CN110577931A - Intermittent hypoxia treatment stem cell source exosome and application thereof in myocardial tissues - Google Patents
Intermittent hypoxia treatment stem cell source exosome and application thereof in myocardial tissues Download PDFInfo
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Abstract
An exosome derived from mesenchymal stem cells is prepared by performing intermittent hypoxia stimulation on the mesenchymal stem cells. The exosome prepared by the invention has stronger tissue organ ischemia-reperfusion injury protection effect, improves the biological effect of a unit exosome, and is more beneficial to the treatment of ischemic heart diseases and the like.
Description
Technical Field
The invention relates to an induced extracellular membrane vesicle, in particular to an induced exosome, which has specific composition and content and is suitable for tissue repair, such as: myocardial tissue.
Background
exosomes (exosomes) are of increasing interest as important mediators of paracrine because of their particular biological role. Exosomes are membrane vesicles secreted by living cells, which have the functions of performing protein, nucleic acid transport, specifically targeting receptor cells, exchanging proteins and lipids or triggering downstream signaling events, participating in intercellular communication, and the like, and are gaining increasing attention. Since exosomes can exist in the extracellular environment for a long period of time without degradation or dilution, and can be transported to distant target cells by interstitial fluid or blood, and exosomes are almost non-immunogenic, rejection reactions do not occur. Therefore, exosomes as ideal information-transmitting molecules can be used for the regulation of tissue repair, such as: regulating and controlling myocardial ischemia reperfusion injury.
in order to regulate and control myocardial ischemia-reperfusion injury, researchers focus on the repair effect of exosomes derived from mesenchymal stem cells on myocardial ischemia-reperfusion injury, and obtain very remarkable results. Exosomes have very significant clinical transformation advantages. First, exosomes have little antigenicity, and in the current large-scale exosome research, allogenic and xenogenic exosomes are mostly adopted for research. The exosome is not easy to recruit immune cells in a body, can wrap the content in the exosome well, is used as a drug carrier with great potential, can protect the content from being degraded in blood circulation, and has extremely high clinical transformation value.
furthermore, most cells with differentiation potential are currently being discovered, such as: bone marrow mesenchymal stem cells, embryonic stem cells, adipose mesenchymal stem cells (ADMSC), induced pluripotent stem cells and the like all have a protective effect of improving myocardial ischemia-reperfusion injury.
However, the separation technology of exosomes is still under development, so that the low exosome yield is a great problem which hinders the clinical application of exosome transformation. How to obtain a more pronounced unit protection (even if the unit exosomes carry a richer cardiomyocyte protective factor) exosomes becomes a challenge that must currently be faced.
Therefore, through the related researches in recent years, the fact that most of the existing exosomes are modified by adopting lentivirus or adeno-associated virus to over-express certain protein molecules and non-coding RNA which have proved to have protective effect on myocardial ischemia-reperfusion injury in maternal cells or loading the protein molecules and the non-coding RNA into the exosomes through means of liposome, electroporation and the like is found, and the protective effect of the unit exosomes on myocardial injury is further improved. Although, with some success, there are additional obstacles to the clinical transformation of exosomes: on one hand, the biological effect of the exosome is the 'resultant force' generated by the inclusion thereof, and the single molecule is singly overexpressed or knocked-out, so that the biological effect of the exosome cannot be changed to a great extent; on the other hand, the means and technology for extracting exosomes are not mature at present, the yield is not high, and the exosome modification by introducing nucleic acid in a virus infection or electroporation mode and the like can affect the survival state of mesenchymal stem cells, so that the synthesis function of exosomes can be affected. Thirdly, from the clinical transformation point of view, the biological modification means such as virus infection and the like have great biological safety hidden dangers at present, and the cost is too high, thus being not beneficial to clinical transformation.
disclosure of Invention
It is an object of the present invention to provide a stem cell-derived exosome having a more efficient, more significant cardioprotective effect.
The invention also aims to provide a preparation method of the exosome, which aims to change the abundance and expression profile of the cardioprotective molecules, such as miRNA, contained in the exosome secreted by the mesenchymal stem cells by means of physical intervention.
The invention further aims to provide application of exosome obtained by separating adipose-derived mesenchymal stem cells after pretreatment in preparation of a myocardial ischemia-reperfusion injury medicament.
The invention also aims to provide application of exosome obtained by separating adipose-derived mesenchymal stem cells after pretreatment in preparation of a myocardial ischemia-reperfusion injury apparatus.
The invention also aims to provide application of exosome obtained by separating adipose-derived mesenchymal stem cells after pretreatment in preparation of a medicament for regulating and controlling the apoptosis level of myocardial cells in myocardial ischemia-reperfusion injury.
the invention also aims to provide application of exosome obtained by separating adipose-derived mesenchymal stem cells after pretreatment in preparation of a device for regulating and controlling apoptosis level of myocardial cells in myocardial ischemia-reperfusion injury.
Mesenchymal stem cells of various tissue sources may be applied to the present invention. It is contemplated that mesenchymal stem cells are present in higher amounts in adipose tissue than other mesenchymal stem cells, and that the intrinsic biological characteristics of ADMSCs are more stable than other species of MSCs. Adipose tissue is also the most convenient way to extract from exosomes in terms of clinical transformation potential, and is more acceptable to patients than MSCs derived from other tissues, and therefore has incomparable clinical transformation potential compared with MSCs derived from other tissues. In the present invention, it is preferred to select ADMSC as the mother cell for the extraction of exosomes and to obtain exosomes therefrom by means of physical induction.
A mesenchymal stem cell-derived exosome mainly comprises microRNA224-5 p.
The other exosome derived from the mesenchymal stem cells mainly comprises non-coding RNAs such as microRNA224-5p, microRNA141-3p, microRNA 34a-5p, microRNA 23a-3p, microRNA 130a-3p, microRNA 23b-3p, microRNA 200a-3p, microRNA93-5p, microRNA 301a-3p and microRNA 152-3p, and the substances have the effect of protecting cardiac muscle (cells) singly or in combination.
The exosome derived from the mesenchymal stem cells has a remarkable repairing function on myocardial ischemia-reperfusion injury, and has a more efficient and remarkable heart protection effect.
