CN111471647A - Preparation method of adult stem cell exosome and application of adult stem cell exosome in treating Parkinson's disease - Google Patents
Preparation method of adult stem cell exosome and application of adult stem cell exosome in treating Parkinson's disease Download PDFInfo
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Abstract
The invention discloses a preparation method of an adult stem cell exosome and application of the adult stem cell exosome in treating Parkinson's disease, and belongs to the technical field of biomedicine. Bone marrow mesenchymal stem cells, skin epidermal stem cells and adipose stem cells are isolated and cultured in vitro, exosomes in cell culture supernatant is extracted by an ultracentrifugation method, and the drug is administered by a nasal administration mode. The exosomes from the three sources can obviously relieve the relevant symptoms of the Parkinson model mouse, such as restoring the motor function of the mouse and increasing the number of dopaminergic neuron cells at the substantia nigra part in the brain of the mouse.
Description
Technical Field
The invention relates to a preparation method of an adult stem cell exosome and application of the adult stem cell exosome in treating Parkinson's disease, belonging to the technical field of biomedicine.
Background
Parkinson's disease, also known as parkinsonism tremor, is the second major neurodegenerative disease after Alzheimer's disease that severely impacts human quality of life. Statistically, 1 out of every 800 people worldwide will have parkinson, and by 2030, the prevalence of parkinson will double, with over 900 million patients expected due to the accelerated aging process. The direct medical costs to the patient per year are expected to exceed $ 10,000, placing a heavy burden on the home and society. Parkinson's disease is characterized by progressive, multiple and occult onset of disease, and mainly manifests as bradykinesia, myotonia, resting tremor and postural instability. The main pathological features of parkinson are mainly represented by the decrease of dopaminergic neuronal cells in the substantia nigra site. The dopamine-producing cells in the brain gradually lose function affecting the nervous system, limiting the ability of the patient to control the muscles. At present, the molecular mechanism of the Parkinson's disease is still unclear, and the clinical treatment of the Parkinson's disease mainly comprises dopamine replacement therapy, and in addition, non-drug therapies such as deep brain stimulation, stem cell transplantation, gene therapy, rehabilitation therapy and the like. Although these therapies can improve the symptoms of the patients with Parkinson to a certain extent and relieve the pain of the patients, no treatment strategy can completely cure Parkinson at present. Therefore, the molecular mechanism of the occurrence and development of Parkinson is further deeply researched, and the search of a novel therapeutic means for treating Parkinson has very important scientific significance and clinical application value.
Stem cells are a class of cells that have unlimited or immortal self-renewal capacity and are capable of producing at least one type of highly differentiated progeny cells. From the viewpoint of source, stem cells can be classified into embryonic stem cells and adult stem cells. Embryonic stem cells are cells screened from the inner cell mass of an embryo or primordial germ cells by in vitro inhibition culture, have totipotency in development, and theoretically can be induced to differentiate into all kinds of cells in the body. Currently, embryonic stem cells are most attractive for research and application in a variety of stem cells. But because the source has strict requirements on time and space, the method limits the large-scale application of the embryonic stem cells in clinic. Adult stem cells are not limited by the above factors and have been a hot spot in medical research. The adult stem cells include bone marrow mesenchymal stem cells, adipose stem cells, skin epidermal stem cells and the like.
However, stem cell therapy suffers from a number of considerable problems, such as biosafety problems, long-term storage and transportation problems, differentiation for non-therapeutic purposes, and the like. For the above reasons, it is important to find a therapeutic approach to replace stem cells. Exosomes have the biological properties of the source cell and are attracting increasing attention. Exosomes are vesicular vesicles of lipid bilayers, 50-150nm in diameter, produced by cells through active secretion and excreted extracellularly. Exosomes contain a variety of substances including lipid molecules, proteins and nucleic acids (including DNA, RNA and various non-coding RNAs). Exosomes play an important role in intercellular information exchange and substance co-transmission. Currently, more and more studies indicate that exosomes play an indispensable role in early diagnosis, clinical treatment and prognosis of diseases.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art and provide a preparation method of an adult stem cell exosome and application thereof in treating Parkinson's disease, wherein the adult stem cell exosome can obviously relieve relevant symptoms of a Parkinson model mouse, such as restoring the motor function of the mouse and increasing the number of dopaminergic neuron cells at the substantia nigra part in the brain of the mouse.
