradual loss of synapses. ④ Acetylcholine hypothesis: Acetylcholine is an important neurotransmitter in cholinergic neurons; reduced acetylcholine synthesis directly leads to damage to cholinergic neurons.

[0003] Based on the above pathogenic hypotheses, current medications for treating Alzheimer's disease include: cholinesterase inhibitors (galantamine, donepezil, rivastigmine bitartrate, and tetrahydroaminoacridine) and receptor antagonists (memantine—N-methyl-D-aspartate, NMDA). However, these drugs are unlikely to cure or slow disease progression and are accompanied by serious side effects. Subsequently developed anticholinesterase (AChE) inhibitors can alleviate disease symptoms, but the effect is limited, though they have brought hope to Alzheimer's patients.

[0004] Therefore, it is necessary to provide a new pharmaceutical composition with more significant therapeutic effects. Summary of the Invention

[0005] In order to improve the treatment effect of Alzheimer's disease, this application provides an exosome mixture for treating Alzheimer's disease and a method for preparing the same.

[0006] In a first aspect, this application provides an exosome mixture for treating Alzheimer's disease, employing the following technical solution:

[0007] An exosome mixture for treating Alzheimer's disease, the exosome mixture comprising Exo-miR-431 and Exo-miR-140-5p-in; the exosome content in the exosome mixture is (1.5-10)×10 11 per mL.

[0008] By employing the above-described technical approach, the synergistic effect of the two exosomes (Exo-miR-431 and Exo-miR-140-5p-in) can significantly improve Alzheimer's disease. Exo-miR-431 refers to the exosome miR-431, which contains the exogenous gene miR-431; Exo-miR-140-5p-in refers to the exosome miR-140-5p-in, which contains the exogenous gene miR-140-5p-in. These two exosomes work synergistically to inhibit the overactivation of M1 microglia and stimulate M2 microglia to phagocytose Aβ plaques, ultimately achieving a significant therapeutic effect on Alzheimer's disease.

[0009] Optionally, the exosomes have a particle size distribution of 50-140 nm; the median particle size of the exosomes is 59 nm, the D90 is 76 nm, and the D10 is 56 nm.

[0010] Secondly, this application provides a method for preparing the above-mentioned exosome mixture for treating Alzheimer's disease, using the following technical solution:

[0011] A method for preparing the above-described exosome mixture for treating Alzheimer's disease, the method comprising the following steps: preparing a recombinant lentivirus, wherein the recombinant lentivirus includes the gene miR-431, the gene miR-140-5p-in, an resistance gene, and a selection marker gene;

[0012] The recombinant lentivirus and stem cells are brought into contact, cultured at 32-38°C, and the supernatant and exosomes are collected to obtain an exosome mixture.

[0013] By employing the above-described technical solution, the exosomes required in this application are obtained by inserting the genes miR-431 and miR-140-5p-in into lentivirus and then transfecting human adipose-derived mesenchymal stem cells (ADMSCs), followed by culturing these ADMSCs. By inserting the two genes (miR-431 and miR-140-5p-in) into the lentivirus, both genes are highly expressed in this system, resulting in high yields of miR-431 and miR-140-5p-in.

[0014] Optionally, the preparation method further includes a step of magnetizing the exosome mixture.

[0015] Optionally, the magnetization conditions include: a magnetization intensity of 5-8 A / m, a magnetization time of 15-30 min, and a magnetization temperature of 0-10℃.

[0016] By employing the above-mentioned technical solutions and magnetizing exosomes under appropriate conditions, the permeability of the exosome membrane structure and the charge and energy distribution outside the membrane structure can be increased. Ultimately, this allows Exo-miR-431 and Exo-miR-140-5p-in to release exogenous genes more completely and fully into the target cells, or allows Exo-miR-431 and Exo-miR-140-5p-in to better fuse with the target cells and release exogenous genes into the target cells, thereby exerting their effects.

