CN113181216A - Mesenchymal stem cell exosome-AM 1241 complex and application thereof in treating Alzheimer disease - Google Patents
Mesenchymal stem cell exosome-AM 1241 complex and application thereof in treating Alzheimer disease Download PDFInfo
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Abstract
The invention relates to a mesenchymal stem cell exosome-AM 1241 complex and application thereof in treating Alzheimer disease. The invention discovers for the first time that the exosome carries CB2 receptor selective agonist AM1241 which can effectively improve the symptoms of the Alzheimer disease, and animal experiments show that the Exo-AM1241 nano-drug system can basically restore the learning and memory ability of the mouse model of the Alzheimer disease to a normal level; AM1241 is found to be effective in improving the Alzheimer disease for the first time; the improvement effect of the mesenchymal stem cell exosome-AM 1241 complex on the Alzheimer disease is remarkably superior to that of the single AM1241 or exosome, and the obvious difference of the functions and the molecular mechanisms of the three in improving the Alzheimer disease is disclosed.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a mesenchymal stem cell exosome-AM 1241 complex and application thereof in treating Alzheimer disease.
Background
Alzheimer's Disease (AD) is a chronic degenerative central nervous system disease, in which clinical symptoms such as progressive memory loss, cognitive dysfunction, personality changes and language disorders mainly appear, and the social, professional and life functions of patients are seriously affected. Alzheimer's disease not only imposes a heavy burden on patients and family members thereof, but also is a serious social and economic problem. However, the pathogenesis of AD is not clear at present, most of the existing clinical drugs can only delay the progress of dementia exacerbation, and cannot treat the dementia fundamentally, so that a new treatment target and a new drug carrier/new drug dosage form are urgently needed to be searched, and a lot of research works are continuously devoted to searching for a safe and effective novel preparation for the Alzheimer disease.
The nano material as a novel drug delivery system has the advantages of slow release, reduction of degradation of the drug in a non-focal region, good biocompatibility and the like. The exosome as a natural nano material contains a plurality of bioactive compounds, can penetrate through a blood brain barrier, promotes nerve regeneration, and has great potential in treatment of neurodegenerative diseases. But the single exosome material has poor targeting and limited curative effect. The patent entitled application of a compound in preparing a medicament for treating Parkinson's disease at the earlier stage of the subject group (201510210663.0) researches the treatment effect of a cannabinoid receptor agonist AM1241 on the Parkinson's disease, and researches show that the AM1241 activates CB2 expression of substantia nigra and hippocampus and has a remarkable neuroprotective effect. There is also literature (De Carvalho C R, Hoeller A, Franco P L C, et al, the cannabinoid CB2 receptor-specific agonist AM1241 involved in creating a specific differentiation strategy in a Wistar rates [ J ]. Epilepsy Research,2016,127: 160-. However, no report on the effect of AM1241 on the treatment of Alzheimer's disease is found at present, and no report on the utilization of AM1241 as a functional targeting drug to assist an exosome material in the treatment of Alzheimer's disease is found.
Disclosure of Invention
The invention aims to provide a novel medicine for treating Alzheimer disease and a preparation method and application thereof aiming at the defects in the prior art.
In a first aspect, the invention provides a mesenchymal stem cell exosome-AM 1241 complex prepared from a mesenchymal stem cell exosome loaded AM 1241.
As a preferred example of the present invention, AM1241 is loaded on mesenchymal stem cell exosomes by an electrostatic adsorption method.
As another preferred example of the present invention, the mesenchymal stem cell is a bone marrow, umbilical cord blood, amnion, placenta, fat, dental pulp or peripheral blood-derived mesenchymal stem cell.
In a second aspect, the present invention provides a method for preparing a mesenchymal stem cell exosome-AM 1241 complex as described above, comprising a step of loading AM1241 on a mesenchymal stem cell exosome.
In a preferred embodiment of the present invention, the preparation method comprises loading the AM1241 on the mesenchymal stem cell exosome by an electrostatic adsorption method.