In order to solve the above technical problems, the present invention provides a method for preparing an exosome of adult stem cells, comprising: separating and culturing adult stem cells in vitro, and extracting exosomes in cell culture solution supernatant by an ultracentrifugation method.
Preferably, the adult stem cells include bone marrow mesenchymal stem cells, skin epidermal stem cells and adipose stem cells.
Preferably, the method for isolating and culturing the mesenchymal stem cells comprises the following steps:
anaesthetizing a mouse, breaking the neck, killing the mouse, completely extracting femurs and shinbones on two sides of the mouse, sucking DMEM/F-12 culture solution containing 10% fetal calf serum by using an injector, inserting a needle into a marrow cavity, washing out marrow, placing the marrow in a culture dish, and filtering;
centrifuging the filtrate, removing supernatant, resuspending cells with DMEM/F-12 culture solution containing 10% fetal calf serum, transferring the cell suspension to a culture bottle, adding DMEM/F-12 culture solution containing 10% fetal calf serum, and culturing in a cell culture box;
after 72h, the liquid is changed for the first time, the liquid is changed every 2 days, the growth state of the cells is observed under an inverted microscope every day in the cell culture process, and when the bottom of the bottle is full of the cells, the ratio of the growth state to the growth state is 1:2, passage in proportion;
analyzing and screening by a flow cytometer to obtain the bone marrow mesenchymal stem cells.
Preferably, the method for isolating and culturing the skin epidermal stem cells comprises the following steps:
cleaning and soaking fresh foreskin tissue taken down in an operation by PBS (phosphate buffer solution) containing double antibodies under an aseptic condition, separating epidermis, fat and dermis, cutting the epidermis into fragments, transferring the fragments into a small conical flask, adding 0.5% neutral protease, completely soaking tissue blocks, sealing a sealing film, and digesting at 4 ℃ overnight;
peeling epidermis with forceps, removing dermis, cutting epidermis, adding 0.5% pancreatin, digesting in water bath at 37 deg.C for 10min, stopping digestion with serum-containing culture medium, suspending cells, and filtering with cell filter to obtain single cell suspension;
after centrifugation, the precipitated cells were pressed 105Cells/cm2Inoculating the cells into an IV collagen coated culture bottle, placing the culture bottle in a cell culture box for culturing for 10min, sucking the upper non-adherent cells, adding a KSFM culture medium, culturing in the cell culture box, and changing the culture solution every other day.
Analyzing and screening by a flow cytometer to obtain the skin epidermal stem cells.
Preferably, the method for isolating and culturing the adipose-derived stem cells comprises the following steps:
removing surface fascia and blood vessels from subcutaneous adipose tissue taken out by operation, cutting the extracted adipose tissue into pieces after washing with phosphate buffer solution, digesting with collagenase type I at a constant temperature of 37 ℃ for 40min, and adding DMEM medium containing 10% FBS to stop digestion;
centrifuging, removing supernatant, adding 10% FBS-containing DMEM medium, resuspending, filtering, inoculating into culture flask, standing at 37 deg.C and 5% CO2Culturing in an incubator;
changing the culture medium after 24h, removing the non-adherent cells, changing the culture medium every 3 days until the cells grow to 80-90% of fusion, digesting with pancreatin, and carrying out passage at the ratio of 1: 3;
detecting cell phenotype by a flow cytometer to obtain the adipose-derived stem cells.
Preferably, the specific method for extracting exosomes in cell culture solution supernatant comprises the following steps:
filtering the stem cell culture solution, performing gradient centrifugation on the filtrate at 300rpm, 4 deg.C, 5min, 2000rpm, 4 deg.C, 10min, 10000rpm, 4 deg.C, and 30min, respectively, retaining the supernatant, and discarding the cell residue;
collecting supernatant after gradient centrifugation, ultracentrifuging at 100000rpm at 4 deg.C for 2h, and removing supernatant to obtain precipitate as exosome secreted by stem cells;
the obtained exosomes were resuspended and washed with precooled PBS, ultracentrifuged again at 100000rpm at 4 ℃ for 2h, resuspended with 50. mu.l of 4 ℃ PBS, and transferred to a low-adhesion tube to obtain exosomes.