[0017] In this approach, the magnetization intensity and time need to be controlled within a certain range; otherwise, it will have a significant impact on exosomes, causing the functional substances to be destroyed, which may result in negative therapeutic effects or no therapeutic effect at all.

[0018] In some embodiments, the magnetization intensity during magnetization is 5-6.3 A / m, 5.8-7.6 A / m, or 6.7-8 A / m; in some embodiments, the magnetization time during magnetization is 15-19 min, 17-22 min, 20-25 min, 23-27 min, or 26-30 min.

[0019] In some embodiments, the magnetization intensity during magnetization is 5 A / m, 5.7 A / m, 5.7 A / m, 6.1 A / m, 6.7 A / m, 7.5 A / m, or 8 A / m; in some embodiments, the magnetization time during magnetization is 15 min, 16.5 min, 18.4 min, 19.6 min, 20.7 min, 22.6 min, 24.3 min, 26.2 min, 27.1 min, 28.5 min, 29.7 min, or 30 min.

[0020] Optionally, the resistance gene is a puromycin resistance gene;

[0021] The process of bringing recombinant lentiviruses and stem cells into contact and culturing them at 32-38°C also includes a screening culture step using a medium containing puromycin.

[0022] By employing the above-mentioned technical solution, stem cells containing recombinant lentiviruses are screened using resistance genes to achieve purification, thereby further expanding the culture of target stem cells.

[0023] Optionally, the stem cells are selected from either adipose-derived mesenchymal stem cells or umbilical cord blood mesenchymal stem cells.

[0024] Optionally, the method for preparing the recombinant lentivirus includes the following steps:

[0025] Gene recombination: The miR-431 gene, the miR-140-5p-in gene, the resistance gene, and the selection marker gene are linked to the lentiviral gene to obtain recombinant lentivirus;

[0026] Viral packaging: The recombinant lentivirus is brought into contact with host cells and cultured to allow the recombinant lentivirus to be packaged within the host cells. The supernatant is then collected after centrifugation.

[0027] Optionally, the sequence of the gene miR-431 is as shown in SEQ ID NO.1, the sequence of the gene miR-140-5p-in is as shown in SEQ ID NO.2, and the lentiviral gene sequence for preparing the recombinant lentivirus is shown in SEQ ID NO.3.

[0028] Optionally, the sequence of the resistance gene is as shown in SEQ ID NO.4; the sequence of the selection marker gene is as shown in SEQ ID NO.5.

[0029] In summary, this application has the following beneficial effects:

[0030] 1. This application demonstrates that the two exosomes, Exo-miR-431 and Exo-miR-140-5p-in, have excellent synergistic effects in treating Alzheimer's disease, significantly improving the symptoms in dementia mice.

[0031] 2. This application describes a method for transfecting human adipose-derived mesenchymal stem cells with lentiviruses containing both miR-431 and miR-140-5p-in genes simultaneously. The resulting exosomes, Exo-miR-431 / 140-5p-in, exhibited high expression of these genes. When these exosomes were used on dementia-like mice, the synergistic effect of the two genes significantly improved related behaviors and indicators in the mice.

[0032] 3. Before using Exo-miR-431 / 140-5p-in for treatment, this application further magnetizes the exosomes to further improve their therapeutic effect. Attached Figure Description

[0033] Figure 1 This is a graph showing the expression levels of genes miR-431 and miR-140-5p-in in human adipose-derived mesenchymal stem cells and ordinary adipose-derived mesenchymal stem cells in Example 1.

[0034] Figure 2 This is a particle size distribution diagram of exosomes obtained in Example 1;

[0035] Figure 3 These are the behavioral results of exosome treatment in dementia mice in Examples 1, 5, and 6;

[0036] Figure 4 This is a graph showing the gene expression levels of inflammation-related factors in the hippocampus of mice treated with exosomes in Examples 1, 5, and 6 (Comparative Examples 5 and 6).

