CN111944748A - high-IL-10-expression human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction and application thereof - Google Patents
high-IL-10-expression human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction and application thereof Download PDFInfo
- Publication number
- CN111944748A CN111944748A CN202010820947.2A CN202010820947A CN111944748A CN 111944748 A CN111944748 A CN 111944748A CN 202010820947 A CN202010820947 A CN 202010820947A CN 111944748 A CN111944748 A CN 111944748A
- Authority
- CN
- China
- Prior art keywords
- mesenchymal stem
- human adipose
- derived mesenchymal
- stem cell
- exosome
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/5428—IL-10
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Abstract
The invention provides a human adipose-derived mesenchymal stem cell exosome with high IL-10 expression for treating myocardial infarction and application thereof, relating to the technical field of biology. The invention provides a high IL-10 expression human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction, which is an exosome secreted by a human adipose-derived mesenchymal stem cell overexpressing IL-10. The invention has the advantages that the human adipose-derived mesenchymal stem cell exosome for over-expressing IL-10 is prepared by culturing the human adipose-derived mesenchymal stem cell for over-expressing IL-10, contains a large amount of IL-10 and other various RNAs and cytokines, effectively repairs infarcted cardiac muscle cells, enhances cardiac function, reduces compensatory myocardial load, relieves heart failure, and provides a treatment medicament or a treatment strategy for myocardial infarction.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an over-expression IL-10 human adipose-derived mesenchymal stem cell exosome and application thereof.
Background
Coronary atherosclerotic heart disease is caused by abnormal lipid metabolism, and lipid in blood deposits on the originally smooth intima of artery and gradually accumulates to form white plaque, which causes stenosis or obstruction of blood vessel lumen, and can seriously cause coronary artery occlusion and blood flow interruption, so that partial cardiac muscle is locally necrotized due to serious persistent ischemia, and myocardial infarction is caused. After myocardial infarction occurs, partial myocardial cells are difficult to repair due to ischemia and hypoxia, so that the cardiac function is reduced, the compensatory myocardial load is increased, and serious complications such as heart failure and the like can be caused over time. Coronary artery occlusion occurs for 20-30 minutes during coronary heart disease, and this myocardial damage is usually reversible, but the amount of necrotic myocardium increases over time. If the coronary occlusion time is prolonged to 2-6 hours, wavy fibers are formed at the edge of the affected myocardium, and beyond 6 hours to 3 days after infarction, the myocardium undergoes coagulative necrosis, interstitial congestion, edema and neutrophil infiltration. If a sufficient amount of the myocardium is affected, ventricular diastolic and systolic dysfunction may be induced, resulting in a series of hemodynamic changes.
Stem cells are a type of pluripotent cells that have the ability to self-replicate. Due to the high lethality of myocardial infarction and the strong differentiation capacity of stem cells, stem cell-based cell therapy has become a hotspot for myocardial infarction research. Although the treatment of myocardial infarction using stem cells is theoretically safe, it is still to be studied whether the number and retention rate of cells distributed in the damaged area after cell transplantation are sufficient to treat or protect myocardial infarction cells. It is known that cells undergo apoptosis rapidly after transplantation, and it is unclear whether or not an active substance produced during apoptosis is tumorigenic. Therefore, the intensive research on the treatment or protection effect of the stem cells on myocardial infarction and the search of new targets and methods for treating myocardial infarction by the stem cells are of great significance.
The adipose-derived mesenchymal stem cell is one of the stem cells with obvious advantages, and has some special advantages compared with other stem cells: such as convenient material acquisition, wide source, stronger proliferation and differentiation capability, low tumor formation rate and no disputes in the aspects of society, morality and ethics and the like. Multiple researches show that the adipose-derived mesenchymal stem cell secretion contains a large amount of cell factors, proteins and the like, can be used for treating various difficult and complicated diseases, and is a novel biomedical material which has wide sources, and is simple and convenient to obtain materials. However, at present, adipose-derived mesenchymal stem cells also have some common problems of common stem cells, for example, although adipose-derived mesenchymal stem cells contain more bioactive factors, the content of the bioactive factors is low, the properties of the bioactive factors are unstable, and the retention rate of the bioactive factors in a lesion area is low, so that the problems greatly limit the wide clinical application of adipose-derived mesenchymal stem cells.