The exosome derived from the mesenchymal stem cells can regulate the apoptosis level of myocardial cells in myocardial ischemia-reperfusion injury by a cell apoptosis mechanism.
The exosome derived from the mesenchymal stem cells provided by the invention is prepared into a medicament for regulating and controlling the apoptosis level of myocardial cells in myocardial ischemia-reperfusion injury and realizing the repair of the myocardial ischemia-reperfusion injury.
The exosome derived from the mesenchymal stem cells provided by the invention is applied to a carrier (such as but not limited to a bracket made of PLGA (polylactic-co-glycolic acid) and metal and the like) to prepare a medical device, and is used for regulating and controlling the apoptosis level of myocardial cells in myocardial ischemia-reperfusion injury and realizing the repair of the myocardial ischemia-reperfusion injury.
The exosome derived from the mesenchymal stem cells is prepared by performing intermittent hypoxia (namely, hypoxia/reoxygenation stimulation for more than 2 times, for example, at least 3 cycles of hypoxia → reoxygenation → hypoxia → reoxygenation) intervention on the mesenchymal stem cells, and has a myocardial protection effect.
A preparation method of exosome derived from mesenchymal stem cells comprises the following steps:
When the confluence rate of the mesenchymal stem cells of the generations P2-P3 reaches 85% -90%, carrying out intermittent hypoxia stimulation on the cells in a container (such as a culture dish) through a hypoxia chamber, namely, after 3 cycles of hypoxia → reoxygenation, replacing the cell culture medium with a DMEM-F12 complete culture medium containing 5v/v fetal calf serum without exosomes, incubating for 24-36 hours in the culture box, collecting the culture medium, obtaining cell culture supernatant, carrying out membrane filtration (such as 0.22 mu m), and collecting filtrate; and then placing the filtrate at the rotating speed of 300 g-100000 g for differential centrifugation to obtain a precipitate, namely the exosome.
And (2) hypoxia, namely placing the adipose-derived mesenchymal stem cells into a hypoxia chamber, replacing a cell culture medium with a sugar-free and serum-free DMEM culture medium, introducing mixed gas of 80 v/v% -95 v/v% nitrogen and 0 v/v% -5 v/v% carbon dioxide into the chamber, detecting the oxygen concentration of the gas in the hypoxia chamber by an oxygen battery until the oxygen concentration is 0 v/v%, and then closing a ventilation valve of the chamber to isolate sugar in the cells and supplying oxygen for 45-60 minutes.
Reoxygenation, i.e., opening the hypoxia chamber valve and replacing the medium with DMEM-F12 medium with sugar, and transferring to an incubator with 5 v/v% carbon dioxide at 37 ℃ for 1 hour.
In order to improve the enrichment efficiency of exosomes, a specific differential centrifugation mode is to centrifuge at the rotating speeds of 300g, 2,000g, 10,000g, 100,000g and 100,000g at 4 ℃, and finally obtain precipitates as exosomes.
In another specific embodiment of differential centrifugation, centrifugation is carried out at a rotation speed of 300g, 2,000g, 10,000g and 100,000g for 10-60 minutes at 4 ℃, the obtained precipitate is re-suspended and then centrifuged at 100,000g for 70 minutes, and finally the obtained precipitate is exosome.
In another specific differential centrifugation implementation mode, firstly, centrifugation is carried out for 10 minutes at the rotating speed of 300g at the temperature of 4 ℃, and supernate is taken; then centrifuging at 4 ℃ for 10 minutes at the rotating speed of 2,000g, and taking supernatant; centrifuging at 4 deg.C and 10,000g speed for 60 min, and collecting supernatant; centrifuging at 100,000g rotation speed at 4 deg.C for 70 min, discarding supernatant, and resuspending the precipitate with PBS; finally, the exosome is obtained after centrifugation for 70 minutes at the rotating speed of 100,000g at the temperature of 4 ℃.
the exosome obtained after intervention of the adipose mesenchymal stem cells by the intermittent hypoxia pretreatment method provided by the invention has a more remarkable cell and tissue repair effect than an exosome derived from the mesenchymal stem cells without intervention, and can be prepared into a medicament or a medical apparatus for cell and tissue repair, in particular to application in myocardial cell injury repair and myocardial tissue ischemia-reperfusion injury protection.
The technical scheme of the invention has the following beneficial effects:
The Exosome (INTEXO) secreted by the adipose-derived mesenchymal stem cells intervened by the pretreatment method provided by the invention uses the adipose-derived mesenchymal stem cells from the aspect of tissue or cell sources, the tissue is easily obtained, the enrichment degree of the mesenchymal stem cells is high, the mesenchymal stem cells are easily accepted by people in clinical application, and the adipose-derived mesenchymal stem cells have good clinical transformation potential. In the aspect of a pretreatment method, the invention abandons a method for changing the content of the exosome by using biological means such as virus, plasmid and the like, and pretreats the adipose-derived mesenchymal stem cells by using transient, intermittent and non-invasive hypoxia/reoxygenation stimulation through physical means, so that the adipose-derived mesenchymal stem cells generate feedback to the hypoxia stimulation, and further, more abundant miRNA with the myocardial protection effect is loaded into the exosome, thereby generating more remarkable protection effect on myocardial cells and myocardial tissue ischemia reperfusion injury.
the exosome prepared by the method has a repairing effect on cells and tissues, particularly has an important improving effect on proliferation and damage repair of myocardial cells, has a clinical transformation potential for treating myocardial infarction, tissue organ embolism and reperfusion injury after vascular recanalization, and has high safety, clinical practicability and feasibility.
