CN115300529A - Preparation method of extracellular vesicles derived from young osteocytes and application of extracellular vesicles in preparation of medicine for treating Alzheimer's disease - Google Patents
Preparation method of extracellular vesicles derived from young osteocytes and application of extracellular vesicles in preparation of medicine for treating Alzheimer's disease Download PDFInfo
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Abstract
The invention is applicable to the technical field of medicines, and provides a preparation method of extracellular vesicles derived from young bone cells and application of the extracellular vesicles in preparation of a medicine for treating Alzheimer's disease. The application of the extracellular vesicles derived from young osteocytes in preparing the medicine for treating the Alzheimer's disease is proved through experiments. Also provides a preparation method of the extracellular vesicles derived from young osteocytes. The 4-month-old APP/PS1 mice intervened by the extracellular vesicles derived from the young osteocytes at the age of 2 months show better cognitive function, reduced Abeta plaques in brain tissues, reduced synaptic damage and reduced neuron loss; while the 16-month-old osteocyte-derived extracellular vesicle stem group showed no significant difference between in vitro and in vivo experiments, indicating that the protective effect of osteocyte-derived extracellular vesicles decreased with age. In conclusion, the extracellular vesicles derived from the young osteocytes can be applied to preparation of the medicine for treating the Alzheimer's disease.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a preparation method of extracellular vesicles derived from young bone cells and application of the extracellular vesicles in preparation of a medicine for treating Alzheimer's disease.
Background
Alzheimer's Disease (AD) is a neurodegenerative disease with hidden and progressive disease, and is also the most common dementia subtype in the elderly, and its main clinical features are progressive decline of cognitive function mainly due to situational memory. Osteoporosis (OP) is a systemic metabolic disease characterized by progressive reduction in bone mass, deterioration in bone microarchitecture, increased bone fragility, and susceptibility to fracture. Both AD and OP are common degenerative diseases associated with aging, and are closely related in clinical epidemiology. Several studies report that alzheimer patients have a lower Bone density (BMD) and a higher incidence of osteoporosis, a low BMD also being associated with an increased risk of AD development. Currently, there is little research on the transfer of signals between the brain and the bone. In recent years, researches show that Extracellular Vesicles (EVs) are a main factor for mediating the biological signal exchange between prokaryotes and higher eukaryotes, and Extracellular vesicles (Osteocytic-derived Extracellular vesicles, OCY-EVs) derived from osteocytes can mediate the functional regulation and control between bones and non-skeletal organs. This study will explore the role of OCY-EVs in information transfer between the brain and bone, and the role of OCY-EVs on AD.
Disclosure of Invention
The embodiment of the invention provides a preparation method of extracellular vesicles derived from young osteocytes and application of the extracellular vesicles in preparation of a medicine for treating Alzheimer's disease, and aims to solve the problem that blood brain barriers exist when AD medicines enter the brain in the prior art, and the treatment of AD can only delay symptoms.
In order to achieve the purpose, the invention provides the following technical scheme:
application of extracellular vesicles derived from young bone cells in preparation of medicines for treating Alzheimer's disease.
The young osteocyte-derived extracellular vesicles are 2-month-old osteocyte-derived extracellular vesicles.
The preparation method of the extracellular vesicles derived from the young osteocytes comprises the following steps:
1) Culturing primary osteocytes in an osteocyte exosome-free culture medium; changing the liquid every other day, collecting cells, and preserving at-80 deg.C;
2) Centrifuging at 300 Xg for 10 min at 4 deg.C, and collecting the first supernatant; centrifuging the first supernatant at 2000 Xg for 30 min at 4 deg.C, and collecting the second supernatant; centrifuging at 4 deg.C at 10000 × g for 30 min, and collecting the third supernatant;
3) Filtering the third supernatant with a sterile 0.22 μm filter to remove residual cell debris;
4) Transferring the filtered supernatant into a super-separation tube, centrifuging at 100000 Xg for 10 hours at 4 ℃, collecting the sediment at the bottom of the tube, and discarding the supernatant; resuspending the pellet with PBS; young osteocyte-derived extracellular vesicles (OCY-EVs) were obtained.
The bone cell culture medium without exosomes in the step 1) comprises the following specific components: 44.5mL of alpha-MEM cell culture medium; 5mL of FBS without exosome; 0.5mL of P/S double antibody.
If the extracellular vesicles derived from the young osteocytes obtained in the step 4) are not used immediately, the extracellular vesicles can be packaged and stored at-80 ℃ to avoid repeated freeze thawing.