[0037] Figure 5 These are histological examination results of the hippocampus in mice treated with exosomes in Examples 1, 5, and 6. Detailed Implementation

[0038] The present application will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that: unless otherwise specified in the following embodiments, the conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from commercially available sources.

[0039] In this application, miR is an abbreviation for microRNA; Exo is an abbreviation for exosome; and in miR-140-5p-in is an abbreviation for inhibitor.

[0040] Preparation Example

[0041] The following preparation examples 1 to 7 are examples of preparation of recombinant lentiviruses.

[0042] Preparation Example 1

[0043] The preparation method of recombinant lentivirus is as follows:

[0044] Step 1: Design the gene miR-431, the gene sequence of which is shown in SEQ ID NO.1; design the gene miR-140-5p-in, the gene sequence of which is shown in SEQ ID NO.2; prepare lentivirus, the gene sequence of which is shown in SEQ ID NO.3; design the puromycin resistance gene, the gene sequence of which is shown in SEQ ID NO.4; design the green fluorescent protein selection marker gene, the gene sequence of which is shown in SEQ ID NO.5.

[0045] The genes miR-431 and miR-140-5p-in, the green fluorescent protein selection marker gene, and the puromycin resistance gene were linked to a lentiviral gene to obtain a recombinant lentivirus. The preparation of this recombinant lentivirus was commissioned to Nanjing Zebrafish Biotechnology Co., Ltd.

[0046] Step II: Add the recombinant lentivirus obtained in Step I to a culture flask for culturing 293T cells. The recombinant lentivirus will continuously package in the 293T cells. Then, centrifuge and collect the supernatant of the 293T cell culture medium (containing a large amount of packaged recombinant lentivirus), and store it at -20℃ for later use.

[0047] Preparation Example 2

[0048] The difference between this preparation example and Preparation Example 1 is that the gene miR-431 is replaced by the gene miR-17-92. The sequence of the gene miR-17-92 is shown in SEQ ID NO.6. Otherwise, it is the same as Preparation Example 1.

[0049] Specifically, the preparation method of recombinant lentivirus is as follows:

[0050] Step I: Design the gene miR-17-92, the gene sequence of which is shown in SEQ ID NO.6; design the gene miR-140-5p-in, the gene sequence of which is shown in SEQ ID NO.2; the lentivirus, puromycin resistance gene and green fluorescent protein selection marker gene are the same as those in Preparation Example 1.

[0051] The genes miR-17-92 and miR-140-5p-in, the green fluorescent protein selection marker gene, and the puromycin resistance gene were linked to a lentiviral gene to obtain a recombinant lentivirus. The preparation of this recombinant lentivirus was commissioned to Nanjing Zebrafish Biotechnology Co., Ltd.

[0052] Step II is the same as in Preparation Example 1.

[0053] Preparation Example 3

[0054] The difference between this preparation example and Preparation Example 1 is that the gene miR-140-5p-in is replaced with the gene miR-17-92. The sequence of the gene miR-17-92 is shown in SEQ ID NO.6. Otherwise, it is the same as Preparation Example 1.

[0055] Specifically, the preparation method of recombinant lentivirus is as follows:

[0056] Step I: Design gene miR-17-92, the gene sequence of which is shown in SEQ ID NO.6; design gene miR-431, the gene sequence of which is shown in SEQ ID NO.1; lentivirus, puromycin resistance gene and green fluorescent protein selection marker gene are the same as in preparation example 1.

[0057] Genes miR-17-92 and miR-431, a green fluorescent protein selection marker gene, and a puromycin resistance gene were linked to a lentiviral gene to obtain a recombinant lentivirus. The preparation of this recombinant lentivirus was commissioned to Nanjing Zebrafish Biotechnology Co., Ltd.

[0058] Step II is the same as in Preparation Example 1.