Drawings
Fig. 1 is a flow identification diagram of adipose mesenchymal stem cell surface markers;
FIG. 2 is an electron microscope identification of exosomes derived from adipose derived mesenchymal stem cells;
FIG. 3 is a diagram of Western Blot for identifying exosome surface antigen molecules;
FIG. 4 is a graph of particle size analysis and identification of exosome NTA derived from adipose-derived mesenchymal stem cells;
FIG. 5A is a Western Blot result chart of apoptosis of cells generated in an in vitro myocardial cell hypoxia reoxygenation injury model by exosomes (INTEXO) generated by induction and Exosomes (EXO) derived from non-stem adipose-derived mesenchymal stem cells due to the expression change of a apoptosis pathway signal molecule after myocardial cell hypoxia/reoxygenation; wherein, the graph A is the expression change condition of the apoptosis pathway signal molecules after the hypoxia/reoxygenation intervention of the myocardial cells; panel "B" is a statistical analysis, representing P < 0.05 compared to the IR group; CON represents normal cardiomyocytes; IR represents the group of cardiomyocytes prognostic of hypoxic reoxygenation; IR + EXO represents the group of hypoxic reoxygenation-prognostic cardiomyocytes added to the group of ADMSC-derived exosomes; IR + INTEXO represents the group of myocardial cells with the prognosis of hypoxia-reoxygenation stem added with the group of exosomes derived from ADMSC after the intermittent hypoxia treatment;
FIG. 5B is a Western Blot result graph of apoptosis generated by exosomes (INTEXO) generated by induction and Exosomes (EXO) derived from non-stem preadipocyte mesenchymal stem cells in an in vitro myocardial cell hypoxia/reoxygenation injury model due to apoptosis pathway signal molecule expression change after myocardial cell hypoxia/reoxygenation; wherein, the graph A is the expression change condition of the apoptosis pathway signal molecule after the hypoxia/reoxygenation intervention of the cardiac muscle cell; panel "B" is a statistical analysis, representing P < 0.05 compared to the IR group; CON represents normal cardiomyocytes; IR represents the group of cardiomyocytes prognostic of hypoxic reoxygenation; IR + EXO represents the group of hypoxic reoxygenation-prognostic cardiomyocytes added to the group of ADMSC-derived exosomes; IR + INTEXO represents the group of myocardial cells with the prognosis of hypoxia-reoxygenation stem added with the group of exosomes derived from ADMSC after the intermittent hypoxia treatment;
FIG. 5C is a graph of the AnnexinV-PI flow results of the protective effect of INTEXO and EXO on cardiomyocyte apoptosis in an in vitro myocardial cell hypoxia-reoxygenation injury model; wherein "a" panel CON represents normal cardiomyocytes; "B" panel IR represents the group of cardiomyocytes prognostic of hypoxic reoxygenation; "C" panel IR + EXO represents the addition of the group of hypoxic-reoxygenation-prognostic cardiomyocytes to the group of ADMSC-derived exosomes; "D" panel IR + INTEXO represents the group of hypoxic-reoxygenation-prognostic cardiomyocytes added to the group of ADMSC-derived exosomes after intermittent hypoxic treatment; the "E" plot is a statistical analysis; the "A" panel, "the" B "panel, the" C "panel and the" D "panel, the upper left quadrant of which indicates the proportion of dead cells; the upper right quadrant represents the proportion of late apoptotic cells; the lower left quadrant represents the proportion of normal viable cells; the lower right quadrant represents the proportion of early apoptotic cells; p < 0.05 compared to IR;
FIG. 6A is a graph of Western Blot results of INTEXO and EXO on myocardial cell apoptosis and apoptosis in an in vivo mouse myocardial Ischemia Reperfusion (IR) model; wherein, the graph A is the expression change condition of a tar death pathway signal molecule after the myocardial ischemia reperfusion operation of the mice; panel "B" is a statistical analysis, representing P < 0.05 compared to the IR group; CON represents normal mouse myocardial tissue group; IR for mouse post-myocardial ischemia reperfusion surgery group; IR + EXO represents the mice post-myocardial ischemia reperfusion surgery group injected via tail vein into ADMSC-derived exosome group; IR + INTEXO represents that the tail vein of the mouse after myocardial ischemia reperfusion operation is injected into an exosome group with the ADMSC source after intermittent hypoxia treatment;
FIG. 6B is a graph of Western Blot results of INTEXO and EXO on myocardial cell apoptosis and apoptosis in an in vivo mouse myocardial Ischemia Reperfusion (IR) model; wherein, the graph A is the expression change condition of a tar death pathway signal molecule after the myocardial ischemia reperfusion operation of the mice; panel "B" is a statistical analysis, representing P < 0.05 compared to the IR group; CON represents normal mouse myocardial tissue group; IR for mouse post-myocardial ischemia reperfusion surgery group; IR + EXO represents the mice post-myocardial ischemia reperfusion surgery group injected via tail vein into ADMSC-derived exosome group; IR + INTEXO represents that the tail vein of the mouse after myocardial ischemia reperfusion operation is injected into an exosome group with the ADMSC source after intermittent hypoxia treatment;
FIG. 6C is a graph of the Evenblue/TTC staining results of INTEXO and EXO in evaluating the infarct rate of myocardial tissue in various groups of mice in an in vivo mouse IR model; wherein, IR is myocardial ischemia reperfusion group; IR + EXO is an exosome group derived from ADMSC injected after myocardial ischemia reperfusion; IR + intaxo is an ADMSC-derived exosome group pretreated by interjection hypoxia following myocardial ischemia reperfusion; AAR, Area in risk is the Area of danger; IS IS infarct size; total Area is the Total Area; p < 0.05 compared to the IR group;
FIG. 6D is a graph of cardiac ultrasound results of INTEXO and EXO in evaluating systolic function in mice in vivo in a mouse IR model; wherein, the heart function of the mice is 3 days after the "A" picture; mouse cardiac function on day 7 post "B" mapping; IR myocardial ischemia reperfusion group; IR + EXO is an exosome group derived from ADMSC injected after myocardial ischemia reperfusion; IR + intaxo is an ADMSC-derived exosome group pretreated by interjection hypoxia following myocardial ischemia reperfusion; EF: left ventricular ejection fraction; FS: left ventricular minor axis contraction; p < 0.05 compared to Wild (WT) mice;
FIG. 6E is a graph of the results of TUNEL staining of INTEXO and EXO in vivo in mouse IR models to assess in situ apoptosis in the hearts of various groups of mice; wherein, IR is myocardial ischemia reperfusion group; IR + EXO is an exosome group derived from ADMSC injected after myocardial ischemia reperfusion; IR + intaxo is an ADMSC-derived exosome group pretreated by interjection hypoxia following myocardial ischemia reperfusion; the graph A is the change of in-situ myocardial cell apoptosis in myocardial tissues detected by Tunnel staining after myocardial ischemia reperfusion; the "B" diagram is a statistical analysis of the apoptosis case; p < 0.05 compared to the IR group;
FIG. 7 shows the result of differential sequencing of miRNA expression in INTEXO and EXO two groups of exosomes; wherein EXO represents a panel of ADMSC-derived exosomes; INTEXO stands for interstitial hypoxia-pretreated ADMSC-derived exosomes; the specific sample unit numerical value is the relative expression quantity of the Z-SCORE data after the Z-SCORE data is normalized; the right column is miRNA name, and the lower column is sample name of each group;
FIG. 8 shows the results of the dual-luciferase reporter gene binding verification of miRNA224-5p with the most significant difference in expression between INTEXO and EXO and its target site TXINIP; wherein 3 'UTR-NC represents a negative control for a TXNIP 3' UTR overexpression plasmid; 3 'UTR-WT represents a TXNIP 3' UTR overexpression plasmid; 3 'UTR-Mut represents a TXNIP 3' UTR mutant overexpression plasmid; MiR224-5p represents MicroRNA224-5p plasmid; MiR224-5 p-NC stands for the negative control of MicroRNA224-5p plasmid; p < 0.05 compared to the other groups.