The acquisition of the primary bone cells in the step 1) specifically comprises the following steps:
(1) Soaking the mouse in 75% ethanol for 2 min, and placing femur, tibia and humerus in a sterile cell culture dish containing 1%P/S PBS;
(2) Removing the muscle attached to the bone with forceps and scissors; removing periosteum with surgical blade; cutting off two ends of epiphysis, extruding by using a mortar, and repeatedly flushing by using PBS (phosphate buffer solution) to remove bone marrow;
(3) Cutting bone into pieces as much as possible to obtain bone pieces with size of 0.8-1.2 mm; repeatedly washing with PBS until the liquid turns colorless, and removing residual bone marrow;
(4) Adding II type collagenase digestive juice into the bone slices, placing the bone slices in a shaking table at 37 ℃ and digesting the bone slices in the dark for 30 minutes at the speed of 80 rpm/min; discarding collagenase II digestive juice, and washing with PBS for 3 times;
(5) Repeating the step (4) twice;
(6) Adding an EDTA solution with the pH value of 5mM not less than 7.4, placing the mixture in a shaker at 37 ℃ and digesting the mixture for 30 minutes in the dark at the speed of 80 rpm/min; PBS washing for 3 times;
(7) Sequentially repeating the digestion treatment according to the conditions of the step (4) and the step (6) for twice;
(8) Adding 3mL of I-type rat tail collagen solution into a cell culture bottle for plating, and putting the cell culture bottle into a cell culture box at 37 ℃ for incubation for 30 minutes;
(9) Adding II type collagenase digestive juice into the bone slices, placing the bone slices in a shaking table at 37 ℃ and digesting the bone slices in the dark for 30 minutes at the speed of 80 rpm/min; discarding II type collagenase digestive juice, washing 3 times with PBS; removing PBS, and cutting bone fragments into pieces;
(10) Transferring the bone slices to a cell culture bottle after plating, adding 6mL of bone cell complete culture medium, shaking the bone slices uniformly, putting the bone slices into a cell culture box, and standing for 7 days;
(11) And taking out the T25 cell culture flask after 7 days, and observing that bone cells creep out around the bone fragments under an optical microscope to obtain primary bone cells.
The type II collagenase digestive juice in the step (4) comprises the following specific components: α -MEM cell culture 48.5mL; 1mL of FBS; 500 mu L of type II collagen zymogen liquid; the type II collagenase stock solution is 1mg/mL.
The I type rat tail collagen solution in the step (8) comprises the following specific components: 48mL of PBS; 60 mu L of glacial acetic acid; 2mL of I-type rat tail collagen stock solution; the amount of the type I rat tail collagen stock solution is 3.59mg/mL.
The bone cell complete culture medium in the step (10) comprises the following specific components: 44.5mL of alpha-MEM cell culture medium; 2.5mL of FBS; CS 2.5mL; 0.5mL of P/S double antibody.
The conditions of the cell culture chamber in step (10) are 37 ℃,5% CO 2 And saturation humidity.
The invention achieves the following beneficial effects:
1. the experimental results of the invention show that 4-month-old APP/PS1 mice intervened with extracellular vesicles derived from young osteocytes (2 months old) exhibit better cognitive function, reduced Abeta plaques in brain tissue, reduced synaptic damage and reduced neuronal loss; whereas the 16-month-old osteocyte-derived extracellular vesicle intervention group showed no significant difference between in vitro and in vivo experiments, indicating that the protective effect of osteocyte-derived extracellular vesicles decreased with age. In conclusion, the extracellular vesicles derived from the young osteocytes can be applied to preparation of the medicine for treating the Alzheimer's disease.
2. The treatment methods of AD which are clinically applied at present are relatively limited, and the research result of the invention provides a new idea for the treatment medicine of AD. With the development of engineered EV technology, we can artificially synthesize extracellular vesicles, and generate a novel therapeutic agent by precisely packaging key components of young OCY-EV into artificial EV. Due to the characteristics that the EV can penetrate through a blood brain barrier and be targeted and positioned, the EV is expected to be an efficient and targeted AD treatment drug.
Drawings
FIG. 1 is a graph of the results of the behavioural studies after OCY-EVs intervention in 4M APP/PS1 mice; wherein: a: experimental design schematic, wherein time points of intervention and detection are noted; B-E: OCY 2M -EVs、OCY 16M Water maze (B-C), Y maze (D), OLT and NORT (E) result plots for EVs, solvent-intervened 4M APP/PS1 mice and WT mice; each group n =4-6; black arrows indicate plateaus; **** P<0.0001, *** P<0.001, ** P< 0.01, * P<0.05。
FIG. 2 is a diagram showing the results of ELISA assay for detecting Abeta after OCY-EVs intervene in 4M APP/PS1 mice; wherein: A-C: OCY 2M -EVs、OCY 16M -the ratio (C) of a β 42 level (a), a β 40 level (B) and a β 42/40 in brain homogenate supernatants of EVs, solvent-intervened 4M APP/PS1 mice and WT mice; each group n =4-6; * P<0.01,*P<0.05; WT, solvent, OCY in histogram 2M -EVs、OCY 16M Differentiation of EVs can be distinguished with reference to fig. 1B.