In a third aspect, the invention provides an application of the mesenchymal stem cell exosome-AM 1241 complex in preparation of a medicine for treating Alzheimer's disease.
In a fourth aspect, the invention provides the use of AM1241 in the manufacture of a medicament for the treatment of alzheimer's disease.
As a preferred example of the invention, the medicament is in the form of injection or oral preparation.
As another preferred embodiment of the present invention, the medicament is administered by intravenous, arterial, oral, parenteral, buccal, vaginal, rectal, inhalation, insufflation, sublingual, intramuscular, subcutaneous, topical, intranasal, intraperitoneal, or intracranial routes.
In a fifth aspect, the present invention provides a pharmaceutical composition for treating alzheimer's disease, comprising the mesenchymal stem cell exosome-AM 1241 complex as described above, and a pharmaceutically acceptable carrier.
The invention has the advantages that:
1. the exosome loaded CB2 receptor selective agonist AM1241 is found for the first time to be capable of effectively improving the symptoms of the Alzheimer disease, and animal experiments show that the Exo-AM1241 nano-drug system can enable the learning and memory ability of an Alzheimer disease model mouse to be basically restored to a normal level, so that unexpected technical effects are achieved.
2. AM1241 was found for the first time to be effective in ameliorating the symptoms of Alzheimer's disease.
3. Related pathways of activation of CB2 receptors and the functions and molecular mechanisms of targeting Exosome materials in improving Alzheimer's disease in the process of treating Alzheimer's disease by Exo-AM1241 are disclosed, and the difference of signal pathways of AM1241 or an Exosome treatment group is analyzed. The result shows that compared with single exosomes and AM1241, Exo-AM1241 can more effectively inhibit the neuronal apoptosis caused by amyloid deposition and toxicity thereof, and meanwhile, the expression levels of apoptosis-related proteins clear caspase3 and clear caspase9 are also obviously reduced. The single exosome generates a treatment effect by influencing the expression of calcium signal pathway related kinases such as calcium/calmodulin dependent protein kinase 2A (Camk II alpha), inositol 1,4,5 triphosphate 3 kinase A (ITPKA) and the like; AM1241 alone produces therapeutic effects primarily by affecting neural tissue ligand-receptor interactions, including upregulation of dopamine receptor D1(Drd1) and the expression of transcription factor Lhx 8; the Exo-AM1241 nano-drug preparation influences the expression of related genes of a downstream calcium signal channel and an Erk signal channel by activating a CB2 receptor, and the related genes comprise a neuropeptide Y receptor 2(Npy2R) and a transcription factor NKX 2.1, promote the regeneration of neurons and improve the pathological response of the Alzheimer disease.
Drawings
FIG. 1 is a transmission electron microscope image of exosomes and Exo-AM 1241.
FIG. 2 shows the measurement of the intracerebral drug concentration after administration of AM1241 and Exo-AM 1241.
FIG. 3 is a graph of experimental results of behavioral improvement of Alzheimer's disease model mice by exosome/AM 1241/Exo-AM1241 treatment groups.
FIG. 4 is a graph of the effect of exosome/AM 1241/Exo-AM1241 treatment groups on the area of amyloid deposition in the brains of mice model Alzheimer's disease.
FIG. 5 is a graph of the effect of exosome/AM 1241/Exo-AM1241 treatment groups on neuronal apoptosis in Alzheimer's disease model mouse brain.
FIG. 6 shows the signal pathway changes in brain tissue following exosome/AM 1241/Exo-AM1241 treatment in Alzheimer's disease model mice.
Detailed Description
The present invention is further described below in conjunction with specific embodiments, but it should be understood by those skilled in the art that the following embodiments are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.