Detailed Description
The technical scheme of the invention is described in detail in the following with reference to the accompanying drawings. Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.
Other reagents used in the examples of the invention were purchased from Sigma-Aldrich, unless otherwise specified.
The method for obtaining the adipose-derived mesenchymal stem cells of the P2-P3 generations in the embodiment is as follows:
Step 1: obtaining a clinical fat sample or extracting adipose tissues at the groin of an experimental animal;
Step 2: cutting adipose tissue into small pieces;
And step 3: adding an equal volume of collagenase IV for digestion (collagenase dissolved in DMEM/F12 complete medium);
And 4, step 4: placing in a constant temperature shaking table at 37 ℃, and fully oscillating;
and 5: centrifuging, collecting upper layer oil, middle layer culture medium, tissue, and collagenase mixed solution, completely culturing the precipitated cells in the basal layer with DMEM/F12, separating to obtain adipose-derived mesenchymal stem cells, and inoculating (for example, in T25 em)2Type cell culture flasks) and subjected to primary culture and subculture.
Step 6: taking P2-P3 cells to carry out flow type surface antigen identification;
And 7: inoculating P2-P3 cells and culturing the cells;
And 8: when the confluent rate of the adipose-derived mesenchymal stem cells from P2 to P3 reaches 85 to 90 percent, ADMSC is subjected to intermittent anoxic drying, and the supernatant is collected after incubation for 24 hours.
The final concentration of collagenase is 0.1 w/v% -0.2 w/v%, the digestion time is 100 minutes-120 minutes, the digestion temperature is 37 ℃ plus or minus 1 ℃, and the oscillation with the speed of 200 rpm-220 rpm is also matched.
And replacing the culture medium every 3 days in the mesenchymal stem cell culture process until the cell healing degree reaches 85-90%, and collecting the culture medium.
The antibodies used for flow surface antigen identification were the following murine anti-human monoclonal antibodies: CD29, CD45 and CD90 are used as positive indexes, CD34, CD105 and CD106 are used as negative markers, and the same species and labeled non-specific antibody are used as isotype control.
Example 1 isolation preparation, intermittent hypoxia treatment and identification of adipose-derived mesenchymal stem cells
Taking SD rats of 4-6 weeks old; sacrifice (anesthesia sacrifice) without clipping; soaking in 75% alcohol for 10mins is not suitable for long time to avoid affecting cell activity.
preparation of digestive juice (DMEM-F12 complete medium with double antibody and 10% FBS, collagenase): collagenase (tested, collagenase I, II and collagenase IV can be used in the step) is added into an F-12 complete culture medium according to the proportion of 0.075-0.1%, generally, the number of rats in one experiment is 1 and only 5ml of digestive juice, and so on, after being uniformly mixed, the mixture is filtered by a 0.22um filter screen and is reserved after being filtered.
Taking the rat out of the alcohol solution in a fume hood, placing the rat in a sterile culture dish in a prone position, cutting back skin from a position 1cm above the tail of the rat along a back coronary axis to be horizontal to a chest cavity, clamping two sides of skin by using two 16cm bent forceps clamps, tearing the back skin of the rat towards two sides, tearing the back skin to the root of the lower limb of the rat to expose the subcutaneous tissue of the groin, showing that the groin at two sides of the rat is close to the back side and has a white cellulite about 1cm square, and carefully peeling the back skin by using ophthalmic forceps without puncturing the peritoneum to avoid pollution; placing the collected fat mass in a sterile culture dish, dripping a few drops of complete F12 culture medium on the fat mass to prevent the fat tissue from drying, shearing the fat tissue into a superfine minced meat sample by an ophthalmic scissors or an ultrasonic tissue disruptor, placing the sheared fat tissue in the previously prepared collagenase culture medium, shaking the collagenase culture medium on a shaker at 37 ℃ for 90 minutes at a rotating speed of 180-200 ℃, and then showing that the whole collagenase culture medium is milky white.
After the solution is centrifuged at 37 ℃ for 5mins at 1000rpm by a centrifuge, three layers of layers can be seen, namely a cotton-like fat layer, a culture medium layer, free cells (including adipose-derived stem cells) and a larger tissue block from top to bottom. Carefully sucking out the cotton-like fat layer by using a Pasteur tube (the first layer can only be sucked out and can not be poured out, otherwise, the cotton-like fat layer can be adhered to the tube wall), pouring out the second layer of culture medium after the first layer of fat is sucked out, then completely suspending the third layer of cell layer by using F12 culture medium, and transferring the third layer of cell layer into a culture dish (namely, spreading cells according to two 10cm dishes of a rat); the cells are cultured by using the F12 complete culture medium for 3-5 days, and the cells are not required to move within 3 days (for example, when the cells are taken out and seen from a microscope, the wall adhesion of adipose-derived mesenchymal stem cells (ADMSC) can be influenced by the fluctuation of liquid during movement, the wall adhesion condition of the cells can be observed after 3 days, if the cells are found to be adhered to the wall, the liquid can be changed for the first time, if the cells are found to be less adhered to the wall, the cells can be continuously cultured for 2 days, the liquid can be changed after 5 days after extraction, the cells can generally and rapidly grow after the liquid is changed for the first time, and the adipose-derived stem cells with the purity of more than 95 percent can be obtained by the general P2.