[0059] Preparation Example 4

[0060] The difference between this preparation example and Preparation Example 1 is that the gene miR-140-5p-in is replaced with the gene miR-30d-5p. The sequence of the gene miR-30d-5p is shown in SEQ ID NO.7. Otherwise, it is the same as Preparation Example 1.

[0061] Specifically, the preparation method of recombinant lentivirus is as follows:

[0062] Step I: Design the gene miR-30d-5p, whose gene sequence is shown in SEQ ID NO.6; design the gene miR-431, whose gene sequence is shown in SEQ ID NO.1; the lentivirus, puromycin resistance gene, and green fluorescent protein selection marker gene are the same as those in Preparation Example 1.

[0063] The genes miR-30d-5p and miR-431, the green fluorescent protein selection marker gene, and the puromycin resistance gene were linked to a lentiviral gene to obtain a recombinant lentivirus. The preparation of this recombinant lentivirus was commissioned to Nanjing Zebrafish Biotechnology Co., Ltd.

[0064] Step II is the same as in Preparation Example 1.

[0065] Preparation Example 5

[0066] The difference between this preparation example and Preparation Example 1 is that the gene miR-431 is replaced by the gene miR-30d-5p. The sequence of the gene miR-30d-5p is shown in SEQ ID NO.7. Otherwise, it is the same as Preparation Example 1.

[0067] Specifically, the preparation method of recombinant lentivirus is as follows:

[0068] Step I: Design the gene miR-30d-5p, whose gene sequence is shown in SEQ ID NO.7; design the gene miR-140-5p-in, whose gene sequence is shown in SEQ ID NO.2; the lentivirus, puromycin resistance gene, and green fluorescent protein selection marker gene are the same as those in Preparation Example 1.

[0069] The genes miR-30d-5p and miR-140-5p-in, the green fluorescent protein selection marker gene, and the puromycin resistance gene were linked to a lentiviral gene to obtain a recombinant lentivirus. The preparation of this recombinant lentivirus was commissioned to Nanjing Zebrafish Biotechnology Co., Ltd.

[0070] Step II is the same as in Preparation Example 1.

[0071] Preparation Example 6

[0072] The difference between this preparation example and Preparation Example 1 is that the gene miR-140-5p-in is not inserted into the lentivirus; instead, the recombinant lentivirus is prepared by inserting the gene miR-431 into the lentivirus.

[0073] Specifically, the preparation method of recombinant lentivirus is as follows:

[0074] Step I: The miR-431 gene, lentivirus, puromycin resistance gene, and green fluorescent protein selection marker gene were prepared in the same way as in Example 1.

[0075] The miR-431 gene, the green fluorescent protein selection marker gene, and the puromycin resistance gene were linked to a lentiviral gene to obtain a recombinant lentivirus. The preparation of this recombinant lentivirus was commissioned to Nanjing Zebrafish Biotechnology Co., Ltd.

[0076] Step II is the same as in Preparation Example 1.

[0077] Preparation Example 7

[0078] The difference between this preparation example and Preparation Example 1 is that the miR-431 gene is not inserted into the lentivirus; instead, the miR-140-5p-in gene is inserted into the lentivirus to prepare the recombinant lentivirus.

[0079] Specifically, the preparation method of recombinant lentivirus is as follows:

[0080] Step I: The miR-140-5p-in gene, lentivirus, puromycin resistance gene, and green fluorescent protein selection marker gene were prepared in the same way as in Example 1.

[0081] The miR-140-5p-in gene, the green fluorescent protein selection marker gene, and the puromycin resistance gene were linked to a lentiviral gene to obtain a recombinant lentivirus. The preparation of this recombinant lentivirus was commissioned to Nanjing Zebrafish Biotechnology Co., Ltd.

[0082] Step II is the same as in Preparation Example 1.