The exosome is a membrane vesicle released into extracellular matrix after an intracellular multivesicular body is fused with a cell membrane, and comprises a plurality of bioactive substances such as DNA fragments, mRNA, functional proteins, transcription factors and the like, the membrane structure of the exosome can also express a plurality of antigens and antibody molecules, and the bioactive substances enter target cells and can participate in regulating a plurality of physiological and biochemical processes in the target cells to further generate various biological effects. The discovery of the exosome provides a thought for the further application of the stem cells and the adipose tissue-derived mesenchymal stem cells in myocardial infarction. Although research shows that the exosome derived from the stem cells has a remarkable effect on myocardial infarction, the exosome can inhibit apoptosis of cardiac cells, promote angiogenesis and improve the function of the myocardial cells after myocardial infarction. However, because the bioactive substances in the exosomes expressed by stem cells from different sources are greatly different, whether the exosomes from adipose mesenchymal stem cells express the active substances capable of treating myocardial infarction needs more research to prove.
Interleukin-10 (IL-10) is a multi-cellular, multifunctional cytokine that is involved in mediating cell growth and differentiation, in inflammatory and immune responses, and is a currently recognized validating and immunosuppressive factor. Research shows that IL-10 can promote the expression of IL-1 receptor antagonist, is favorable to resisting atherosclerosis and may play certain role in treating coronary heart disease. Exogenous IL-10 supplementation can be used as a new way for myocardial infarction caused by coronary atherosclerotic heart disease. However, the retention rate of exogenous IL-10 in the damaged area and the rejection of the body can influence the function of the damaged area, and further research on how to make IL-10 play a role stably in the damaged area of myocardial infarction is needed.
According to the invention, the SD rat is constructed to verify the protective effect of the exosome derived from the adipose-derived mesenchymal stem cell over-expressing IL-10 on the myocardial cell, and the adipose-derived mesenchymal stem cell line is constructed by using the SD rat, so that technical support is provided for the exosome derived from the adipose-derived mesenchymal stem cell over-expressing IL-10 to treat myocardial infarction.
Disclosure of Invention
The invention aims to provide a human adipose-derived mesenchymal stem cell exosome with high IL-10 expression for treating myocardial infarction and application thereof.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
In one aspect, the embodiment provides a high IL-10 expression human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction, wherein the exosome is secreted by a human adipose-derived mesenchymal stem cell overexpressing IL-10.
In some embodiments of the present invention, the above-mentioned human adipose-derived mesenchymal stem cell exosome for expressing IL-10 is prepared by the following steps: culturing human adipose mesenchymal stem cells over expressing IL-10, and collecting cell culture solution; and (3) centrifuging the cell culture solution at a differential speed, collecting supernatant, ultracentrifuging the supernatant to obtain target microspheres, and filtering, purifying and sterilizing the target microspheres to obtain the exosome.
In some embodiments of the present invention, the above-mentioned human adipose-derived mesenchymal stem cell exosome with high expression of IL-10 is prepared by the following steps: preparing an IL-10 overexpression vector, transfecting the IL-10 overexpression vector into the human adipose mesenchymal stem cells, and screening and identifying to obtain the human adipose mesenchymal stem cells overexpressing IL-10.
In some embodiments of the present invention, the above-mentioned exosomes for high-expression IL-10 human adipose-derived mesenchymal stem cells, the means for transfecting the IL-10 overexpression vector into the human adipose-derived mesenchymal stem cells include adenovirus, lentivirus, Lip2000 and Lip 3000.
In some embodiments of the invention, the identification and screening of the human adipose-derived mesenchymal stem cell exosome with high IL-10 expression comprises adding 700 μ G/mL of G418 into the human adipose-derived mesenchymal stem cell, and culturing for 10-14 days.
In some embodiments of the invention, the human adipose-derived mesenchymal stem cell exosome for highly expressing IL-10 is prepared by digesting human adipose tissues, inoculating and subculturing, wherein the enzymes for digesting the human adipose tissues comprise 0.06-0.16% by volume of collagenase type IV and 0.015-0.02% by mass of BSA.
In some embodiments of the invention, the human adipose-derived mesenchymal stem cell exosome for high expression of IL-10 is obtained from the generation P2-P8.
In some embodiments of the invention, the enzyme for digesting the human adipose tissue is collagenase type IV 0.1% by volume and BSA 0.02% by mass, and the digestion time is 30-35 min.
In some embodiments of the present invention, the culture solution for culturing the human adipose-derived mesenchymal stem cell exosome with high expression of IL-10 is 10% by volume of fetal bovine serum culture medium without exosome; the inoculation density is 2-6 multiplied by 104/cm2(ii) a The differential centrifugation comprises centrifugation at 1500g for 10min and then at 10000g for 10 min.