Example 1
The research route of the invention is as follows:
(1) constructing an Exo-AM1241 nano-drug system, identifying the nano-drug system by using a transmission electron microscope and an immunoblotting method, and comparing the brain-entering efficiency of the nano-drug system and the brain-entering efficiency of the single AM1241 by using a high performance liquid chromatography;
(2) the Exo-AM1241 is used for treating Alzheimer disease model mice, and the influence of the Exo-AM1241 on the learning and memory ability of the animals is analyzed;
(3) analyzing the inhibition effect of Exo-AM1241 on amyloid deposition and neuron apoptosis by means of immunofluorescence, immunoblotting and the like;
(4) RT-qPCR was used to explore the potential mechanism of Exo-AM 1241/exosomes/AM 1241 for treating Alzheimer's disease.
The experimental contents comprise:
1. preparation of Exo-AM1241 nano-drug system
And (3) recovering the Mesenchymal Stem Cells (MSC) of the mouse, taking out the cryopreservation tube in which the MSC is frozen from the liquid nitrogen tank, and quickly putting the tube into a water bath kettle at 37 ℃ for shaking to accelerate the melting of the culture solution containing the cells. After thawing, the cell suspension was transferred toCentrifuging 5ml 37 deg.C preheated culture solution (containing 10% MSC special fetal calf serum, 1% double antibody F12 DMEM culture solution) at 1000rpm for 5min, removing supernatant, re-suspending with prepared culture solution, transferring to T25 culture flask, placing at 37 deg.C, and adding 5% CO2A cell culture box. When the cell density reaches about 90%, the cells are passaged. Discarding the culture solution, washing with PBS, adding a proper amount of pancreatin containing EDTA (covering the bottom of the culture bottle/dish), digesting for 2min until the cells shrink and become round, adding the culture solution to stop digestion, slightly blowing the culture bottle/dish, transferring to a centrifuge tube, centrifuging at 1000rpm for 5min, discarding the supernatant, resuspending the cells with the preheated culture solution, transferring to a new culture bottle/dish, and placing into an incubator for culture.
And gradually removing cells, cell debris and soluble protein in the overnight culture solution by a density gradient centrifugation method to finally obtain the exosome material with higher purity. Culturing MSC until cell density reaches 80%, discarding FBS-containing culture solution, washing with PBS 3 times, adding 1% double antibody-containing F12 DMEM culture solution, culturing for 12 hr, and transferring the culture solution to a centrifuge tube. Centrifuging for 10min at 300g, taking the supernatant into a new centrifuge tube, centrifuging for 10min at 2000g, taking the supernatant, centrifuging for 30min at 10000g, taking the supernatant, centrifuging for 70min at 100000g, discarding the supernatant, re-suspending the precipitate with PBS, centrifuging for 70min at 100000g, re-suspending the precipitate with PBS, and obtaining the suspension containing the enriched exosome material.
The exosome material carries a small molecule drug AM1241, 1ml of exosome is taken to be mixed with 1mg/ml AM 12411 ml, and the mixture is put into a preheated incubator at 37 ℃ and mixed for 2 hours by a shaking table. Centrifuging at 100000g for 70min to obtain precipitate, which is the exosome-AM 1241 nano drug-carrying system successfully constructed.
Observing an exosome material and Exo-AM1241 (shown in figure 1) by using a transmission electron microscope, dripping the material on a 200-mesh copper mesh, observing the morphology characteristics of the material by using a transmission electron microscope and 200kV accelerating voltage after air drying, wherein the exosome material and the Exo-AM1241 both have a saucer-like structure with a central depression of about 100nm in diameter.
2. Exosomes improve AM1241 brain targeting
Detecting the drug loading capacity of the exosome, preparing AM1241 solutions (200, 400, 600, 800 and 1000 mu g/ml) with different concentrations, and measuring the ultraviolet light absorption value to obtain a standard curve corresponding to the concentration and the light absorption value. And (3) carrying the medicine on the exosome material, centrifuging, taking the precipitate to measure the light absorption value, and contrasting with the working curve of the AM1241 to obtain the concentration of the AM1241 in the exosome.