[0083] Preparation Example 8

[0084] The preparation method of adipose-derived mesenchymal stem cells is as follows:

[0085] (1) Obtaining a cell cluster resuspension of adipose-derived mesenchymal stem cells:

[0086] Sterile adipose tissue was obtained from a cosmetic surgery clinic. The tissue was first rinsed with 0.01 mol / L sterile PBS buffer until it was bloodless. Then, the adipose tissue was cleaned with sterile ophthalmic scissors and forceps and cut into pieces approximately 1 mm in size. 3 The adipose tissue was divided into small, block-sized pieces. Then, an appropriate amount of 0.1 wt% type I collagenase solution was added to the block-shaped adipose tissue for digestion, and the digestion was carried out with shaking at 37°C for 30 min. Digestion was then terminated with Gibco cell culture medium containing 10 vol.% fetal bovine serum, and centrifuged at 1000 rpm for 15 min. The supernatant of the centrifuged suspension was discarded. The incompletely digested adipose tissue blocks and the underlying cell clusters were resuspended in mesenchymal stem cell basal culture medium, mixed thoroughly, and the cell cluster resuspension was obtained.

[0087] (2) Expanded culture: The cell clusters obtained in step (1) were resuspended and cultured in a 37°C, 5 Vol.% CO2 incubator. After 3 days of adherent culture, adipose-derived mesenchymal stem cells were obtained and expanded in large quantities using a cell factory for later use.

[0088] Example

[0089] Example 1

[0090] The preparation method of the exosome mixture is as follows:

[0091] Step 1: Add serum-free basal medium for human mesenchymal stem cells (AM-V Serum-Free Medium; catalog number SC-2013-GA, Tianjin Haoyang) to a T175 culture flask, and transfer the human adipose mesenchymal stem cells prepared in Example 8 to the flask. Add 1 wt% penicillin and an appropriate amount of recombinant lentivirus prepared in Example 1, and then culture at 37°C for 24 h.

[0092] Step 2: Then, replace the serum-free basal medium for human adipose-derived mesenchymal stem cells in bottle T175 with serum-free selective medium for human adipose-derived mesenchymal stem cells, and add 2 μg / mL puromycin. Continue screening and culturing at 37°C until all human adipose-derived mesenchymal stem cells show obvious green fluorescence under a fluorescence microscope.

[0093] Step 3: Continue to expand with serum-free selective medium for human mesenchymal stem cells and start continuously collecting culture supernatant. Concentrate the culture supernatant using the 3D Flo Trix viva EXO exosome harvesting system (produced by Beijing Huakan Company) to obtain an exosome mixture.

[0094] Example 2

[0095] The difference between this embodiment and Embodiment 1 is that, after step three, a fourth step is included. Specifically, step four involves magnetizing the exosome mixture obtained in step three under the following conditions: magnetization for 20 minutes at a magnetization intensity of 6 A / m and a temperature of 0°C. All other conditions are the same as in Embodiment 1.

[0096] Comparative Example

[0097] Comparative Examples 1-6

[0098] The difference between Comparative Examples 1-6 and Example 1 lies in the selection of recombinant lentiviruses.

[0099] Specifically:

[0100] Comparative Example 1 replaces the recombinant lentivirus of Example 1 with an equal amount of the recombinant lentivirus prepared in Preparation Example 2.

[0101] Comparative Example 2 replaces the recombinant lentivirus of Example 1 with an equal amount of the recombinant lentivirus prepared in Preparation Example 3.

[0102] Comparative Example 3 replaces the recombinant lentivirus of Example 1 with an equal amount of the recombinant lentivirus prepared in Preparation Example 4.

[0103] Comparative Example 4 replaces the recombinant lentivirus of Example 1 with an equal amount of the recombinant lentivirus prepared in Preparation Example 5.

[0104] Comparative Example 5 replaces the recombinant lentivirus of Example 1 with an equal amount of the recombinant lentivirus prepared in Preparation Example 6.