after the supernatant of ADMSC is removed, a proper amount of sugar-free and serum-free DMEM medium is added. The cells were then placed in an anoxic reoxygenation chamber and the lid was fastened. Connecting two air inlets of the oxygen-poor reoxygenation small chamber to nitrogen and carbon dioxide air outlets respectively, introducing the nitrogen and carbon dioxide gas (the ratio is 95: 5) for several minutes at the same time according to the reading instruction displayed by a flowmeter until the oxygen is completely discharged from the air outlet, closing the nitrogen and carbon dioxide air outlets after the oxygen concentration of the air outlet is measured to be zero by adopting an oxygen electrode, and clamping all pipelines of the oxygen-poor reoxygenation small chamber to keep a closed state. Then placing the hypoxia reoxygenation chamber into a 37 ℃ incubator to continue the hypoxia culture for 1 hour, then opening the hypoxia reoxygenation chamber, taking out the cell plate and placing the cell plate into 5% CO at 37 DEG C2And (3) continuing reoxygenation culture for 1 hour in the incubator, repeating the steps for 3 times, replacing the sugar-free and serum-free culture medium with a complete culture solution of F12-DMEM + 10% FBS (without exosome serum), namely finishing intermittent hypoxia stimulation on ADMSC, collecting supernatant after 24 hours, and extracting exosomes.
When the growth of the primary adipose tissue-derived mesenchymal stem cells is close to 80 percent of the growth of the bottom of the bottle, sucking the supernatant,Adding 0.125% trypsin, digesting at 37 deg.C, observing cell morphology, removing pancreatin when cells are spherical and are not adhered, adding fresh culture solution to resuspend cells, centrifuging at 4 deg.C and 3000rpm for 3min, removing supernatant, adding culture solution to resuspend cells, repeatedly blowing, mixing, inoculating into new culture flask at a ratio of 1: 2, placing in 5% CO2And continuing culturing at 37 ℃ in an incubator with the saturation humidity of 95 percent, changing the liquid every day until the cells are fused into sheets by adherence, repeating the operation when the cell is close to 80 percent of the bottom of the bottle, and subculturing again.
The 3 rd generation cells were digested with 0.125% trypsin, centrifuged, washed with PBS to harvest the cells, counted and adjusted to a cell density of 5X 106Taking 100 mul of cell suspension to respectively react with CD29, CD34, CD45, CD90, CD105 and CD106 monoclonal antibodies at room temperature for 30min in a light-blocking way, washing the cell suspension for 2 times by using a washing solution, centrifuging the cell suspension, and sending the cell suspension to a flow cytometer to detect surface markers (see figure 1), wherein the result is visible, and positive controls are CD29, CD45 and CD 90; and negative controls CD34, CD105, CD106, wherein the positive proportion of CD29 is 98.6%; the positive rate of CD45 is 98.7; the positive rate of CD90 was 97.7%. In the negative control, the positive proportion of CD34 is 99.2%; the CD105 positive ratio is 99.7; the CD106 positive rate is 87.2%.
therefore, as shown by the flow results, the proportion of the positive control to the negative control is higher than 70%, which indicates that the purity and phenotype of the ADMSC extracted in the research are both in accordance with the requirements of the experiment, and exosomes can be extracted from the ADMSC for further research.
Example 2 extraction and identification of adipose-derived mesenchymal stem cell exosomes
Extraction of exosomes: cell supernatants were extracted and transferred to a new tube for centrifugation (200g, 20min, 4 ℃); carefully re-move the supernatant to a new tube and centrifuge (10,000g, 30min, 4 ℃) to remove larger vesicles; samples at this stage can be filtered through a 0.22 μm syringe sieve (Millipore) and centrifuged at 110,000g (Sorvall WXULTRA SERIES, rotor F65L) for 2 hours and 4 deg.C, resuspended in cold PBS and then ultracentrifuged again (110,000g, 1 hour, 4 deg.C) to carefully dry and resuspend the exosomes in cold PBS, which should be used immediately or refrigerated at-80 deg.C.
electron microscopy identification of exosomes: mixing the exosome solution and 4% paraformaldehyde according to a ratio of 1: 1 (the total volume is enough to be 10-20 mu l), dripping the exosome solution on a clean plastic film to form liquid drops, then buckling the front surface of an electron microscope carbon mesh on the liquid drops, and standing for 20 min; the carbon net was placed on 100ul PBS drop and washed twice, 3min each time; the carbon net was placed on 100. mu.l of 50mM glycine drop for 3min, repeated 3 times; and (3) sealing: placing the carbon net on 100 μ l of 5% BSA blocking solution, and blocking for 10 min; diluting the primary antibody with a primary antibody diluent, and incubating the carbon net on 20 μ l of the primary antibody drop for 30 min; placing the carbon net on 100 μ l wash buffer drop, washing for 3min for 6 times; diluting secondary antibody (anti-mouse, rabbit, etc. corresponding to the primary antibody) labeled with colloidal gold at a ratio of 1: 20, and placing carbon net thereon for 30 min; placing the carbon net in 100 μ l drop of 0.5BSA, washing for 3min for 6 times; the carbon net was placed on 100. mu.l PBS drop for 2min and washed 6 times; placing carbon net on 100 μ l of 1% glutaraldehyde solution drop, and standing for 2 min; placing the carbon net on 100 μ l deionized water drop, washing for 6 times for 2 min; 10 mul uranyl acetate was negative-stained for 90s, baked on carbon net, and tested on machine (see FIG. 2).