Mixing 1ml of exosome with 1mg/ml of AM 12411 ml, placing into a preheated incubator at 37 ℃, and mixing for 2h by a shaker. 100000g were centrifuged for 1h, the pellet was taken and finally resuspended in 300. mu.l PBS. Mice were fixed with a tail vein syringe, the tail vein of the mice was dilated with hot water, and 200. mu.l of the drug was injected. Different time points are set, namely 10min, 2h, 4h, 6h, 12h and 24 h. Mice were sacrificed by cervical dislocation and brain tissue was removed and weighed. Adding 200 μ l water into brain tissue, homogenizing at low temperature, adding 400 μ l acetonitrile, vortex shaking, and 13.3 × 103Centrifuging at 4 deg.C for 30min, and collecting supernatant. Detecting by High Performance Liquid Chromatography (HPLC) under the conditions of 10% acetonitrile and 90% double distilled water for the first 10min, 70% acetonitrile and 30% double distilled water for the last 17min, and detecting at 270 nm. The measured peak area was subjected to relative peak area calculation according to the brain tissue weight. Fig. 2 is a schematic diagram showing the enrichment of AM1241 and exosome-AM 1241 in brain tissue after administration in this example. The experiment shows that the exosome-AM 1241 can efficiently enter the brain to play a role by penetrating through a blood brain barrier, the brain drug concentration is improved, when the drug concentration reaches the peak value in 12 hours, the brain drug concentration of the Exo-AM1241 group is about 2.3 times of that of the AM1241 group alone, and the exosome-AM 1241 has an obvious brain targeting effect compared with the AM1241 alone.
3. Influence of Exo-AM1241 on learning and memory ability of Alzheimer disease model animals
APP/PS1 transgenic Alzheimer's disease model mice were prepared, randomized into groups, and exosomes, AM1241, Exo-AM1241 and PBS were administered, while normal control groups were set. Preparing drug and material drug-loaded solution, and carrying out tail vein injection on mice, wherein the administration dosage of AM1241 and Exo-AM1241 in the AM1241 group is 6 mg/kg; the dosing was performed for two weeks, once every two days.
And at the dosing end point, performing a water maze experiment to analyze the learning and memory abilities of the model animal. The experimental procedure for the water maze included: a location navigation experiment (place navigation) for detecting the memory acquisition ability of the mouse in the water maze learning process, observing and recording a route map and required time for the mouse to find and climb to a platform; and (3) probe test for evaluating the capability of the animal to maintain the spatial position memory of the platform after learning to find the platform. After the positioning navigation experiment is finished, the platform is removed from the water maze system, animals are put into water from the same water inlet point, and parameters such as the time required for the animals to reach the original platform for the first time, the times of crossing the original platform and the like are recorded. As shown in fig. 3, the platform route found in the group administered exosome-AM 1241 was substantially restored to the level of normal control mice. The number of times of table crossing of the exosome-AM 1241 treatment group was significantly increased compared to the disease model mice injected with PBS (p ═ 0.025), which was 1.8 times that of exosome alone and 1.6 times that of AM1241 alone; exosome-AM 1241 treatment groups also had significant differences in residence time in the target quadrant over the control placebo group (p ═ 0.0008). Although the exosome alone and the AM1241 alone can also improve the learning and memory ability of the animals to some extent, the differences from the placebo group are not significant. It is proved that the Exo-AM1241 nano-drug system can more effectively improve the symptoms of the Alzheimer disease with the decline of learning and memory abilities compared with the independent exosome or AM 1241.