[0105] Comparative Example 6 replaces the recombinant lentivirus of Example 1 with an equal amount of the recombinant lentivirus prepared in Preparation Example 7.

[0106] Effects characterization

[0107] 1. Real-time PCR detection

[0108] The human adipose-derived mesenchymal stem cells infected with recombinant lentivirus in Example 1 and ordinary adipose-derived mesenchymal stem cells were subjected to Real-time-PCR detection. The results are shown in [Figure 1]. Figure 1 .from Figure 1 The results showed that, after transfection with recombinant lentivirus, the expression intensity of the human adipose-derived mesenchymal stem cells in Example 1 was significantly increased at the mRNA level for the genes miR-431 and miR-140-5p-in. Specifically, the expression intensity of miR-431 at the mRNA level increased by 35-fold, and the expression intensity of miR-140-5p-in at the mRNA level increased by 46-fold. This result fully demonstrates that this method can simultaneously achieve efficient expression of both miR-431 and miR-140-5p-in at the mRNA level.

[0109] 2. The concentration of the exosome mixture obtained after concentration in Example 1 was determined using nanoparticle tracking analysis (NTA). Specific results are shown in [link to results]. Figure 2 . Figure 2 The results showed that the particle size of Exo-miR-431 / 140-5p-in was 53-135 nm, with a median particle size of 59 nm, a D90 of 76 nm, and a D10 of 56 nm. The detection results of Comparative Examples 1 and 2 were similar.

[0110] 3. Animal experiments

[0111] Six-month-old APP / PS1 mice exhibiting dementia symptoms were selected and divided into three treatment groups (n=10 per group): Exo-miR-431 (administered with exosomes from Comparative Example 5), Exo-miR-130-5p-in (administered with exosomes from Comparative Example 6), and Exo-miR-431 / 140-5p-in (administered with exosomes from Example 1). Ten six-month-old C57BL / 6 mice served as a normal control group, and ten untreated six-month-old APP / PS1 mice served as a control group. After one month of treatment, the improvement in dementia-related symptoms was compared across the four groups, and the mechanism of action was explored from the perspective of inflammatory mediators.

[0112] 3.1 Behavioral Assessment

[0113] 3.1.1. Open field test and novel object recognition test were performed on each group of mice. The results are shown in […]. Figure 3 The results showed that the motor function and emotional state of the mice in Example 1 were close to those of the normal group, and significantly better than those of Comparative Example 5, Comparative Example 6 and the control group.

[0114] 3.1.2. The water maze test was performed on each group of mice, and the results are shown in [the table below]. Figure 3In Example 1, the number of times mice crossed the platform, the time required to reach the platform, and the time spent on the platform were all close to those of the normal group, and significantly better than those of Comparative Examples 5, 6, and the control group. Based on a comprehensive evaluation of the behavioral improvements in each group, the exosomes in Example 1 significantly improved the symptoms in mice compared to Comparative Examples 5 and 6.

[0115] 3.2 Detection of inflammatory mediators in the hippocampus

[0116] The relative mRNA expression levels of inflammatory cytokines (IL-1β and TNF-α) in hippocampal tissue were detected by qPCR. See the attached results for details. Figure 4 In Example 1, the mRNA expression levels of inflammatory cytokines in the mouse hippocampus were close to those in the normal group, and significantly lower than those in Comparative Examples 5, 6, and the control group. This indicates that the exosomes in Example 1 can significantly inhibit the gene expression of IL-1β and TNF-α, two cytokines that damage neuronal dendrites and spikes, thereby significantly reducing the damage of IL-1β and TNF-α to neuronal cells, and thus treating Alzheimer's disease.

[0117] Hippocampal tissue was analyzed using an enzyme-linked immunosorbent assay (ELISA). See attached results. Figure 4 The results indicate that the expression levels of mRNA corresponding to the Aβ plaques in Example 1 were close to those in the normal group, and significantly lower than those in Comparative Examples 5, 6, and the control group.