FIG. 3 is a graph showing the results of WB assay for A β after OCY-EVs intervention in 4M APP/PS1 mice; wherein: a: OCY 2M -EVs、OCY 16M Western blot images of insoluble Α β multimers in brain homogenates of EVs, solvent-intervened 4M APP/PS1 mice and WT mice; b: analyzing the grey value of the Western blot image in the A; each group n =4-6; * P<0.01,*P<0.05; WT, solvent, OCY in histogram 2M -EVs、 OCY 16M Differentiation of EVs can be distinguished with reference to fig. 1B.
FIG. 4 is a graph showing the results of immunofluorescence assay for A β plaques after OCY-EVs intervention in 4M APP/PS1 mice; wherein: a: OCY 2M -EVs、OCY 16M -representative fluorescence images of Α β plaques in brain tissue sections of EVs, solvent-intervened 4M APP/PS1 mice; a scale: 100 μm; b: quantitative analysis results of 6E10 immunofluorescence images: the number of a β plaques, the size of a β plaques, and 6E10 fluorescence intensity; each group n =4-6; *** P <0.001, ** P<0.01, * P<0.05; wherein the Solvent and OCY in the histogram 2M -EVs、OCY 16M Differentiation of EVs can be distinguished with reference to fig. 1B.
FIG. 5 is a graph showing the relationship between A β plaques and microglia detected by immunofluorescence after OCY-EVs intervene in 4M APP/PS1 mice; wherein: a: OCY 2M -EVs、OCY 16M Representative fluorescence images of Α β plaques and microglia in brain tissue sections of EVs, solvent-intervened 4M APP/PS1 mice; a scale: 10 mu m; b, 6E10 and Iba1 immunofluorescence image quantitative analysis result: ratio of Iba1 fluorescence intensity and Iba1/6E10 fluorescence intensity around Α β plaques; calculate Iba1 fluorescence intensity within a 100 μm radius of the a β plaques; at least 100 plaques were counted per intervention group; each group n =4-6; **** P<0.0001, *** P<0.001; wherein the Solvent and OCY in the histogram 2M -EVs、OCY 16M Differentiation of EVs can be distinguished with reference to fig. 1B.
FIG. 6 is a graph showing the results of immunofluorescence assay for the detection of synapsin in the cortex and hippocampus following OCY-EVs intervention in 4M APP/PS1 mice; wherein: a: OCY 2M -EVs、OCY 16M Representative fluorescence images of the synaptogrin (Syp) in brain tissue sections of EVs, solvent-intervened 4M APP/PS1 mice; a scale: 10 mu m; b: quantitative analysis of Syp immunofluorescence images in cortex and hippocampusFruit; each group n =4-6; *** P<0.001, ** P <0.01; wherein the Solvent and OCY in the histogram 2M -EVs、OCY 16M Differentiation of EVs can be distinguished with reference to fig. 1B.
FIG. 7 is a graph of the results of immunofluorescence assays for neurons in the cortex and hippocampus following OCY-EVs intervention in 4M APP/PS1 mice; wherein: a: OCY 2M -EVs、OCY 16M Representative fluorescence images of mature neurons (NeuN) in brain tissue sections of EVs, solvent-intervened 4M APP/PS1 mice; a scale: cortex: 10 mu m; sea horse: 50 μm; b: quantitative analysis results of NeuN immunofluorescence images in cortex and hippocampus; each group n =4-6; *** P<0.001, ** P<0.01; wherein the Solvent and OCY in the histogram 2M -EVs、OCY 16M Differentiation of EVs can be distinguished with reference to fig. 1B.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.
At present, aiming at solving the problem that the treatment of blood brain barrier and AD in the prior art when AD drugs enter the brain can only delay symptoms, the invention provides a preparation method of extracellular vesicles from young osteocytes and application of the extracellular vesicles in preparation of drugs for treating Alzheimer's disease.