In another aspect, the embodiment of the application provides a use of the human adipose-derived mesenchymal stem cell exosome with high IL-10 expression in the preparation of a medicament for treating myocardial infarction.
Compared with the prior art, the embodiment of the invention has at least the following advantages or beneficial effects:
the human adipose-derived mesenchymal stem cell exosome with high IL-10 expression provided by the invention is prepared by separating and culturing human adipose-derived mesenchymal stem cells by adopting a specific method, stably overexpressing IL-10 to the human adipose-derived mesenchymal stem cells and collecting exosomes in a specific mode, the whole process is simple and convenient, the obtained exosome has stable effect, and meanwhile, the repair capacity of the prepared exosome is greatly enhanced compared with that of exosomes without overexpressing IL-10 due to the overexpression of IL-10. The infarcted myocardial cells are difficult to repair, so that the heart function is reduced, the compensatory myocardial load is increased, and serious complications such as heart failure and the like are finally caused.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows the result of screening the concentration of an enzymatic hydrolysate I in example 1 of the present invention;
FIG. 2 shows the results II of screening different enzymatic hydrolysates in example 1 of the present invention;
FIG. 3 shows the results of identifying primary cells in example 1 of the present invention;
FIG. 4 is the relative expression of exosomes under different extraction conditions in examples 2 and 5 of the present invention;
FIG. 5 shows the identification results of exosomes of human adipose-derived mesenchymal stem cells from different sources in examples 2 and 5 of the present invention;
FIG. 6 shows the results of the identification of passaged cells in example 3 of the present invention;
FIG. 7 shows the expression of IL-10 in human adipose-derived mesenchymal stem cells stably overexpressing IL-10 in example 4 of the present invention;
FIG. 8 is a graph showing the results of example 7 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to specific examples.
In one aspect, the embodiment of the present application provides a high IL-10 expression human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction, wherein the exosome is secreted by a human adipose-derived mesenchymal stem cell over-expressing IL-10.
In another aspect, the embodiment of the application provides an application of the human adipose-derived mesenchymal stem cell exosome with high expression of IL-10, namely the human adipose-derived mesenchymal stem cell exosome with high expression of IL-10 is used for preparing a medicament for treating myocardial infarction.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The present example aims to isolate and culture human adipose-derived mesenchymal stem cells.
1. Material
Preparing human fat (discarded normal human fat after operation, signed with informed consent), PBS, BSA, collagenase type IV, fetal bovine serum, CD29+、CD44+、CD90+、CD34-And CD45-The monoclonal antibody of (1).
2. Method of producing a composite material
2.1 isolation of human adipose-derived mesenchymal Stem cells
(1) Collecting about 15mL of adipose tissue under aseptic condition, and removing connective tissue envelopes and blood vessels on the surface of the adipose tissue;
(2) rapidly cutting into paste, adding 3mL of a culture medium without exosomes, wherein the culture medium without exosomes comprises IV-type collagenase with a final concentration of 0.06-0.16% and 0.015-0.02% BSA, carrying out oscillatory digestion at a constant temperature of 120r/min at 37 ℃ for 30-35 min, observing the digestion state of the tissue at any time, taking out and uniformly mixing every 5-8 min, filtering by using 100-mesh and 200-mesh screens in sequence after digestion is ended, removing undigested tissues, and collecting filtrate; different digestion conditions have a greater effect on the activity and subsequent proliferation rate of isolated primary cells, and therefore, in this example, the effect of different digestion conditions on cell activity and cell proliferation rate was examined. Digestion conditions were grouped as follows:
TABLE 1 digestion conditions
| Group of | Collagenase type IV (%) | BSA(％) |
| 1 | 0.04 | - |
| 2 | 0.06 | - |
| 3 | 0.8 | - |
| 4 | 0.1 | - |
| 5 | 0.12 | - |
| 6 | 0.14 | - |
| 7 | 0.1 | 0.015 |
| 8 | 0.1 | 0.02 |
(3) Centrifuging the filtrate obtained in the step (2) at 1500r/min for 15min, and removing the supernatant to obtain a precipitate;
(4) gently blowing and cleaning the sediment with PBS (phosphate buffer solution) at 37 ℃ for 2-3 times, adding a culture medium containing 10% volume fraction of exosome-free fetal calf serum into the sediment, and re-suspending the fetal calf serum by 2-6 multiplied by 104cells/cm2Inoculation at 37 ℃ with 5% CO2And culturing in an incubator with 100% relative humidity, after culturing for 12-14 h, changing the liquid for the first time, wherein the liquid changing action needs to be light, so that cells which are just attached to the wall are prevented from being changed, and then changing 1 time of fresh culture medium every 48h and observing the cell morphology.