[0118] 3.3 Histological examination of the hippocampus

[0119] Hippocampal tissue was observed using an electron microscope. See details below. Figure 5 In Example 1, the dendritic and spinous process density, as well as the number and density of neural synapses, of the mice were similar to those of the normal group, and significantly higher than those of Comparative Example 5, Comparative Example 6, and the control group.

[0120] In summary, Exo-miR-431 / 140-5p-in is significantly more effective than Exo-miR-431 and Exo-miR-140-5p-in alone in treating Alzheimer's disease.

[0121] Based on the above research, this application further investigated the therapeutic effects of exosomes in Example 2 and Comparative Examples 1-4. The specific process and results are as follows.

[0122] Six-month-old APP / PS1 mice exhibiting dementia symptoms were selected and divided into five treatment groups (n=10 per group): Exo-miR-431 / 140-5p-in with magnetization (administered with exosomes from Example 2), Exo-miR-17-92 / 140-5p-in (administered with exosomes from Comparative Example 1), Exo-miR-431 / 17-92 (administered with exosomes from Comparative Example 2), Exo-miR-431 / 30d-5p (administered with exosomes from Comparative Example 3), and Exo-miR-30d-5p / 140-5p-in (administered with exosomes from Comparative Example 4). Ten six-month-old C57BL / 6 mice served as the normal control group, and ten untreated six-month-old APP / PS1 mice served as the control group. After one month of treatment, the improvement in dementia-related symptoms was compared among the four groups.

[0123] 4.1 Conduct behavioral assessment experiments using the methods described above. The specific results are shown in Table 1.

[0124] Table 1 Results of behavioral assessment experiments on mice using different protocols

[0125]

[0126]

[0127] Note: The data in Table 1 are averages.

[0128] The data in Table 1 show that treating mice with Exo-miR-431 / 140-5p-in (Examples 1 and 2) significantly improved the behavioral characteristics of dementia mice. Comparing Examples 1 and 2, it was found that magnetizing Exo-miR-431 / 140-5p-in before treatment of dementia mice resulted in better efficacy. This may be because magnetization itself has a certain effect on exosomes, improving their charge and energy distribution, and also influencing their membrane structure, thus affecting membrane permeability. This makes it easier for substances within the exosomes to be released or to fuse with target cells, thereby better releasing exogenous genes into or between target cells. Therefore, this ultimately leads to better therapeutic effects.

[0129] However, it was also observed that transfecting human adipose-derived mesenchymal stem cells with recombinant lentiviruses obtained by randomly inserting two microRNA genes, and then treating dementia mice with exosomes prepared from these exosomes, did not necessarily yield good therapeutic effects. Exo-miR-17-92 has a neuronal repair function, and Exo-miR-30d-5p reverses M1 microglia, which have neuronal-damaging effects, into M2 microglia, which have neuronal-repairing functions. However, data from Comparative Examples 1, 4, and 1 showed that the synergistic effect of Exo-miR-140-5p-in with Exo-miR-17-92 or Exo-miR-140-5p-in with Exo-miR-30d-5p was insufficient to achieve excellent behavioral improvements in dementia-like mice. The reasons may be as follows: when the genes miR-17-92 and miR-140-5p-in are simultaneously inserted into lentivirus and transfected into human adipose-derived mesenchymal stem cells, they are difficult to express efficiently, resulting in insufficient expression of Exo-miR-140-5p-in / 17-92, which makes it difficult to exert a good effect; it may also be because when the genes miR-17-92 and miR-140-5p-in are added at the same time to treat dementia mice, the exogenous genes miR-17-92 and miR-140-5p-in have ineffective or negative regulation on the mice's own genes, thus making it difficult to achieve the effect of synergistically improving the behavior of dementia mice. However, the data from Comparative Examples 2, 3, and 1 indicate that Exo-miR-431, when synergistic with Exo-miR-17-92 or Exo-miR-431, when synergistic with Exo-miR-30d-5p, also fails to achieve a superior effect in improving the behavior of dementia-like mice. This may be related to the expression level or regulatory effect of exogenous genes.