Example 1
Firstly, the acquisition of primary bone cells specifically comprises the following steps:
(1) Soaking the mouse in 75% ethanol for 2 min, and placing femur, tibia and humerus in a sterile cell culture dish (10 cm) containing 1%P/S PBS;
(2) Moving to a clean bench, and removing the muscle attached to the bone by using forceps and scissors; removing periosteum with surgical blade; the two epiphyseal ends were cut off (cartilage removed), squeezed with a mortar, and washed repeatedly with PBS to remove bone marrow;
(3) The bone is cut into pieces as much as possible to form bone slices with the size of about 1 mm; repeatedly washing with PBS until the liquid turns colorless, and removing residual bone marrow;
(4) Adding type II collagenase digestive juice into the bone slices, placing the bone slices in a shaking table (80 rpm/min) at 37 ℃ to digest for 30 minutes in a dark place; discarding type II collagenase digestive juice by a bus straw, and washing 3 times by PBS;
(5) Repeating the step (4) twice;
(6) 5mM EDTA solution (pH = 7.4) was added and the mixture was digested in a 37 ℃ shaker (80 rpm/min) in the dark for 30 minutes; PBS washing for 3 times;
(7) Sequentially carrying out digestion treatment according to the step (4) and the step (6), and repeating twice;
(8) Adding 3mL of I-type rat tail collagen solution into a T25 cell culture bottle for plating, and putting the cell culture bottle into a 37 ℃ cell culture box for incubation for 30 minutes;
(9) Adding type II collagenase digestive juice into the bone slices, placing the bone slices in a shaking table (80 rpm/min) at 37 ℃ to digest for 30 minutes in a dark place; discarding type II collagenase digestive juice by a bus straw, and washing 3 times by PBS; removing PBS, and cutting the bone into pieces;
(10) Transferring the bone fragments to a plated T25 cell culture flask, adding 6mL of bone cell complete medium, shaking the bone fragments, and placing in a cell culture chamber (37 ℃,5% CO) 2 Saturated humidity), standing for 7 days;
(11) And taking out the T25 cell culture flask after 7 days, and observing that bone cells climb out around the bone fragments under an optical microscope to obtain primary bone cells.
The type II collagenase digestive juice in the step (4) comprises the following specific components: α -MEM cell culture 48.5mL; 1mL of FBS; 500 mu L of type II collagen zymogen liquid; the type II collagenase stock solution is 1mg/mL.
The I type rat tail collagen solution in the step (8) comprises the following specific components: 48mL of PBS; 60 mu L of glacial acetic acid; 2mL of I-type rat tail collagen stock solution; the amount of the type I rat tail collagen stock solution is 3.59mg/mL.
The bone cell complete culture medium in the step (10) comprises the following specific components: 44.5mL of alpha-MEM cell culture medium; 2.5mL of FBS; CS 2.5mL; 0.5mL of P/S double antibody.
A preparation method of extracellular vesicles derived from young osteocytes comprises the following steps:
1) Replacing a bone cell complete culture medium of the primary bone cells with a bone cell exosome-free culture medium; changing the liquid every other day, collecting cell culture medium, and storing at-80 deg.C;
2) Centrifuging at 300 Xg for 10 min at 4 deg.C, and collecting the first supernatant; centrifuging the first supernatant at 2000 Xg for 30 min at 4 deg.C, and collecting the second supernatant; centrifuging at 4 deg.C at 10000 × g for 30 min, and collecting the third supernatant;
3) Filtering with sterile 0.22 μm filter in a clean bench to remove residual cell debris;
4) Transferring the filtered supernatant into a super-separation tube, centrifuging at 100000 Xg for 10 hours at 4 ℃ to see that the bottom of the tube has sediment, and discarding the supernatant; resuspend pellet with PBS (add appropriate volume of PBS depending on pellet size); obtaining young bone cell-derived extracellular vesicles (OCY-EVs); if not used immediately, it can be stored in-80 deg.C (to avoid multiple freeze thawing) in separate packages.
The bone cell culture medium without exosomes in the step 1) comprises the following specific components: 44.5mL of alpha-MEM cell culture medium; 5mL of FBS without exosome; 0.5mL of P/S double antibody.
Collecting basic information and bone density results of 88 AD patients and 104 age-matched healthy controls with normal cognitive function, and analyzing the correlation between the cognitive function and the bone density; plasma EVs of AD patients and normal controls were extracted by ultracentrifugation and analyzed for OCY-EVs content by western blot. The bone loss (T-Score ≦ -1.0) was found to be 92.0% in AD patients, significantly higher than the normal control (80.8%, P = 0.025), and positively correlated with cognitive function (MMSE Score) (correlation coefficient r =0.165, P = 0.012). AD patients contained more OCY-EVs in their plasma relative to the cognitively normal control population (P < 0.01).
(II) detecting whether OCY-EVs can reach the brain in a physiological state by using an APP/PS1 transgenic model mouse (Dmp 1Cre; cd63 fl/-mouse) which is obtained by homologous recombination of CRISPR/Cas9 and Cre/LoxP recombination technology at the early stage of a subject group and can trace OCY-EVs by using a fluorescence imaging method; fluorescence-labeled OCY-EVs are injected into APP/PS1 transgenic mice through a medullary cavity and a tail vein, and a living fluorescence imaging method is used for detecting whether OCY-EVs can reach the brain in a pathological state. It was found that OCY-EVs fluorescence signal was observed in the brain of mice in both transgenic model mice traceable with OCY-EVs (Dmp 1Cre; cd63 fl/-mice) and APP/PS1 transgenic mice injected with fluorescence-labeled OCY-EVs in the medullary cavity.