2.2 cell proliferation Rate analysis
mu.L of MTT solution (5mg/mL) was added to the cells at a fixed time, and after incubation in an incubator at 37 ℃ for 4h, the supernatant was aspirated, and 150. mu.L of dimethyl sulfoxide was added to each well. Shaking for 10min, and detecting Optical Density (OD) value of each well by enzyme-linked immunoassay cell with 490nm wavelength. Calculating the mean value, taking time as abscissa (optical density at specific time point-optical density at day 0)/optical density at day 0 x 100%, namely the proliferation rate of cells at different time points, and drawing a growth curve by taking the proliferation rate as ordinate.
2.3 detection of cell surface marker proteins
Observing cell state and cell fusion degree under microscope, digesting cells when cell state is good and fusion degree exceeds 95%, stopping digestion by adding culture medium after cell edge begins to separate from culture plate, centrifuging, taking precipitate, re-suspending cells with fresh culture medium to adjust cell concentration, preparing multiple centrifuge tubes, adding into 1 × 10 suspension tubes respectively6Respectively adding adhesion molecule receptor expression marker CD29 into cell suspension of individual cells+And CD44+Stem cell marker CD90+And the hematopoietic cell marker CD34-And CD45-And performing computer detection after incubation.
3. Results
3.1 cellular Activity of h-MSCs obtained under different digestion conditions
As shown in fig. 1, it is seen from fig. 1 that the cellular activity of the 0.1% type IV collagenase group (P <0.05) is significantly higher than that of the 0.12% type IV collagenase, and is significantly higher than that of the other groups (P <0.01), when 0.015 to 0.02% BSA is added for digestion, the cellular activity of the obtained h-MSCs is improved to some extent by 0.015% BSA, but the statistical significant difference is not observed, and the cellular activity of the h-MSCs digested by the 0.1% type IV collagenase is improved significantly by 0.02% BSA (P < 0.05).
3.2 cell proliferation Rate of h-MSCs obtained under different digestion conditions
The results of cell proliferation rate are shown in FIG. 2 (except for groups 4, 7 and 8, other groups are not shown), compared with the 0.1% type IV collagenase group, although the cell activity of the h-MSCs is not statistically improved when 0.015-0.02% BSA is added for digestion, the early proliferation (1-3 d, especially 1-2 d) (P <0.01) of the h-MSCs at the corresponding time point can be remarkably improved by adding 0.02% BSA, the early proliferation (1-3 d, especially 1-2 d) (P <0.05) of the h-MSCs can be remarkably improved by adding 0.015% BSA, and the logarithmic phase of the h-MSCs can be prolonged to some extent by adding a certain amount of BSA.
From this, the collagenase type IV concentration in the digestive enzyme solution is preferably 0.1% collagenase type IV, and the BSA concentration is preferably 0.02%.
3.3 h-MSCs identification results
The cell morphology observed by an electron microscope is that the cell is a fibroblast and is in a vortex shape. Taking cells cultured for 5 days by 0.1% collagenase IV + 0.02% BSA group, and measuring the h-MSCs surface marker protein CD29 by flow+、CD44+、CD90+、CD34-And CD45-The results are shown in FIG. 3, from which it can be seen that the cell highly expresses adhesion molecule receptor expression marker CD29+And CD44+Stem cell marker CD90+Low expression of the hematopoietic marker CD34-And CD45-The results meet the identification criteria of MSCs by the international society for cell therapy. Thus, the cells isolated in this example were h-MSCs.
Example 2
The present embodiment aims to isolate and purify human adipose-derived mesenchymal stem cell exosomes, comprising the following steps:
1. material method
Human fat (normal human fat discarded after surgery, signed with an informed consent), PBS, BSA, collagenase type IV, fetal bovine serum, CD9, CD63, CD81, monoclonal antibodies to α -actin-4 and CD40, and dimethyloxalylglycine were prepared.