The invention also provides application of the adult stem cell exosome prepared by the method in treating Parkinson's disease.
The invention achieves the following beneficial effects: the invention separates and cultures the marrow mesenchymal stem cell, the skin epidermal stem cell and the adipose stem cell in vitro, extracts the exosome in the cell culture supernatant by an ultracentrifugation method, and administers the drug by a nasal administration mode. The exosomes from the three sources can obviously relieve the relevant symptoms of the Parkinson model mice, such as restoring the motor function of the mice and increasing the number of dopaminergic neuron cells at the substantia nigra part in the brain of the mice, and can be used for treating Parkinson diseases.
Drawings
FIG. 1 is a graph of the effect of three adult stem cell exosomes on the rotarod capacity of PD model mice.
Grouping: 1- -control group; 2- - -PD model group; 3- - -bone marrow mesenchymal stem cell exosome group; 4- - -adipose-derived stem cell exosome group; 5- -epidermal stem cell exosome group. P <0.001 compared to group 1; # P <0.05 vs group 2.
FIG. 2 is the effect of three adult stem cell exosomes on the rod-climbing ability of PD model mice.
Grouping: 1- -control group; 2- - -PD model group; 3- - -bone marrow mesenchymal stem cell exosome group; 4- - -adipose-derived stem cell exosome group; 5- -epidermal stem cell exosome group. P <0.001 compared to group 1; # P <0.05 vs group 2.
FIG. 3 is a graph of the effect of three adult stem cell exosomes on suspension resting time of PD model mice.
Grouping: 1- -control group; 2- - -PD model group; 3- - -bone marrow mesenchymal stem cell exosome group; 4- - -adipose-derived stem cell exosome group; 5- -epidermal stem cell exosome group. P <0.001 compared to group 1; # P <0.05, # P <0.01 compared to group 2.
FIG. 4 is a graph of the effect of three adult stem cell exosomes on the smell of PD model mice.
Grouping: 1- -control group; 2- - -PD model group; 3- - -bone marrow mesenchymal stem cell exosome group; 4- - -adipose-derived stem cell exosome group; 5- -epidermal stem cell exosome group. P <0.001 compared to group 1; # P <0.01, # # P <0.001 compared to group 2.
FIG. 5 is a graph of the effect of three adult stem cell exosomes on Tyrosine Hydroxylase (TH) -positive cells at the substantia nigra locus of mice in the PD model.
Grouping: 1- -control group; 2- - -PD model group; 3- - -bone marrow mesenchymal stem cell exosome group; 4- - -adipose-derived stem cell exosome group; 5- -epidermal stem cell exosome group. P <0.001 compared to group 1; # P <0.05, # P <0.01 compared to group 2.
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
Example 1
The invention provides a preparation method of an adult stem cell exosome, which comprises the following steps: separating and culturing adult stem cells in vitro, and extracting exosomes in cell culture solution supernatant by an ultracentrifugation method. The adult stem cells comprise bone marrow mesenchymal stem cells, skin epidermal stem cells and adipose-derived stem cells. The method specifically comprises the following steps:
1. bone marrow mesenchymal stem cell isolation and culture
Male C57B L/6J mice at 4 weeks of age were anesthetized and sacrificed by cervical dislocation, bilateral femurs and bilateral femurs of the mice were extracted completelyTibia, washed with sterile saline and 75% ethanol solution, respectively. The attached muscle, tissue, is removed from the bone surface and the bone ends are cut with surgical scissors to expose the marrow cavity. Sucking DMEM/F-12 culture solution containing 10% fetal calf serum by using an injector, inserting a needle into a marrow cavity, washing out bone marrow, placing the bone marrow in a culture dish, blowing and beating repeatedly to fully mix the bone marrow and the culture solution, and filtering by using a 200-mesh stainless steel filter screen. The filtrate was centrifuged at 1000rpm for 5min, and the supernatant was removed and the cells