4. Effect of Exo-AM1241 on brain amyloid deposition in Alzheimer's disease model animals
The mice were removed 2min in advance and were anesthetized by intraperitoneal injection of pentobarbital sodium solution (45 mg/kg). Fixing limbs after the mouse is paralyzed, opening the chest cavity to expose the heart, cutting a small opening on the right auricle, inserting a pin on the left ventricle, slowly infusing PBS (PBS) of about 20ml until the liquid is not bright red, and then slowly infusing 4% Paraformaldehyde (PFA) until the body of the mouse is hardened. The brain tissue of the mice was completely removed and placed on ice, and the hippocampal tissue of the mice was dissected off. The dissected hippocampus was placed in PFA and shaken overnight in a shaker at room temperature. PFA was discarded and washed 2 times with PBS for 15 min. PBS was discarded, and a 15% by mass sucrose solution was added and left overnight at 4 ℃. The 15% sucrose solution was discarded, replaced with a 30% sucrose solution and left at 4 ℃ overnight, and after overnight, replaced with a new 30% sucrose solution and stored at 4 ℃. Taking out the processed Hippocampus, placing into embedding box, adding encapsulant, freezing at-20 deg.C, and standing at-80 deg.C overnight. The block was removed from the cassette, cut to 15 μm thickness, mounted on positive charge-modified slides and stored at-80 ℃.
Taking out the required tissue section, washing with PBS for 10-15min until the encapsulant is rinsed, and only the tissue is pasted on the glass slide and dried. The immunohistochemical pen circled the tissue and the permeation solution (PBS containing 1% triton X) was dropped for 15 min. The permeate was removed and blocking solution (PBS containing 5% coat serum and 0.3% triton X) was added and blocked at 37 ℃ for 60 min. Preparing a primary antibody: amyloid β (1:200 diluted with blocking solution) was added dropwise to brain tissue. Slides were placed in wet boxes with PBS and incubated overnight at 4 ℃. The primary antibody was discarded and washed three times with PBS, one time for 10 min. The secondary antibody is diluted at a ratio of 1:200, and the secondary antibody is added dropwise and incubated for 1h at 37 ℃. The secondary antibody was removed and washed three times with PBS for 10 min. DAPI staining solution is added dropwise and incubated for 10min at room temperature. PBS was washed three times for 10 min. After the glass slide is dried, the mounting agent is dripped, and a cover glass is covered. Fluorescence signals were detected using laser confocal. As shown in fig. 4, the area of amyloid deposition in brain tissue of Exo-AM1241 treated group was approximately 1/5 in the model group, the area of amyloid deposition in brain tissue of AM1241 treated group was reduced by 1.67 times compared to the model group, and the area of amyloid deposition in brain tissue of exosome-treated group alone was reduced by half compared to the model group. Compared with the single exosome and AM1241, the Exo-AM1241 nano-drug preparation can inhibit amyloid deposition in brain tissues more effectively, the deposition area of amyloid in an exosome treatment group is about 2.5 times that of Exo-AM1241, and the deposition area of amyloid in an AM1241 treatment group is about 3 times that of Exo-AM 1241.
5. Effect of Exo-AM1241 on brain neuron apoptosis in Alzheimer's disease model animals
The TUNEL kit is used for detecting the apoptosis of the cerebral neurons of different treatment groups of animals. Taking out the required tissue section, washing with PBS for 10-15min until the encapsulant is rinsed, and only the tissue is pasted on the glass slide and dried. The immunohistochemical pen circled the tissue and the permeation solution (PBS containing 1% triton X) was dropped for 15 min. The appropriate amount of TUNEL detection solution was prepared according to the TUNEL apoptosis detection kit instructions, mixed well, 50. mu.l of TUNEL detection solution was added to the sample, and incubated at 37 ℃ for 60 minutes in the absence of light. TUNEL staining solution was discarded and washed three times with PBS for 10 min. DAPI staining solution is added dropwise and incubated for 10min at room temperature. PBS was washed three times for 10 min. After the glass slide is dried, the mounting agent is dripped, and a cover glass is covered. Fluorescence signals were detected using laser confocal. The results show that the apoptosis rate in the brain tissue of the model animal is 4.8 times that of the Exo-AM1241 treatment group, 3.6 times that of the AM1241 treatment group and 1.2 times that of the single exosome treatment group. Compared with the single exosome and AM1241, the Exo-AM1241 nano-drug preparation can more effectively inhibit the neuronal apoptosis in brain tissue, the apoptosis rate in the brain tissue sample of the single exosome treatment group is about 3.9 times of that of Exo-AM1241, and the apoptosis rate in the brain tissue sample of the single AM1241 treatment group is about 1.3 times of that of Exo-AM1241 (figure 5).