2. Collecting human adipose-derived mesenchymal stem cell exosomes
(1) Collecting human adipose-derived mesenchymal stem cells, inoculating, culturing with exosome-free serum culture medium at 37 deg.C and 5% CO2Starting culture under the condition;
(2) after 12h of culture, changing a serum culture medium of the human adipose-free mesenchymal stem cell exosome containing 300 mu mol/L dimethyloxalyl glycine, continuing to culture for 36h, and collecting a cell culture solution;
(3) performing differential centrifugation on the cell culture solution obtained in the step (2) at 4 ℃, namely centrifuging for 10min under the condition of 1500g, and centrifuging for 10min at 10000g to remove cell debris and macromolecular proteins to obtain supernatant;
(4) performing ultracentrifugation on the supernatant obtained in the step (3), namely performing ultracentrifugation for 2 hours at 100000g, and collecting precipitates to obtain target microspheres;
(5) adding physiological saline into the target microspheres in the step (4) for resuspension to obtain a resuspension solution, filtering the resuspension solution by using a 0.22-micron filtering membrane to remove apoptotic bodies and microbubbles, purifying the filtered target microspheres to obtain purified human adipose-derived mesenchymal stem cell Exosomes (EVs), and sterilizing the purified human adipose-derived mesenchymal stem cell exosomes to obtain the human adipose-derived mesenchymal stem cell exosomes;
(6) identifying the human adipose-derived mesenchymal stem cell exosomes by an electron microscope; and identifying the obtained human adipose-derived mesenchymal stem cell exosomes by using Elisa.
3. Results
3.1 this example packet information
In this example, the effect of the addition of dimethyloxalylglycine and differential centrifugation on exosome content was explored in groups as follows, "+" indicates that dimethyloxalylglycine was added or differential centrifugation was performed, and "-" indicates that dimethyloxalylglycine was not added or centrifugation was performed in a conventional manner. Specifically, as shown in table 2:
table 2 packet information
| Group of | Dimethyloxalylglycine | Differential centrifugation |
| A | - | - |
| B | + | - |
| C | - | + |
| D | + | + |
3.2 Excreta expression and Electron microscopy results
As can be seen from fig. 4, compared to the group a without diformylglycine, the group D with diformylglycine and differential centrifugation can effectively increase the content of human adipose-derived mesenchymal stem cell exosomes (P < 0.01). And (3) identifying the form of the human adipose-derived mesenchymal stem cell exosome obtained in the step (6) by using an electron microscope, wherein the result shows that the human adipose-derived mesenchymal stem cell exosome is circular or elliptical, has a complete thin film structure and has a diameter of 40-120 nm.
3.3 Elisa results
The obtained human adipose-derived mesenchymal stem cell exosomes were identified by Elisa, the EVs markers comprise CD9, CD63, CD81, alpha-actin-4 and CD40, and the detection result shows that the human adipose-derived mesenchymal stem cell exosomes can express exosome markers CD9, CD63, CD81, alpha-actin-4 and CD40 (shown in figure 5). Therefore, the exosome prepared in the embodiment is an exosome derived from human adipose mesenchymal stem cells.
Note that, in this example, the same procedure as in example 1 was used where the human adipose-derived mesenchymal stem cell culture procedure was not described.
Example 3
The aim of this example was to culture h-MSCs cell lines.
1. Material
Preparing human body fat (discarded normal human body fat after operation, signed with know-howBook), PBS, BSA, collagenase type IV, fetal calf serum, CD29+、CD44+、CD90+、CD34-And CD45-The monoclonal antibody of (1).
Passage of h-MSCs
(1) Adipose tissues were collected as shown in example 1, digested with the digestion conditions of the group of 0.1% collagenase IV + 0.02% BSA, and then cells were cultured to 80% to 90% confluency according to the method described in example 1, digested with 0.25% pancreatin-0.02% EDTA, and centrifuged to obtain cell pellets;
(2) resuspending the cell sediment by using a fresh culture medium without exosomes, carrying out passage according to the ratio of 1: 2-4, culturing to P10, wherein each generation of cells are respectively named as P1-h-MSCs, P2-h-MSCs, P3-h-MSCs, P4-h-MSCs, P5-h-MSCs, P6-h-MSCs, P7-h-MSCs, P8-h-MSCs, P9-h-MSCs and P10-h-MSCs;
(3) collecting P2-h-MSCs, P5-h-MSCs, P8-h-MSCs and P10-h-MSCs, digesting respectively to prepare single cell suspension, and detecting cell surface molecular marker CD29 by flow cytometry+、CD44+、CD90+、CD34-And CD45-Expression of (2).
3. Results
3.1 Effect of different passage ratios on cell proliferation Rate
The cell passage time and cell proliferation rate under different passage conditions are shown in table 3, and it can be seen from table 3 that the proliferation rate of the cells is significantly greater than that of the cells obtained by the same passage at the ratio of 1:2 (P <0.05) and significantly greater than that of the cells obtained by the same passage at the ratio of 1:4 (P <0.01) after passage at the ratio of 1:3, and thus it can be seen that the optimal passage time interval and cell proliferation rate can be obtained by passage at the ratio of 1:3, which is beneficial for the later period of the experiment.