were resuspended in DMEM/F-12 medium containing 10% fetal bovine serum. Transferring the cell suspension to a culture flask, adding DMEM/F-12 culture solution containing 10% fetal bovine serum, and culturing in a cell culture box (37 deg.C, 5% CO)2). After 72h, the solution was changed for the first time, and every 2 days thereafter. And (3) observing the growth state of the cells under an inverted microscope every day in the cell culture process, and when the cells basically grow to fill the bottom of the bottle, performing the steps of 1: and (5) carrying out passage at a ratio of 2. The bone marrow mesenchymal stem cell surface antigen was identified by the following method. The old culture solution in the culture flask was poured into a waste solution bottle, 2ml of PBS solution was pipetted into the culture flask, and the remaining culture solution was washed. The PBS wash was decanted, digested with tryptic digest and then digested by addition of DMEM/F-12 medium containing 10% fetal bovine serum to stop the digestion and form a cell suspension. Transferring the cell suspension into a centrifuge tube, centrifuging at 1000rpm for 5min, removing supernatant, washing with PBS solution, centrifuging for 2 times, preparing cell suspension with PBS, and counting cells with cell counting plate to 1x 106and/L, subpackaging the cell suspension into 3 tubes, wherein each tube is 0.1ml, adding 20 mu l of PE labeled CD44 and CD45 antibodies in a dropwise manner under a light-proof condition respectively, adding PBS in the rest 1 tube serving as a blank control, incubating at room temperature for 30min in a light-proof manner, centrifuging at 1000rpm for 5min, pouring out supernatant, washing each tube with PBS solution for 2 times, diluting to 1ml with the PBS solution, analyzing and screening by a flow cytometer, and obtaining bone marrow mesenchymal stem cells for subsequent experiments.
2. Obtaining and culturing adipose-derived stem cells
Washing subcutaneous adipose tissue, removing surface fascia and blood vessel, cutting the extracted adipose tissue after washing with Phosphate Buffer Solution (PBS), digesting with collagenase type I (3mg/m L) at 37 deg.C for 40min, adding DMEM medium containing 10% FBS to stop digestion, and centrifuging at 1000rpmRemoving supernatant after 5min, adding 10% FBS-containing DMEM medium, resuspending, filtering with 200 mesh screen, and inoculating to 25cm2In a culture flask. Placing at 37 ℃ and 5% CO2Culturing in an incubator. After 24h, the culture medium is replaced, nonadherent cells are removed, the culture medium is replaced every 3 days until the cells grow to 80-90% fusion, the cells are digested by pancreatin (0.25%), and the cells are passaged according to the proportion of 1: 3. Selecting the 2 nd generation adipose-derived stem cells, digesting with pancreatin (0.25%) when the cells grow and fuse to 80% -90%, centrifuging, washing with PBS for 2 times, counting the cells, and taking 10 cells from each tube6The method comprises the following steps of (1) resuspending each cell in 100 mu L PBS, adding flow antibodies APC-CD44, APC-CD45 and FITC-CD31 respectively, incubating for 30min at 4 ℃, washing off unlabeled antibodies by PBS, adding 300 mu L PBS to resuspend the cells, taking no antibody as a negative control, detecting cell phenotype by a Beckman flow cytometer, and performing cell clone formation experiment of the adipose-derived stem cells, namely selecting 3 rd generation adipose-derived stem cells, inoculating the cells into a 6-well plate according to the density of 100 cells/well, changing the solution every 2 days, removing the culture medium after conventional culture for 14 days, washing the cells for 2 times by PBS, fixing the cells by 4% paraformaldehyde solution for 15min, staining for 15min by 0.1% crystal violet, taking pictures after washing by PBS, marking colonies of more than 50 cells as 1 clone, and determining the ratio of the number of the clone in each well to the number of the inoculated cells as.