Western Blot is used for detecting the expression condition of the apoptosis-related protein in brain tissues of different treatment groups of animals. Extracting animal brain tissue protein by using a protein extraction kit, adding 5 × loading buffer into the whole protein extracting solution to finally dilute the loading buffer to 1 × and heating at 95 ℃ for 5min, and storing at-80 ℃ for later use. Preparing separation gel and concentrated gel in advance, adding a certain amount of protein and protein merker into comb holes, putting into an electrophoresis tank, adding electrophoresis liquid, carrying out 80V electrophoresis for 30min, and then carrying out 120V electrophoresis until bromophenol blue just runs out. PVDF membrane in methanol activation for 3 min. Prying the glass plate open, cutting off the concentrated gel, stripping the residual separation gel, and then putting the separation gel, the sponge, the filter paper and the activated PVDF membrane into a container with a rotating membrane buffer. Opening the film transferring clamp, sequentially laying sponge, filter paper, glue PVDF film, filter paper and sponge, expelling bubbles, putting the clamp into a film transferring groove, adding a film transferring buffer, and performing electrophoresis at 110V for 70 min. TBST solution containing 5% BSA was prepared as a blocking solution, and the membrane was removed and then blocked for 1 hour on a shaker. TBST washing 3 times, once for 5 min. Primary anti-clear-caspase 3, caspase3, clear-caspase 9, caspase9 and beta-actin were diluted with blocking solution. Shaking table incubation was performed overnight at 4 ℃. TBST washing 3 times, once for 5 min. The secondary antibody was diluted with blocking solution and incubated at room temperature for 1 h. TBST washing 3 times, once for 5 min. The membrane is soaked with a color developing solution and imaged in a chemiluminescence imager. The results show that the expression levels of apoptosis-related proteins clear caspase3 and clear caspase9 in brain tissues of treated animals are reduced, wherein the expression change of Exo-AM1241 treatment histone is more obvious (FIG. 5).
6. exosome/AM 1241/Exo-AM1241 differences in signal pathways for treating alzheimer's disease
And detecting the gene change condition in the brain tissue of the treated Alzheimer disease model animal by using real-time fluorescent quantitative PCR (RT-qPCR). As shown in fig. 6, exosomes/AM 1241/Exo-AM1241 were able to significantly affect the expression of brain tissue genes compared to placebo, while the signaling pathways primarily affected were different. The single exosome has a treatment effect by influencing the expression of calcium signal channel related kinases such as calcium/calmodulin-dependent protein kinase 2A (Camk II alpha), inositol 1,4,5 triphosphate 3 kinase A (ITPKA) and the like, and has no obvious influence on the expression of dopamine receptor D1(Drd1), transcription factor Lhx8, neuropeptide Y receptor 2(Npy2R) and transcription factor NKX 2.1; AM1241 alone produces therapeutic effects primarily by affecting neuronal tissue ligand-receptor interactions, including upregulation of dopamine receptor D1(Drd1) and the expression of transcription factor Lhx8, with no significant effect on the expression of calcium/calmodulin-dependent protein kinase 2A (Camk II α), inositol 1,4,5 triphosphate 3 kinase a (itpka), neuropeptide Y receptor 2(Npy2R), and transcription factor NKX 2.1; the Exo-AM1241 nano-pharmaceutical preparation influences the expression of related genes of a downstream calcium signal pathway and an Erk signal pathway by activating a CB2 receptor, comprises a neuropeptide Y receptor 2(Npy2R) and a transcription factor NKX 2.1, promotes neuron regeneration, improves the pathological response of the Alzheimer disease, and has no obvious influence on the expression of calcium/calmodulin-dependent protein kinase 2A (Camk II alpha), inositol 1,4,5 triphosphate 3 kinase A (ITPKA), dopamine receptor D1(Drd1) and transcription factor Lhx 8.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.