(III) constructing AD cell models of SH-SY5Y-APPswe and HT22-APPswe by using a virus transfection or plasmid transfection technology; OCY-EVs (OCY M-EVs or OCY M-EVs) are extracted from culture supernatant of young or old primary osteocytes by an ultracentrifugation method, and two AD cell models are intervened, and the levels of A beta 42 and A beta 40 and the apoptosis level are detected. Extracellular vesicles derived from osteocytes (OCY 2M-EVs) extracted from 2-month-old C57BL/6 mice were found to improve AD pathology in AD cell models. In SH-SY5Y-APPsw cell line, the concentration of A beta 42 (8.29 +/-3.19 pg/mL) and the ratio of A beta 42/A beta 40 (0.13 +/-0.08) are obviously reduced compared with a control group (39.69 +/-23.21 pg/mL,0.66 +/-0.34, P < -0.05), and the proportion of apoptotic cells (6.47 +/-1.80%) is obviously reduced compared with the control group (15.58 +/-2.94%) (P < 0.001); however, extracellular vesicles derived from osteocytes (OCY M-EVs) extracted from 16-month-old C57BL/6 mice were not able (P > 0.05).
(IV) injecting young or old OCY-EVs into the tail vein of AD transgenic mice with the age of 4 months and the age of 6 months, and evaluating the cognitive function of the mice through behavioral detection; detecting the levels of mouse brain tissues Abeta 42 and Abeta 40 by an ELISA method; mice brains were examined for Α β plaques, synaptic lesions and neuronal loss by immunofluorescence. It was found that OCY 2M EVs can also obviously improve cognitive impairment and AD pathological changes of APP/PS1 transgenic mice with the age of 4 months: behavioral (water maze, Y maze and new object identification experiments) results improved;the number of A beta plaques (6.02 +/-2.39 plaques/mm < 2 >) is remarkably reduced compared with that of a control group (12.72 +/-2.98 plaques/mm < 2 >) 2 ，P<0.05 ); the fluorescence intensity of synapsin in cortex and hippocampus regions is significantly increased compared with that in the control group (both P)<0.01 ); the number of neurons in the cortex and hippocampus was significantly increased compared to the control group (P)<0.01 And P<0.001 ); but OCY 16M EVs then not (P)>0.05). Further, OCY 2M The cognitive impairment and AD pathology improvement effects of EVs on 6-month-old APP/PS1 transgenic mice were similar to those of the 4-month-old group.
And fifthly, injecting Rab27a-shRNA adenovirus into a marrow cavity of a young C57BL/6 mouse to construct a mouse model for selectively inhibiting bone-derived EVs secretion, injecting A beta pathogenic protein through bilateral hippocampus to construct an AD mouse model on the basis, and evaluating the change of the cognitive function of the AD mouse model through behavioural detection. It was found that inhibition of EVs secretion from bone by intramedullary injections of Rab27a-shRNA adenovirus could potentiate the cognitive dysfunction in young AD model mice induced by Α β 40, as shown by the poorer behavioural outcome of the intervention group.
And (VI) analyzing the OCY-EVs of the young and the old by a proteomic analysis method, and further performing GO and KEGG functional analysis. The results of proteomics show that OCY 16M Compared with the EVs, OCY M-EVs enrich various protective factors in the AD pathway, such as ADAM10 (main component of α -secretase), NCSTN and PSEN2 (main component of β -secretase), NEP (a β -degrading enzyme), and various mitochondrial energy metabolism-related proteins. And KEGG pathway analysis showed that the first 5 pathways, which are most protein rich, are pathways for alzheimer's disease, parkinson's disease, neurodegenerative disorders-multiple diseases, prion disease and huntington's disease, respectively.
In summary, AD patients have lower levels of bone density than the cognitively normal control population. Young OCY-EVs can enter the brain and improve cognitive impairment and Α β pathology in AD mice; inhibition of bone-derived EVs secretion exacerbates cognitive dysfunction in AD mice. In addition, functional factors related to A beta degradation and mitochondrial energy metabolism are highly enriched in young OCY-EVs, and A beta deposition and neuronal damage in AD pathogenesis can be synergistically avoided. In conclusion, the extracellular vesicles derived from the young osteocytes can be applied to preparation of the medicine for treating the Alzheimer's disease.
The experiment of the invention is to inject OCY into tail vein of 4-month-old APP/PS1 mice 2M -EVs、OCY 16M EVs or solvent (injection of OCY-EVs, 2.4X 10, respectively, according to mouse body weight 6 The concentration of each granule per gram of body weight is 1.2 × 10 6 Particles/. Mu.L), intervene once a week for two months, constituting the interference and solvent groups. Behavioural tests were performed within 2 weeks after the end of the intervention, respectively the Morris water maze, the Y maze and the new object identification test. Then, after sacrifice, brain tissue and femur were harvested for subsequent testing: homogenizing the left brain of each mouse, extracting supernatant, detecting soluble Abeta 42 and Abeta 40, extracting protein from the residual homogenate, and detecting insoluble Abeta; the right brain of each mouse was frozen and sectioned for immunofluorescence analysis of A β plaques, synapses, and neuronal related antibodies.