Flow detection of exosome markers: this example uses a BD accuri C6 flow cytometer instrument to analyze the expression of two specific antibodies, microparticles CD63 and CD81, in a sample of ADMSC-derived exosome solution. The exosome solution is concentrated as much as possible through an ultrafiltration tube, each exosome detected in a flow-type manner needs to contain 500ng of protein, 100ul of solution is taken each time, and an isotype control sample is prepared; 5ng of CD63-APC and CD81-APC flow antibody was added to the sample tube; adding 5ng of corresponding isotype control antibody into a control tube, keeping out of the sun, continuously mixing uniformly at 4 ℃, incubating for 1 hour, and then detecting on a machine.
as shown in Table 1, the exosomes obtained after staining are positive in the expression conditions of two specific antibodies of CD63 and CD81, and the positive rate is 76.8% -81.7%. Among them, CD63 and CD81 were expressed in the solution microparticles at a higher ratio, and therefore exosome was considered to be successfully extracted (see table 1 for details).
TABLE 1 exosome flow identification of the proportion of surface markers CD63, CD81 positive microparticles
and additionally, ADMSC, and exosome surface marker TSG101 and CD9 molecules derived from the ADMSC, and an exosome negative control calcixin are identified by a western blot method. As shown in FIG. 3, TSG101 and CD9 are expressed in the exosome group, and Calcexin as a negative control of exosome is negative, so western blot results also indicate that exosome is successfully extracted.
particle size detection of exosome NTA: the exosome is dissolved in 500 mu L of exosome, fully blown and uniformly mixed, 300 mu L of exosome is taken out and put into a machine, and a NanoSigt LM10-HS nanoparticle analyzer is adopted to analyze the particle size and the concentration. 450 μ l of PBE resuspended sample was added and the machine tested. As shown in FIG. 4, the particle diameter distribution curve of the exosome solution is a unimodal curve, which indicates that the particles in the exosome solution are uniformly distributed and have less impurities, and the particles with 121.7nm diameter account for 98.2% of the total number of the total particles, and the inner diameter is within 30nm to 15nm, which indicates the exosome extraction result in the present study, and the impurities in the exosome solution are less, which meets the experimental conditions.
EXAMPLE 3 study of INTEXO and EXO myocardial cell protective Effect in vitro
The experiment was divided into four groups, which were: normal cell group (NC group), hypoxia/reoxygenation group (IR), hypoxia/reoxygenation + exosome group (IR + EXO) and hypoxia/reoxygenation + exosome group derived from ADMSC with intermittent hypoxic pretreatment (IR + intaxo)
Expression changes of proteins related to focal death and apoptosis pathway: adding 30 mu g of ADMSC and exosome secreted by the ADMSC pretreated by the invention into a corresponding rat myocardial cell culture medium which is subjected to anoxic treatment in a 6-well plate, incubating for 12 hours, and collecting cells; preparing a protein lysate: taking a clean centrifuge tube, adding 10ml of RIPA strong lysis solution, adding 1 protease inhibitor and 1 phosphatase inhibitor, fully dissolving and mixing uniformly, subpackaging into a plurality of EP tubes, and storing in a refrigerator at-20 ℃ for later use.
cell lysis: the cell culture plate was taken out of the 37 ℃ incubator, the cell supernatant in the 6-well plate was discarded, 1ml of 1 XPBS was added to each well, the cultured cells were washed, then the washing solution was discarded, and the washing was repeated twice. After the last washing, PBS must be removed as clean as possible without residue. Then, 200. mu.l of RIPA strong lysate was added to each cell well, the 6-well plate was placed on ice for 5 minutes, the cell lysate was scraped off with a cell scraper, and all the liquid was transferred to a new EP tube and placed on ice for continued lysis for 20 minutes, during which time the cells were lysed thoroughly with shaking once every 10 mins. Then centrifuging at 12000g at 4 ℃ for 10 minutes, extracting supernatant to another EP tube, adding 5 xSDS loading buffer, and boiling at 95 ℃ for 5 minutes; protein samples can be used for WesternBlot detection or stored at-80 ℃; western Blot identifies myocardial cell apoptosis and apoptosis pathway related proteins.
Western Blot detection of molecular signals related to the apoptosis pathway was performed on each group of exosomes derived from ADMSC after hypoxia/reoxygenation of cardiomyocytes. The results are shown in FIG. 5A, and the myocardial cells TXNIP, NLRP3, cleared caspase-1 and IL-1. beta. in the IR group are all significantly increased compared with those in the CON group after hypoxia for 1 hour and reoxygenation for 2 hours. In the IR + EXO and IR + INTEXO groups, TXNIP, cleared caspase-1 and IL-1 beta levels were significantly reduced compared to the IR group, and the IR + INTEXO group was more significantly reduced. In addition, the NLRP3 levels in both groups with exosomes added were downward trended compared to the IR group, but were not statistically different. ASC levels were not significantly different in the four groups.
Western Blot detection of apoptosis pathway-related molecular signals was performed on groups of exosomes derived from ADMSC after hypoxia/reoxygenation of cardiomyocytes. As shown in FIG. 5B, the BAX and cleared caspase-3 expression levels in IR cardiomyocytes were significantly increased compared to CON cells at 1 hr of hypoxia and 2 hr of reoxygenation, while the GATA4 and Bcl-2 levels were significantly decreased. In the IR + EXO and IR + INTEXO groups, the BAX and cleared caspase-3 expression levels are obviously reduced compared with the IR group, and the IR + INTEXO group has more obvious reduction trend; the GATA4 and Bcl-2 levels were significantly increased, and the IR + INTEXO group was more strongly elevated.
annexinV-PI detection of myocardial apoptosis levels: digesting four groups of cells with pancreatin, counting the cells, and taking about 1 × 106cells/ml, 1000rpm, 5 minutes at room temperature, discard the supernatant; 1ml of cold PBS was addedWashing the cells, and slightly shaking to suspend the cells; centrifuging at 1000rpm for 5min at room temperature, and discarding the supernatant; repeating twice; resuspend cells in 200. mu.l Bindingbuffer; adding 5ul annexin V-FITC and mixing gently; reacting for 15 minutes at room temperature in dark place; mu.l BindingBuffer (total reaction volume 500. mu.l) and 5. mu.l PI were added and tested on the machine over 1 hour. As shown in fig. 5C, the early apoptosis rate of cardiomyocytes after undergoing hypoxia-reoxygenation was 30.133% ± 2.579%, which was significantly increased compared to the control group; after the H9C2 cells added with the EXO are subjected to hypoxia reoxygenation, the apoptosis ratio is reduced to 21.067% +/-2.838%, and the apoptosis ratio is obviously reduced. The apoptosis of cardiac muscle cells added with INTEXO in the process of hypoxia reoxygenation is more obviously reduced to 15.700 +/-1.539%. This result demonstrates that the induced exosomes of this example exert their protective effect on myocardial ischemia-reperfusion injury.