TABLE 3 cell passage time and cell proliferation Rate for different passage conditions
3.2 expression of cellular markers in different generations of cells
As shown in FIG. 6, the cell morphology of the human adipose-derived mesenchymal stem cells cultured in this example was good, and CD29 was found in P2-P8+、CD44+And CD90+High expression, CD34-And CD45-Essentially non-expressed, CD29 in P10+、CD44+And CD90+Expression began to decline, but CD34-And CD45-Still not expressed. Therefore, the isolated culture method and the passage method provided by the embodiment can obtain stable human adipose-derived mesenchymal stem cells, and at least the stable human adipose-derived mesenchymal stem cells can be successfully transferred to P8.
In the embodiment, a simple and efficient method for separating and culturing the human adipose-derived mesenchymal stem cells is formed by selecting a proper digestion system to digest adipose tissues and performing adherent screening. The results show that the specific ratio of collagenase concentration and digestion conditions in this example can effectively separate fibrous connective tissue from adipose tissue, and increase the concentration, purity and activity of primary cells, so that the primary cells can be stably passaged at least to P8.
Example 4
The purpose of this example was to construct human adipose mesenchymal stem cells stably overexpressing IL-10.
1. Construction of IL-10 overexpression vectors
(1) Searching a coding region of human IL-10 in an NCBI database, designing a primer by using primer 5.0, and synthesizing the primer by sending the primer to Huada gene to obtain a primer group;
(2) extracting human leukocyte mRNA by using an RNA kit, synthesizing cDNA (complementary deoxyribonucleic acid) by using a reverse transcription kit, using cDAN as a template, amplifying an IL-10 coding region by RT-PCR (reverse transcription-polymerase chain reaction) by using the primer group obtained in the step (1), carrying out enzyme digestion to recover a target fragment of the IL-10 coding region, cloning the target fragment to a eukaryotic expression vector pcDNA3.1/Myc-his (A), screening positive clones, sending the positive clones to a biological company for sequencing, and storing bacterial liquid of the positive clones with correct sequencing for later use;
(3) inoculating the bacterial liquid obtained in the step (2) into a fresh culture medium according to the volume ratio of 1:50, performing shake culture at 37 ℃ overnight, collecting the bacterial liquid, centrifuging the bacterial liquid, taking the precipitate to obtain a bacterial precipitate, extracting the plasmid in the bacterial precipitate by using a plasmid extraction kit according to the instruction, and identifying the obtained plasmid by using PCR (polymerase chain reaction) to obtain the correct plasmid, namely the IL-10 overexpression vector pcDNA3.1-IL-10;
2. human adipose-derived mesenchymal stem cell G418 concentration screening
(1) Taking the human adipose-derived mesenchymal stem cells in the embodiment 3, preferably selecting the P2-P8 generation, thawing, recovering, and performing 2-6 × 104cells/cm2Inoculation at 37 ℃ with 5% CO2Culturing in an incubator with 100% relative humidity;
(2) after the cells in the step (1) are cultured to be adherent, the cells are divided into pcDNA3.1-IL-10 transfection group (over-expressing IL-10) and pcDNA3.1 no-load group, fresh culture mediums containing 0 mu G/mL, 200 mu G/mL, 400 mu G/mL, 700 mu G/mL, 1100 mu G/mL and 1500 mu G/mL G418 are respectively changed in, the fresh culture mediums with corresponding concentrations are changed in every 3 days, and the observation is continuously carried out for 7 days, wherein the 700 mu G/mL group causes the cells of the untransfected group to be completely dead, so that the subsequent screening can use 700 mu G/mL as the screening concentration.