3. Obtaining and culturing of epidermal stem cells
Cleaning fresh foreskin tissue taken out from operation with PBS containing double antibody under aseptic condition, soaking for 1-2 hr, separating epidermis, fat and dermis, and cutting epidermis into 1x 0.5cm2Transferring the fragments into a small conical flask, adding 0.5% neutral protease, completely soaking the tissue blocks, sealing with a sealing film, and digesting at 4 ℃ overnight. Peeling epidermis with forceps, discarding dermis, cutting epidermis with ophthalmic scissors, adding 0.5% pancreatin, digesting in water bath at 37 deg.C for 10min, stopping digestion with serum-containing culture medium, suspending cells, and filtering with 70 μm cell filter to obtain single cell suspension. Centrifuging at 1000rpm for 5min, and mixing the precipitated cells at 10%5Cells/cm2Inoculating to IV collagen coated culture bottle, culturing in cell culture box for 10min, sucking upper layer non-adherent cells, adding appropriate amount of KSFM culture medium, and culturing in cell culture box(37℃，5％CO2) And changing the liquid every other day. Sorting the epidermal stem cells by a flow cytometer, comprising the following steps. Taking out cultured primary epidermal cells, removing culture medium by suction, washing with PBS for 2 times, adding 0.05% pancreatin-0.02% EDTA digestive solution, digesting at 37 deg.C for 2min, adding 10ml digestive stop solution to stop digestion, blowing to remove cell wall, centrifuging at 1000rpm for 5min, discarding supernatant, adding precooled PBS, suspending cells, centrifuging at 1000rpm for 5min, adding PBS to resuspend cells, counting cells, and counting according to 106Cells/tube were packed, antibodies (CD71-FITC and CD49f-PE) were added, and epidermal stem cell cultures were screened for flow cytometry analysis for subsequent experiments.
4. Exosome preparation
And extracting exosome secreted by the stem cells by an ultracentrifugation method. Filtering the stem cell culture solution with 0.22 μm screen, and performing gradient centrifugation on the filtrate at 300rpm, 4 deg.C, 5min, 2000rpm, 4 deg.C, 10min, 10000rpm, 4 deg.C, and 30 min. The above steps are all performed by reserving the supernatant and discarding the cell residues. Collecting supernatant after gradient centrifugation, ultracentrifuging at 100000rpm at 4 deg.C for 2h, and removing supernatant to obtain precipitate as exosome secreted by stem cells. The obtained exosomes were then resuspended and washed with pre-cooled PBS and again ultracentrifuged at 100000rpm, 4 ℃ for 2 h. Finally, the suspension was resuspended in 50. mu.l of 4 ℃ PBS and transferred to a low-adhesion tube. The obtained exosome is observed in shape and size through an electron microscope, and the marker protein of the exosome is detected by a Western method, so that the obtained precipitate is confirmed to be a cell exosome. And finally, confirming the concentration of the exosome by using a nanoparticle tracking analyzer for subsequent experiments.
Example 2
The adult stem cell exosome prepared in the example 1 is used for treating a Parkinson model mouse in a nasal administration mode, the ethological detection is carried out, after the ethological detection is finished, the mouse is killed after anesthesia, the substantia nigra part is separated, and the histomorphological detection is carried out.
1. Preparation of a Parkinson animal model:
a PD model mouse is induced by an 8-week-old C57B L/6J male mouse by an intraperitoneal injection method of neurotoxin MPTP (N-methyl-4-phenyl-l,2,3, 6-tetrahydropyridine). three days before administration, the mouse is subjected to a series of behavioural training such as rod turning and climbing every day.
2. Exosome dosing:
in this example, a Parkinson model mouse was treated by nasal administration. PD model mice prepared by the above method were administered 1.0X 10/mouse10Each nanoparticle exosome was dissolved in physiological saline at a volume of 20 μ l per administration, administered every other day, for a total of 3 times. Control mice were given equal volumes of saline. And after the treatment is finished, performing ethological detection, after the ethological detection is finished, killing the mouse after anesthesia, separating a substantia nigra part, and performing histomorphological detection.
3. Behavioural testing
Rotarod experiment (rotarod test): the experiment needs the animal to keep balance and continuously move on the accelerated rotating rod, and is widely used for detecting the movement coordination. The rods were rotated about 2cm in diameter, and the mice were then placed on the rods with the head oriented perpendicular to the long axis of the rods, the rotation speed of the rods was gradually increased from 5rpm to 40rpm within 5min, acclimatized for 3 days before tail vein injection, 5min apart for each test, and the average was taken 3 consecutive times. The total score is calculated from the latency time for the animal to remain balanced on the rotating rod without falling. The results of the detection are shown in FIG. 1.
Pole climbing experiment (pole test): a pole of 1cm diameter and 50cm length with a cork pellet of 1.5cm diameter fixed at the top is made and gauze is wrapped to increase friction, the pole is placed upright, a mouse is placed on the pellet at the top of the home made climbing pole, then the time from the beginning of the animal's movement to the complete turning of the head down until the animal returns to the bottom of the pole is recorded, 5min intervals are detected each time, and the average value is taken 3 times. Three days before tail vein injection, the mice are trained by climbing poles at the same time every day, and the mice with larger differences are removed to reduce experimental errors. The results of the detection are shown in FIG. 2.