Example 4 study of the protective Effect of INTEXO and EXO on myocardial cells in vivo
The mouse myocardial ischemia reperfusion injury model is used for predicting the expression change of the scorching and apoptosis pathway related proteins after exosome intervention: randomly selecting mice in IR group, IR + EXO group and IR + INTEXO group, taking 3 mice for each mouse, establishing a model of myocardial ischemia reperfusion injury in vivo, after chest opening and heart exposure, ligating the left anterior descending branch of coronary artery to cause myocardial ischemia for 30min, and then extracting nodes to perform reperfusion for 2 hours. After myocardial ischemia reperfusion time is over, anesthetizing the mouse again, fixing, opening the chest, exposing the heart, picking off the whole heart by using forceps and scissors, carefully cutting off myocardial tissues in a white infarct area below a left ventricular ligature, rinsing the picked myocardial tissues by using PBS (phosphate buffer solution), placing the rinsed myocardial tissues in a freezing storage tube, rapidly cooling by using liquid nitrogen, immediately extracting proteins of the myocardial tissues, or placing the rinsed myocardial tissues in a refrigerator at the temperature of minus 80 ℃ for storage for later use. The results show that Western Blot detection of molecular signals related to the apoptosis pathway is carried out on groups of mouse exosomes from ADMSC after myocardial ischemia reperfusion operations and after tail vein injection. The results showed that the myocardial cells TXNIP, NLRP3, cleared caspase-1 and IL-1. beta. in the IR group were significantly elevated compared to the CON group after 30min of ischemia and 2 h of reperfusion. In the IR + EXO and IR + INTEXO groups, TXNIP, cleared caspase-1 and IL-1 beta levels were significantly reduced compared to the IR group, and the IR + INTEXO group was more significantly reduced. In addition, in both groups with exosomes added, NLRP3 levels were significantly reduced in the IR + integexo group compared to the IR group, whereas NLRP3 was reduced in the IR + EXO group compared to the IR group, but with no statistical difference. ASC levels were not significantly different in the four groups (see fig. 6A and 6B).
Evans Blue/TTC staining was used to detect changes in the area of ischemic and infarcted myocardium in mice. The experiments were divided into 3 groups: ischemia 30 min/reperfusion 2 hr group (IR group), ischemia 30 min/reperfusion 2 hr group + ADSC exosome 200 μ g group (IR + EXO), ischemia 30 min/reperfusion 2 hr group + exosome 200 μ g secreted by intermittent hypoxia pretreated ADSC group (IR + intaxo) group, male, age-matched, 5 each. An in vivo myocardial ischemia-reperfusion injury model was established using WT mice. After 30 minutes of ischemia and 4 hours of reperfusion, the difference of the areas of the ischemic myocardial and myocardial infarction of the two groups of mice is observed and compared by an Evans Blue/TTC staining method. As shown in FIG. 6C, the ratio of the area of the risk zone (ischemic area + infarcted area) in the IR group to the total myocardial area was 60.129% + -4.509%, that in the IR + EXO group was 60.206% + -2.517%, and that in the IR + INTEXO group was 58.732% + -5.686%, the areas of the risk zones were comparable, all around 60% (P > 0.05), indicating that the models made by the three groups were comparable. Comparing the infarct size to the ratio of the area at risk, it was found that 56.000% + -5.292% of the infarct size in the IR group was significantly greater (P < 0.05) than 45.667% + -4.041% in the IR + EXO group and 31.667% + -4.059% in the IR + INTEXO group, and the infarct size in the IR + INTEXO group was smaller than that in the IR + EXO group, which was statistically significant.
Detecting the change of the heart function of the mouse by the heart color ultrasound: the experimental groups are as above, and the mice establish a model of ischemia-reperfusion injury of heart muscle in vivo. Cardiac color ultrasound was applied 3 days after reperfusion surgery and 7 days after reperfusion surgery, respectively, to assess cardiac functional differences between the two groups of mice: namely, the Acuson Sequoia 512 small animal ultrasonic imaging system (siemens, germany) and 707B (15MHz) ultrasonic probe were used to detect changes in left ventricular Ejection Fraction (EF) and left ventricular short axis contraction Fraction (FS) of two groups of mice. The results are shown in fig. 6D, the left ventricular ejection fraction EF and the minor axis shrinkage FS of the three groups of mice after 3 days of reperfusion were higher in the two groups injected with exosomes than in the myocardial ischemia reperfusion injury model group, while EF and FS of IR + intaxo group were both higher than in the IR + EXO group, and the difference was statistically significant; EF and FS of three groups of mice are increased in different degrees on the 7 th day after operation, while EF and FS of IR + EXO and IR + INTEXO groups are higher than those of the IR group, and the difference has statistical significance. In two groups injected with exosomes after 7 days post-surgery, EF and FS were elevated in the IR + intaxo group compared to the IR + EXO group, but were not statistically different.
TUNEL assay of mouse in situ cardiomyocyte apoptosis changes: fluorescein-labeled dUTP can be attached to the 3' -OH end of fragmented DNA in apoptotic cells under the action of deoxyribonucleotide terminal transferase and specifically bind to HRP-labeled fluorescein antibody, which in turn reacts with HRP substrate to produce a dark brown color. As shown in a of fig. 6E, four groups of mice were subjected to in situ cardiomyocyte apoptosis assay using Tunel method after 30min reperfusion after myocardial ischemia for 3 hours, comparing the ratio of apoptotic cardiomyocytes in each group to total cells. As shown in B in FIG. 6E, it was found that the in situ ratio of cardiomyocyte apoptosis in the IR group was 13.533% + -0.837%, while the IR + EXO and IR + INTEXO groups were 8.267% + -1.888% and 4.933% + -1.201%, respectively, which were significantly reduced compared with the IR group, and the difference was statistically significant. The apoptosis ratio of the IR + INTEXO group is lower than that of the IR + EXO group, and the difference has statistical significance.