Claims (10)
1. The mesenchymal stem cell exosome-AM 1241 complex is characterized in that the mesenchymal stem cell exosome-AM 1241 complex is prepared by loading AM1241 on a mesenchymal stem cell exosome.
2. The mesenchymal stem cell exosome-AM 1241 complex according to claim 1, wherein AM1241 is loaded on mesenchymal stem cell exosomes by electrostatic adsorption method.
3. The mesenchymal stem cell exosome-AM 1241 complex according to claim 1, wherein the mesenchymal stem cell is a bone marrow, umbilical cord blood, amnion, placenta, adipose, dental pulp or peripheral blood derived mesenchymal stem cell.
4. The method for preparing a mesenchymal stem cell exosome-AM 1241 complex according to claim 1, comprising a step of loading AM1241 on a mesenchymal stem cell exosome.
5. The method according to claim 4, wherein the AM1241 is loaded on mesenchymal stem cell exosomes by electrostatic adsorption.
6. Use of the mesenchymal stem cell exosome-AM 1241 complex of claim 1 in the preparation of a medicament for treating alzheimer's disease.
Application of AM1241 in preparing a medicament for treating Alzheimer's disease.
8. The use according to claim 6 or 7, wherein the medicament is in the form of an injection or an oral formulation.
9. The use according to claim 6 or 7, wherein the medicament is administered by intravenous, arterial, oral, parenteral, buccal, vaginal, rectal, inhalation, insufflation, sublingual, intramuscular, subcutaneous, topical, intranasal, intraperitoneal or intracranial routes.
10. A pharmaceutical composition for treating alzheimer's disease, comprising the mesenchymal stem cell exosome-AM 1241 complex of claim 1 and a pharmaceutically acceptable carrier.
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Citations (3)
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|---|---|---|---|---|
| CN106138053A (en) * | 2015-04-28 | 2016-11-23 | 上海市同济医院 | The application in Parkinsonian medicine is treated in preparation of a kind of compound |
| CN109260227A (en) * | 2018-08-29 | 2019-01-25 | 瑞太医药科技（杭州）有限公司 | It is a kind of for treating the autologous fat mesenchymal stem cell injection preparation method of Alzheimer's disease |
| CN109913409A (en) * | 2019-04-11 | 2019-06-21 | 中国科学院苏州纳米技术与纳米仿生研究所 | Dimensional culture source for mesenchymal stem cells excretion body and the preparation method and application thereof |
-
2021
- 2021-06-17 CN CN202110673865.4A patent/CN113181216B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106138053A (en) * | 2015-04-28 | 2016-11-23 | 上海市同济医院 | The application in Parkinsonian medicine is treated in preparation of a kind of compound |
| CN109260227A (en) * | 2018-08-29 | 2019-01-25 | 瑞太医药科技（杭州）有限公司 | It is a kind of for treating the autologous fat mesenchymal stem cell injection preparation method of Alzheimer's disease |
| CN109913409A (en) * | 2019-04-11 | 2019-06-21 | 中国科学院苏州纳米技术与纳米仿生研究所 | Dimensional culture source for mesenchymal stem cells excretion body and the preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| ESTER ASO 等: "CB2 Cannabinoid Receptor As Potential Target against Alzheimer"s Disease", 《FRONT NEUROSCI.》 * |
| JUN SHI 等: "AM1241 alleviates MPTP-induced Parkinson"s disease and promotes the regeneration of DA neurons in PD mice", 《ONCOTARGET.》 * |
| 史敬璞: "大麻素Ⅱ型受体调节阿尔茨海默病样小鼠学习记忆能力的神经源性炎症机制研究", 《万方数据知识服务平台》 * |
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