[0130] 4.2. Histological examination of the hippocampus was performed using the methods described above. The specific results are shown in Table 2.

[0131] Table 2. Histological examination results of the mouse hippocampus under different protocols.

[0132]

[0133] Note: The data in Table 2 are average values.

[0134] Similarly, differences in the expression levels and regulatory effects of exogenous genes can lead to varying effects of different exosomes on relevant indicators in the hippocampus of dementia-affected mice. The data from Comparative Examples 1, 4, and 1 in Table 4 indicate that the synergistic effects of Exo-miR-140-5p-in with Exo-miR-17-92 or Exo-miR-140-5p with Exo-miR-30d-5p are unlikely to achieve significant behavioral improvements in dementia-affected mice. Similarly, the data from Comparative Examples 2, 3, and 1 indicate that the synergistic effects of Exo-miR-431 with Exo-miR-17-92 or Exo-miR-431 with Exo-miR-30d-5p are also unlikely to achieve significant behavioral improvements in dementia-affected mice.

[0135] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. An exosome mixture for treating Alzheimer's disease, characterized in that, The exosome mixture comprises Exo-miR-431 and Exo-miR-140-5p-in; the exosome content in the exosome mixture is (1.5-10)×10⁻⁶. 11 cells / mL; The preparation method includes the following steps: Prepare a recombinant lentivirus, wherein the recombinant lentivirus includes the gene miR-431, the gene miR-140-5p-in, an resistance gene, and a selection marker gene; The recombinant lentivirus and stem cells were brought into contact, cultured at 32-38°C, and the supernatant and exosomes were collected to obtain an exosome mixture. The stem cells are adipose-derived mesenchymal stem cells; The sequence of the gene miR-431 is shown in SEQ ID NO.1, and the sequence of the gene miR-140-5p-in is shown in SEQ ID NO.2; the lentiviral gene sequence for preparing the recombinant lentivirus is shown in SEQ ID NO.

3.

2. The exosome mixture for treating Alzheimer's disease according to claim 1, characterized in that, The exosomes have a particle size distribution of 50-140 nm; the median particle size of the exosomes is 59 nm, the D90 is 76 nm, and the D10 is 56 nm.

3. The method for preparing an exosome mixture for treating Alzheimer's disease according to claim 1, characterized in that, The preparation method further includes the step of magnetizing the exosome mixture.

4. The method for preparing an exosome mixture for treating Alzheimer's disease according to claim 3, characterized in that, The magnetization conditions include: a magnetization intensity of 5-8 A / m, a magnetization time of 15-30 min, and a magnetization temperature of 0-10℃.

5. The method for preparing an exosome mixture for treating Alzheimer's disease according to claim 1, characterized in that, The resistance gene is a puromycin resistance gene; The process of bringing recombinant lentiviruses and stem cells into contact and culturing them at 32-38°C also includes a screening culture step using a medium containing puromycin.

6. The method for preparing an exosome mixture for treating Alzheimer's disease according to claim 1, characterized in that, The method for preparing the recombinant lentivirus includes the following steps: Gene recombination: The miR-431 gene, the miR-140-5p-in gene, the resistance gene, and the selection marker gene are linked to the lentiviral gene to obtain recombinant lentivirus; Viral packaging: The recombinant lentivirus is brought into contact with host cells and cultured to allow the recombinant lentivirus to be packaged within the host cells. The supernatant is then collected after centrifugation.

7. The method for preparing an exosome mixture for treating Alzheimer's disease according to claim 1, characterized in that, The sequence of the resistance gene is as shown in SEQ ID NO.4; the sequence of the selection marker gene is as shown in SEQ ID NO.5.

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