Example 5 study of mesenchymal stem cell-derived exosome-associated molecular mechanism with intermittent hypoxia intervention
Screening of INTEXO and EXO inter-differential mirnas: extracting the total RNA of the exosome INTEXO generated by the invention and the exosome EXO secreted by ADMSC under the normal culture state, and performing quantitative and quality control → cRNA synthesis, construction marking of a sequencing library → anchoring bridging → pre-amplification → single base extension sequencing → differential screening → unsupervised hierarchical clustering; the Go and KEGG analysis steps were performed for sequencing analysis, and the results are shown in fig. 7. Then, according to the sequencing result, miR-224-5p with the highest expression fold difference is selected as a research object in the embodiment, firstly, a MicroRNA224-5p plasmid and TXNIP gene over-expression wild type and mutant type plasmids containing luciferase genes are constructed, then, co-transfection is carried out on a tool cell 293T, and the dual-luciferase reporter gene verifies whether the two plasmids are combined and whether the MicroRNA224-5p can inhibit the expression of the TXNIP. The invention adopts the ratio multiple of the fluorescence of the firefly to the fluorescence of the internal reference Renilla to compare the fluorescence intensity of each group. According to the research results shown in FIG. 8, it can be seen that the firefly fluorescence/sea cucumber fluorescence 4.463 + -0.324 of the group co-transfected by MicroRNA224-5p and Med1-3 'UTR-WT is significantly reduced compared with 10.688 + -1.650 of the group co-transfected by MicroRNA224-5p + Med 1-3' UTR-MUT, 10.726 + -0.376 of the group co-transfected by MicroRNA224-5p + Med1-3 'UTR-NC, 8.704 + -1.518 of the group co-transfected by MicroRNA224-5p + Med 1-3' UTR-NC, 11.677 + -23 of the group co-transfected by MicroRNA224-5 p-NC +3 'UTR-NC, and 9.809 + -1.388 of the group co-transfected by MiR-224-5p-NC + 3' UTR-MUT, and the difference has statistical significance; with no significant change between the other five groups. This result indicates that MicroRNA224-5p binds to TXNIP-3 'UTR-WT and the target mRNA fragment is either untranslated or degraded, so luciferase production in the TXNIP-3' UTR-WT plasmid is reduced and fluorescence is excited by the absence of luciferase degradation even with the addition of luciferin. And when the TXNIP-3' UTR is mutated, the mutant can not be combined with MicroRNA224-5p, and the luciferase expression is not influenced, so that the fluorescence intensity is not obviously changed.
Claims (10)
1. An exosome derived from mesenchymal stem cells is characterized in that the exosome is prepared by carrying out intermittent hypoxia stimulation on the mesenchymal stem cells.
2. the mesenchymal stem cell-derived exosome according to claim 1, which mainly comprises microRNA224-5p, or comprises one or a combination of microRNA224-5p and microRNA141-3p, microRNA 34a-5p, microRNA 23a-3p, microRNA 130a-3p, microRNA 23b-3p, microRNA 200a-3p, microRNA93-5p, microRNA 301a-3p, and microRNA 152-3 p.
3. Mesenchymal stem cell-derived exosome according to claim 1 or 2, characterised by having a stronger tissue organ ischemia-reperfusion injury protective effect.
4. Use of a mesenchymal stem cell-derived exosome according to claim 1 or 2 in the preparation of a medicament or device for ischemia-reperfusion injury repair in a tissue organ.
5. A composition for repairing ischemia-reperfusion injury of a tissue or organ, comprising the mesenchymal stem cell-derived exosome according to claim 1 or 2 as an active ingredient.
6. A medical device for repairing ischemia-reperfusion injury of a tissue or organ, comprising the mesenchymal stem cell-derived exosome according to claim 1 or 2 as an active ingredient.
7. a method for preparing the exosome derived from the mesenchymal stem cell of 1 or 2 is characterized by comprising the following steps:
When the confluence rate of the mesenchymal stem cells of the generations P2-P3 reaches 85% -90%, carrying out intermittent hypoxia stimulation on the cells in the container through the hypoxia chamber, namely after 3 cycles of hypoxia → reoxygenation → hypoxia → reoxygenation, replacing a cell culture medium with a DMEM-F12 complete culture medium containing 5v/v fetal calf serum without exosomes, incubating for 24 hours in an incubator, collecting the culture medium, obtaining cell culture supernatant, carrying out membrane filtration, and collecting filtrate; and then placing the filtrate at the rotating speed of 300 g-100000 g for differential centrifugation to obtain a precipitate, namely the exosome.
8. The method according to claim 7, wherein the hypoxia is performed by placing the mesenchymal stem cells in a hypoxia chamber, replacing the cell culture medium with a sugar-free and serum-free DMEM medium, introducing a mixed gas of 80 v/v% to 95 v/v% nitrogen and 0 v/v% to 5 v/v% carbon dioxide into the chamber, detecting the oxygen concentration of the gas in the hypoxia chamber by an oxygen battery until the oxygen concentration is 0 v/v%, and closing a vent valve of the chamber to isolate the sugar content of the cells and supplying the oxygen for 45 to 60 minutes.
9. The method according to claim 7, wherein the reoxygenation is carried out by opening the anoxic chamber valve and replacing the medium with DMEM-F12 medium with sugar and then transferring the medium into an incubator with 5 v/v% carbon dioxide at 37 ℃ for 1 hour.
10. The method of claim 7, wherein said differential centrifugation is as follows:
Firstly, centrifuging at the rotating speed of 300g at the temperature of 4 ℃ for 10 minutes, and taking supernatant; then centrifuging at 4 ℃ for 10 minutes at the rotating speed of 2,000g, and taking supernatant; centrifuging at 4 deg.C and 10,000g speed for 60 min, and collecting supernatant; centrifuging at 100,000g rotation speed at 4 deg.C for 70 min, discarding supernatant, and resuspending the precipitate with PBS; finally, the exosome is obtained after centrifugation for 70 minutes at the rotating speed of 100,000g at the temperature of 4 ℃.
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