First, the Morris Water Maze (MWM), Y-maze, object Localization Test (OLT), and New object identification test (NORT) were used to assess the cognitive function of the APP/PS1 mouse trunk prognosis. The Morris water maze is an assay for evaluating the learning and memory abilities of a test space. In Morris Water maze day 2 training, OCY 2M The time to plateau for EVs intervention group APP/PS1 mice was 22.13 + -1.85 s, for solvent group APP/PS1 mice was 30.27 + -7.06s, OCY 16M EVs intervention group APP/PS1 mice are 41.39 ± 13.27s; in the 3 rd day training, OCY 2M The time to reach the platform was 22.69. + -. 9.05s for EVs intervention group APP/PS1 mice, 29.29. + -. 6.47s for solvent group APP/PS1 mice, OCY 16M -EVs intervention group APP/PS1 mice at 36.84 ± 6.44s; in the day 6 trial (platform withdrawal), OCY 2M The number of passes of APP/PS1 mice in EVs intervention group through the platform is 3.33 +/-1.21 times, the number of passes of APP/PS1 mice in solvent group is 2 +/-0.82 times, and OCY 16M EVs intervention group APP/PS1 mice were 2.2. + -. 0.84 times. It can be seen that OCY was in the training phase (days 2 and 3) and the test phase (day 6) compared to the solvent group 2M Trend towards improved cognitive abilities in APP/PS1 mice in EVs intervention group (FIGS. 1A-C).
Another test for assessing spatial memory isThe Y maze. During the test period, mice with good spatial memory capacity will tend to explore unexplored arms (i.e. explore new arms longer), this capacity being strongly dependent on the hippocampus. OCY 2M The ratio of the time to explore the new arm for EVs-intervened APP/PS1 mice (35.17. + -. 5.09%) was significantly longer than the solvent group (25.00. + -. 1.83%, P)<0.05 Or OCY 16M EVs intervention group (25.33. + -. 6.59%, P)<0.05 OCY, indicating 2M EVs improved spatial memory in APP/PS1 mice, while OCY 16M EVs have no equivalent effect (FIG. 1D).
The Object Localization Test (OLT) and the New Object Recognition Test (NORT) are two effective behavioral tests that use the inherent preference of mice for novelty to respond to memory of previously encountered objects. OLT mainly evaluates spatial learning ability, which depends largely on hippocampal activity. In contrast, NORT primarily assesses the non-spatial learning ability of object recognition, requiring reliance on multiple brain regions. Similar to the results of the first two behavioral tests, OCY% relative to the solvent set (the ratio of the time to explore moving objects and new objects to the total time to explore objects was 44.79 ± 8.59% and 34.58 ± 6.06%, respectively) in the OLT and NORT tests 2M The cognitive function of EVs-intervened APP/PS1 mice was significantly improved (the ratio of the time to explore moving and new objects to the total time was higher, 75.59. + -. 8.93% and 71.23. + -. 7.17%, respectively), the difference was statistically significant (P)<0.001 and P<0.01 OCY), in turn 16M None of the APP/PS1 mice with EVs intervention improved cognitive function (P)>0.05 (FIG. 1E). This indicates OCY 2M EVs improved cognitive function in APP/PS1 mice, while OCY 16M EVs have no equivalent effect.
Secondly, since a β pathology is one of the putative pathogenic mechanisms of AD and also the major pathogenic mechanism in APP/PS1 mouse models, we tested the levels of soluble a β 42 and a β 40 in brain homogenate supernatants of APP/PS1 mice with OCY-EVs dry prognosis by ELISA (fig. 2), and the remaining insoluble a β multimers were tested by western blotting using a β antibody (6E 10) (fig. 3). And solvent group (815.40 +/-46.15 pg/mL) or OCY 16M OCY compared to the EVs intervention group (828.70. + -. 98.17 pg/mL) 2M Significant reduction in A β 42 levels in the EVs intervention group (666.20 ± 64.83pg/mL, P values are P respectively<0.05 and P<0.01; fig. 2A); no significant difference between the groups at Α β 40 levels (fig. 2B); similarly, OCY 2M The A.beta.42/40 ratio and insoluble A.beta.levels were lower in the EVs intervention group than in the solvent group (FIG. 2C, FIGS. 2A-B). In addition, it can be seen that the levels of a β 42, a β 42/40 ratio and insoluble a β in the solvent group were all significantly higher than those in the WT group, confirming that a β levels are high in APP/PS1 mice and that a β pathology is its main pathogenic mechanism (fig. 2, fig. 3).