3. Transfection of human adipose-derived mesenchymal stem cells
(1) Taking the human adipose-derived mesenchymal stem cells in the embodiment 3, preferably selecting the P2-P8 generation, thawing, recovering, and performing 2-6 × 104cells/cm2Inoculation at 37 ℃ with 5% CO2Culturing in an incubator with 100% relative humidity;
(2) when the cell confluence reaches 80-90%, preparing transfection mixed solution, wherein the transfection mixed solution is prepared by dissolving 0.3 mu g of IL-10 in 100 mu L of Opti-MEM serum-free culture medium, uniformly mixing to prepare A liquid, and then dissolving 10 mu L of Lipofectamine 2000TMDissolving in 100 mu L of Opti-MEM serum-free medium, uniformly mixing to obtain solution B, standing at room temperature for 5min, mixing solution A and solution B to prepare a transfection complex, changing the cell culture medium into Opti-MEM serum-free medium, dripping the transfection complex into cells, culturing for 4-6 h, changing into complete culture medium, and continuing to culture for 3 d;
(3) carrying out passage according to the ratio of 1:3 on the cells in the step (2), adding a fresh culture medium containing 700 mu G/mL G418, changing the fresh culture medium with a corresponding concentration every 3 days, screening for 10-14 d, dying the human embryonic mesenchymal stem cells which are not transfected into pcDNA3.1-IL-10, and carrying out resistant cloning on the human embryonic mesenchymal stem cells transfected into pcDNA3.1-IL-10;
(4) carrying out amplification culture after cloning, digesting and diluting in the step (3) to finally obtain the human adipose-derived mesenchymal stem cells stably overexpressing IL-10;
(5) and (3) taking part of the cells subjected to amplification culture to respectively extract total RNA and protein, and detecting the IL-10 expression condition by using RT-qPCR and Western Blotting. The result is shown in figure 7, which shows that the human adipose-derived mesenchymal stem cells stably overexpressing IL-10 are successfully constructed.
Example 5
The purpose of this example was to prepare human adipose mesenchymal stem cell-derived exosomes stably overexpressing IL-10.
1. Material method
PBS, BSA, collagenase type IV, fetal calf serum, CD9, CD63, CD81, monoclonal antibodies to alpha-actin-4 and CD40, dimethyloxalylglycine, and the human adipose mesenchymal stem cells stably overexpressing IL-10 of example 4.
2. Collecting exosomes derived from human adipose-derived mesenchymal stem cells stably overexpressing IL-10
(1) The human adipose-derived mesenchymal stem cells stably overexpressing IL-10 provided in example 4 were collected, seeded, and cultured in exosome-free serum medium at 37 ℃ in 5% CO2Starting culture under the condition;
(2) after culturing for 12h, changing into a serum culture medium without exosome containing 300 mu mol/L dimethyloxalyl glycine, continuously culturing for 36h, and collecting cell culture solution;
(3) performing differential centrifugation on the cell culture solution obtained in the step (2) at 4 ℃, namely centrifuging for 10min under the condition of 1500g, and centrifuging for 10min at 10000g to remove cell debris and macromolecular proteins to obtain supernatant;
(4) performing ultracentrifugation on the supernatant obtained in the step (5), namely performing ultracentrifugation for 2 hours at 100000g, and collecting precipitates to obtain target microspheres;
(5) adding physiological saline into the target microspheres in the step (4) for re-suspension to obtain a re-suspension, filtering the re-suspension by using a 0.22-micron filtering membrane to remove apoptotic bodies and microbubbles, purifying the filtered target microspheres to obtain purified Exosomes (EVs), and sterilizing the purified exosomes to obtain exosomes;
(6) identifying the human adipose-derived mesenchymal stem cell exosomes by an electron microscope; and identifying the obtained human adipose-derived mesenchymal stem cell exosomes by using Elisa.
3. Results
3.1 Excreta expression level and Electron microscopy results
The grouping information is shown in table 2. As can be seen from fig. 4, the content of exosomes of human adipose-derived mesenchymal stem cells stably overexpressing IL-10 (P <0.01) was effectively increased in group a without differential centrifugation and in group D with differential centrifugation in addition of dimethyloxalylglycine, compared to group a without addition of dimethyloxalylglycine.
3.2 Elisa results
The obtained human adipose-derived mesenchymal stem cell exosomes stably overexpressing IL-10 are identified by Elisa, EVs markers comprise CD9, CD63, CD81, alpha-actin-4 and CD40, and detection results show that the human adipose-derived mesenchymal stem cell exosomes stably overexpressing IL-10 can express exosome markers CD9, CD63, CD81, alpha-actin-4 and CD40 (shown in figure 5).
Example 6
The purpose of this example is to construct a myocardial infarction SD rat model.