Tail suspension test (tail suspension test): the rat tail was fixed to the bar, the rats were kept about 1.5m from the ground, and the time each rat was stationary within 5min was recorded and calculated. The resting time includes the shaking time of the whole body when the limbs and the head of the rat are relatively motionless. The results of the detection are shown in FIG. 3.
Olfaction test (smeltest): all mice were dieted 24h before the experiment, during which a short section of cheese strips was placed for familiarity with smell and taste for the mice to search in the experiment. The experimental article was prepared, a clean cage, and then clean padding was laid evenly over the cage, approximately 3cm thick. The experimental cheese strips were then buried under the padding in the middle, approximately 1cm, making the cheese strips completely invisible. The mice were removed from the cage, gently placed at the midpoint of the experimental cage, and the timer was pressed to begin timing. The data was recorded when the mouse found the cheese strip and started eating by pressing a timer to stop the timing. If none of the mice found cheese strips within 300s, the timing was stopped and the time recorded for 300 s. And then, sequentially burying cheese strips below padding materials at the upper left corner, the upper right corner, the lower left corner and the lower right corner, repeating the steps, recording data, and removing the minimum value and the maximum value for statistical analysis. The results of the detection are shown in FIG. 4.
The experimental results show that compared with the control mice, the exosomes from the three sources can relieve the motor dysfunction of the PD model mice to different degrees, and are shown in that the time spent on the rotarod is increased (figure 1), the time spent on the vertical rod climbing to the desktop is shortened (figure 2), the resting time in the suspension experiment is reduced (figure 3), and the smell sense is more sensitive (figure 4).
4. Tissue morphology detection
A histochemical method is used for staining tyrosine hydroxylase positive cells at the substantia nigra part of an experimental mouse, and the main operation steps are as follows: 1. placing the mouse brain tissue obtained from the materials in 4% paraformaldehyde, and placing in a refrigerator at 4 ℃ for 24 h; 2.1 preparing 20% of the X PB and 30% of the sucrose, and sequentially dehydrating for 24h, wherein if the tissue does not sink to the bottom, the time is properly prolonged; 3. treating dehydrated brain tissue slice, adjusting thickness to 12 μm, oven standing at 37 deg.C overnight, and storing at-20 deg.C; 4. baking the slices at 60 ℃ for 2h before dyeing, and washing the slices with PBS for 3 times, wherein each time is 5 min; 6. sealing with sealing liquid at 37 deg.C for 30 min; washing with PBS for 2 times, and blocking endogenous catalase for 5 min; washing with PBS for 2 times, blocking agent for nonspecific staining for 30min, and oven drying at 37 deg.C; 8. incubated with TH antibody (1:200) overnight in a refrigerator at 4 deg.C; washing with PBS for 2 times, adding biotin-labeled goat anti-mouse/rabbit IgG, and washing at 37 deg.C for 1 hr; washing with PBS for 2 times, adding streptavidin-peroxidase, and washing at 37 deg.C for 1 hr; washing with PBS for 2 times, and performing DAB color development; 12. dehydrating with 50% alcohol, 70% alcohol, 80% alcohol, 95% alcohol and 100% alcohol in sequence, each for 5 min; 13. absolute ethyl alcohol, dimethylbenzene and dimethylbenzene are transparent for three times, each time is 5min, and the neutral resin is sealed and then is observed by a microscope to take a picture. The number of TH positive cells was statistically analyzed using Stereo investigerator software.
The histochemical method is used for detecting Tyrosine Hydroxylase (TH) positive cells at the substantia nigra part of a mouse brain, and the result shows that the exosomes from three sources can increase the number of TH positive cells at the substantia nigra part of a PD model mouse (figure 5), which indicates that the exosomes can protect the degeneration and the inactivation of dopaminergic neurons, thereby maintaining the number of dopaminergic neurons at the substantia nigra part of the PD model mouse.
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 variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (7)
1. The preparation method of the adult stem cell exosome is characterized by comprising the following steps: separating and culturing adult stem cells in vitro, and extracting exosomes in cell culture solution supernatant by an ultracentrifugation method.