In addition, frozen sections of brain tissue from APP/PS1 mice that had been intervened with these OCY-EVs were stained by 6E10 and quantitatively analyzed for Α β plaques. The number of A β plaques in the solvent group was 12.72. + -. 2.98 plaques/mm 2 The size of the A beta plaque is 822360 +/-211303 mu m 2 6E10 fluorescence intensity as a percentage of the total area 10.11. + -. 2.41%. OCY relative to solvent set 2M EVs intervention number of A β plaques in the 4-month age group (6.02. + -. 2.39 plaques/mm) 2 ，P<0.05 Size of A beta plaque (169750 + -68211 μm) 2 ， P<0.01 And 6E10 fluorescence intensity as a percentage of the total area (2.36. + -. 0.86%, P)<0.001 All significantly reduced, OCY 16M No significant difference between EVs intervention group and solvent group (P)>0.05; fig. 4). The results of the brain homogenate of A.beta.suggest that we OCY 2M EVs can improve a β pathology in early AD models.
Microglia tend to phagocytose loose a β, forming dense core a β plaques, a protective mechanism for AD that can limit the spread of toxic pre-plaque a β oligomers throughout the brain. The co-immunofluorescence of Iba1 (a marker for microglia) and 6E10 shows the relationship between microglia and Α β plaques. The results show that OCY compares to the solvent group (4.34 ± 0.44) in the 4-month age group 2M The APP/PS1 mice in the EVs intervention group had a smaller number of A β plaques and a smaller number of peripheral microglia in brain tissue, but the ratio of Iba1/6E10 (10.74. + -. 2.30<0.001 OCY) but higher 16M The EVs intervention group was not significantly different from the solvent group (fig. 5).
Synaptic damage and neuronal loss are also pathogenic mechanisms of AD. Using synapsin(Syp) immunofluorescent staining as a marker of synapses showed OCY in the 4-month group compared to the solvent group 2M Significant increase in fluorescence intensity of Syp both in cortex and hippocampus of EVs-intervened APP/PS1 mice (P)<0.01 OCY), in turn 16M The EVs intervention group was not significantly different from the solvent group (fig. 6).
For the evaluation of neuronal loss, the nuclear marker Neu-N of mature neurons was immunofluorescent and quantitatively analyzed. The results show that in the 4 month age group, OCY compared to the solvent group 2M Significant increase in the number of NeuN-positive neurons in both cortex and hippocampus of EVs-intervened APP/PS1 mice (P)<0.01，P<0.001 OCY), in turn 16M The EVs intervention group was not significantly different from the solvent group (fig. 7).
The above results show that the use of OCY 2M EVs-intervened 4-month-old APP/PS1 mice show better cognitive function, reduced Α β plaques in brain tissue, reduced synaptic damage and reduced neuronal loss; and OCY 16M The EVs intervention group showed no significant difference in vitro and in vivo experiments, indicating that the protective effect of OCY-EVs decreases with age. In conclusion, the extracellular vesicles derived from the young osteocytes can be applied to preparation of the medicine for treating the Alzheimer's disease.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. Application of extracellular vesicles derived from young bone cells in preparation of medicines for treating Alzheimer's disease.
2. Use of young bone cell-derived extracellular vesicles according to claim 1, for the preparation of a medicament for the treatment of alzheimer's disease, wherein: the young osteocyte-derived extracellular vesicles are 2-month-old osteocyte-derived extracellular vesicles.
3. The method for producing extracellular vesicles derived from young bone cells according to claim 1, wherein the method comprises: the method comprises the following steps:
1) Culturing primary osteocytes in an osteocyte exosome-free culture medium; changing the liquid every other day, collecting cells, and storing at-80 deg.C;
2) Centrifuging at 300 Xg for 10 min at 4 deg.C to obtain a first supernatant; centrifuging the first supernatant at 2000 Xg for 30 min at 4 deg.C, and collecting the second supernatant; centrifuging at 10000 Xg for 30 min at 4 deg.C, and collecting the third supernatant;
3) Filtering the third supernatant with a sterile 0.22 μm filter to remove residual cell debris;
4) Transferring the filtered supernatant into a super-separation tube, centrifuging at 100000 Xg for 10 hours at 4 ℃, collecting the sediment at the bottom of the tube, and discarding the supernatant; resuspending the pellet with PBS; young bone cell-derived extracellular vesicles are obtained.
4. The method for producing extracellular vesicles derived from young bone cells according to claim 3, wherein: the bone cell culture medium without exosomes in the step 1) comprises the following specific components: 44.5mL of alpha-MEM cell culture medium; 5mL of FBS without exosome; 0.5mL of P/S double antibody.
5. The method for producing extracellular vesicles derived from young bone cells according to claim 3, wherein: the extracellular vesicles derived from the young bone cells obtained in step 4) were stored in separate containers at-80 ℃.
6. The method for producing extracellular vesicles derived from young bone cells according to claim 3, wherein: the acquisition of the primary bone cells in the step 1) specifically comprises the following steps:
(1) Soaking the mouse in 75% ethanol for 2 min, and placing femur, tibia and humerus in a sterile cell culture dish containing 1%P/S PBS;
(2) Removing the muscle attached to the bone with forceps and scissors; removing periosteum with surgical blade; cutting off two ends of epiphysis, extruding by using a mortar, and repeatedly flushing by using PBS (phosphate buffer solution) to remove bone marrow;
(3) Cutting bone into pieces as much as possible to obtain bone pieces with size of 0.8-1.2 mm; repeatedly washing with PBS until the liquid turns colorless, and removing residual bone marrow;
(4) Adding II type collagenase digestive juice into the bone slices, placing the bone slices in a shaking table at 37 ℃ and digesting the bone slices in the dark for 30 minutes at the speed of 80 rpm/min; discarding II type collagenase digestive juice, washing 3 times with PBS;
(5) Repeating the step (4) twice;
(6) Adding an EDTA solution with the pH value of 5mM not less than 7.4, placing the mixture in a shaker at 37 ℃ and digesting the mixture for 30 minutes in the dark at the speed of 80 rpm/min; PBS washing for 3 times;
(7) Sequentially repeating the digestion treatment according to the conditions of the step (4) and the step (6) for twice;
(8) Adding 3mL of I-type rat tail collagen solution into a cell culture bottle for plating, and putting the cell culture bottle into a cell culture box at 37 ℃ for incubation for 30 minutes;
(9) Adding II type collagenase digestive juice into the bone slices, placing the bone slices in a shaking table at 37 ℃ and digesting the bone slices in the dark for 30 minutes at the speed of 80 rpm/min; discarding II type collagenase digestive juice, washing 3 times with PBS; removing PBS, and cutting bone fragments into pieces;
(10) Transferring the bone fragments to a cell culture bottle after plate laying, adding 6mL of bone cell complete culture medium, shaking the bone fragments evenly, putting the bone fragments into a cell culture box, and standing for 7 days;
(11) And taking out the T25 cell culture flask after 7 days, and observing that bone cells creep out around the bone fragments under an optical microscope to obtain primary bone cells.
7. The method for producing extracellular vesicles derived from young bone cells according to claim 6, wherein: the type II collagenase digestive juice in the step (4) comprises the following specific components: 48.5mL of alpha-MEM cell culture medium; 1mL of FBS; 500 mu L of type II collagen zymogen liquid; the type II collagenase stock solution is 1mg/mL.
8. The method for producing extracellular vesicles derived from young bone cells according to claim 6, wherein: the I-type rat tail collagen solution in the step (8) comprises the following specific components: 48mL of PBS; 60 mu L of glacial acetic acid; 2mL of I-type rat tail collagen stock solution; the amount of the type I rat tail collagen stock solution is 3.59mg/mL.
9. The method for producing extracellular vesicles derived from young bone cells according to claim 6, wherein: the bone cell complete culture medium in the step (10) comprises the following specific components: 44.5mL of alpha-MEM cell culture medium; 2.5mL of FBS; CS 2.5mL; 0.5mL of P/S double antibody.
10. The method for producing extracellular vesicles derived from young bone cells according to claim 6, wherein: the conditions of the cell culture chamber in step (10) are 37 ℃,5% 2 And saturation humidity.
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| US20180353548A1 (en) * | 2015-04-28 | 2018-12-13 | The Texas A&M University System | Scalable production of standardized extracellular vesicles, extracellular vesicle preparations and uses thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20180193263A1 (en) * | 2015-06-16 | 2018-07-12 | Fondazione Città Della Speranza - Onlus | Extracellular vesicles derived from osteoblastic lineage cells for therapeutic and diagnostic use |
Non-Patent Citations (3)
| Title |
|---|
| YA-LING JIANG , ZHEN-XING WANG , XI-XI LIU , MEI-DAN WAN , YI-WEI LIU , BIN JIAO , XIN-XIN LIAO , ZHONG-WEI LUO , YI-YI WA: "The Protective Effects of Osteocyte-Derived Extracellular Vesicles Against Alzheimer\'s Disease Diminished with Aging", ADV SCI (WEINH), vol. 9, no. 17, pages 1 * |
| 张玲莉;元宇;雷乐;: "阿兹海默症骨骼研究进展", 中国骨质疏松杂志, vol. 26, no. 09, pages 1399 - 1404 * |
| 徐慧琳;范震;冯成强;: "中药对阿尔兹海默症APP/PS1转基因小鼠的治疗作用研究进展", 实验动物科学, vol. 37, no. 01, pages 69 - 73 * |
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