2. The method for preparing an exosome according to claim 1, wherein the adult stem cell comprises a bone marrow mesenchymal stem cell, a skin epidermal stem cell and an adipose stem cell.
3. The method for preparing an exosome according to claim 2, wherein the method for isolating and culturing the mesenchymal stem cell comprises:
anaesthetizing a mouse, breaking the neck, killing the mouse, completely extracting femurs and shinbones on two sides of the mouse, sucking DMEM/F-12 culture solution containing 10% fetal calf serum by using an injector, inserting a needle into a marrow cavity, washing out marrow, placing the marrow in a culture dish, and filtering;
centrifuging the filtrate, removing supernatant, resuspending cells with DMEM/F-12 culture solution containing 10% fetal calf serum, transferring the cell suspension to a culture bottle, adding DMEM/F-12 culture solution containing 10% fetal calf serum, and culturing in a cell culture box;
after 72h, the liquid is changed for the first time, the liquid is changed every 2 days, the growth state of the cells is observed under an inverted microscope every day in the cell culture process, and when the bottom of the bottle is full of the cells, the ratio of the growth state to the growth state is 1:2, passage in proportion;
analyzing and screening by a flow cytometer to obtain the bone marrow mesenchymal stem cells.
4. The method for preparing an exosome according to claim 2, wherein the method for isolating and culturing the skin epidermal stem cell comprises:
cleaning and soaking fresh foreskin tissue taken down in an operation by PBS (phosphate buffer solution) containing double antibodies under an aseptic condition, separating epidermis, fat and dermis, cutting the epidermis into fragments, transferring the fragments into a small conical flask, adding 0.5% neutral protease, completely soaking tissue blocks, sealing a sealing film, and digesting at 4 ℃ overnight;
peeling epidermis with forceps, removing dermis, cutting epidermis, adding 0.5% pancreatin, digesting in water bath at 37 deg.C for 10min, stopping digestion with serum-containing culture medium, suspending cells, and filtering with cell filter to obtain single cell suspension;
after centrifugation, the precipitated cells were pressed 105Cells/cm2Inoculating into IV collagen coated culture bottle, culturing in cell culture box for 10min, and removing the upper layerSucking out parietal cells, adding a KSFM culture medium, culturing in a cell culture box, and changing the culture solution every other day;
analyzing and screening by a flow cytometer to obtain the skin epidermal stem cells.
5. The method for preparing an exosome according to claim 2, wherein the method for isolating and culturing the adipose-derived stem cells comprises:
removing surface fascia and blood vessels from subcutaneous adipose tissue taken out by operation, cutting the extracted adipose tissue into pieces after washing with phosphate buffer solution, digesting with collagenase type I at a constant temperature of 37 ℃ for 40min, and adding DMEM medium containing 10% FBS to stop digestion;
centrifuging, removing supernatant, adding 10% FBS-containing DMEM medium, resuspending, filtering, inoculating into culture flask, standing at 37 deg.C and 5% CO2Culturing in an incubator;
changing the culture medium after 24h, removing the non-adherent cells, changing the culture medium every 3 days until the cells grow to 80-90% of fusion, digesting with pancreatin, and carrying out passage at the ratio of 1: 3;
detecting cell phenotype by a flow cytometer to obtain the adipose-derived stem cells.
6. The method for preparing an exosome from an adult stem cell according to claim 1, wherein the method for extracting exosome from a supernatant of a cell culture solution comprises:
filtering the stem cell culture solution, performing gradient centrifugation on the filtrate at 300rpm, 4 deg.C, 5min, 2000rpm, 4 deg.C, 10min, 10000rpm, 4 deg.C, and 30min, respectively, retaining the supernatant, and discarding the cell residue;
collecting supernatant after gradient centrifugation, ultracentrifuging at 100000rpm at 4 deg.C for 2h, and removing supernatant to obtain precipitate as exosome secreted by stem cells;
the obtained exosomes were resuspended and washed with precooled PBS, ultracentrifuged again at 100000rpm at 4 ℃ for 2h, resuspended with 50. mu.l of 4 ℃ PBS, and transferred to a low-adhesion tube to obtain exosomes.
7. Use of an adult stem cell exosome prepared according to the method of any one of claims 1 to 6 in the treatment of parkinson's disease.
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