The specific construction method of the myocardial infarction SD rat model is as follows:
(1) feeding SPF SD male rats to 2-3 months of age, wherein the body weight is 2.59 +/-21 g;
(2) performing intraperitoneal injection anesthesia by using chloral hydrate solution (10% w/v), wherein the dosage of the chloral hydrate solution (10% w/v) is 0.3mL/100g when each SD male rat is anesthetized;
(3) adjusting the respiratory frequency of the anesthetized SD male rats to 70 times/minute, the inspiration/expiration ratio to 1:3 and the tidal volume to be about 3mL/100g, and simultaneously carrying out auxiliary breathing by a breathing machine;
(4) coronary artery ligation: the left chest of the anesthetized SD male rat is depilated and disinfected conventionally, the skin, the superficial fascia and the deep fascia are cut in sequence, the junction of the pectoralis major and the anterior serratus is separated bluntly by hemostatic forceps, separating 3 rd to 4 th intercostal space from sternal margin by hemostatic forceps, cutting 3 rd to 4 th costal cartilage, opening intercostal space to expose heart, pulling open two side tissues of operation incision with two small drag hooks with rubber ring, carefully pushing away lung tissue around heart with wet cotton swab, enlarging operation visual field, lifting pericardium wall layer with forceps, carefully cutting pericardium with ophthalmic scissors, pushing away thymus upwards with cotton swab to clearly expose left coronary vein, the left coronary vein is used as a mark, a 7-0 ophthalmic noninvasive tape suture needle is used for penetrating the deep part of the left coronary vein, the depth of the needle insertion is 0.5-1.0 mm, and knotting is carried out, ligating coronary artery between rat left auricle and pulmonary artery, about 3mm from aortic root;
(5) and (3) postoperative treatment: after the operation, the rats in the step (4) are placed into a small rearing cage, the rats are revived at the temperature of 30 ℃, each surviving rat is placed into the small rearing cage to be reared independently (mainly to prevent the rats from fighting each other due to wound or bloody smell after the operation), the rats are reared for 7 days, and the rats are subjected to intramuscular injection of penicillin after the operation to prevent infection and are subjected to intraperitoneal injection of curcumin to treat the infection. The mice raised for 7d were divided into groups for use.
Example 7
The purpose of this example is to verify the effect of exosomes provided in example 5 on infarcted myocardial tissue repair in a myocardial infarcted SD rat model.
The surviving myocardial infarction SD rats of example 6 were equally divided into 3 groups, numbered A, B, C, D, E, and injected with physiological saline, the adipose mesenchymal stem cells provided in example 1, the human adipose mesenchymal stem cell exosomes provided in example 2, the human adipose mesenchymal stem cells stably overexpressing IL-10 provided in example 4, and the exosomes provided in example 5, respectively. Group A is myocardial infarction model group, and 0.5 ml/injection of normal saline each time; group B is a human adipose-derived mesenchymal stem cell group, and the human adipose-derived mesenchymal stem cell suspension provided in example 1 is injected at a rate of 0.5 ml/injection, wherein the cell density of the human adipose-derived mesenchymal stem cell suspension is 5 × 105cells/ml; group C was injected with 0.5 ml/exosome as provided in example 2; group D human adipose-derived mesenchymal stem cells stably overexpressing IL-10, provided in example 4, were injected at 0.5 ml/injection, cell densityDegree of 5X 105cells/ml; group E was injected with 0.5 ml/mouse of exosomes provided in example 5. The five groups A to E are treated 3 times respectively, and treated once every 5 days.
The processing results are shown in fig. 8 and table 4. The groups A to E in FIG. 8 and groups A to E in Table 4 correspond to the groups A to E in this example, respectively.
TABLE 4 results after treatment of myocardial infarction SD rats with different treatment modalities
As can be seen from fig. 8, after the group a was injected with the saline, the infarct size was much larger than that of the group B injected with the adipose tissue-derived mesenchymal stem cells, and the infarct sizes of the group B and the group D were much larger than those of the group C and the group E (p <0.05), and at the same time, the infarct size of the group E was significantly smaller than that of the group C, and in combination with table 4, it can be seen that the exosomes provided in examples 2 and 5 can significantly promote the repair of the myocardial tissue after infarction, and the effect is superior to that of the adipose tissue-derived mesenchymal stem cells, with significant difference, wherein the effect of the exosome provided in example 4 is optimal.
Exosomes, as a membrane vesicle, may play a role through growth factors, chemokines, cytokines, transcription factors, genes, RNA, and the like. Stem cells are biological materials with great medical value, and more stem cells are applied to heart diseases such as myocardial infarction and psychological failure and heart-related diseases, and the current research still faces some obstacles, such as low retention rate of cells in an injury area and low survival rate of the cells implanted into a human body. Moreover, in fact most studies have stayed only at the cellular level, which, due to its technical drawbacks, such as the limitations of certain technical parameters, has made it impossible to enter animal level to verify the effect of mental protection. However, the adipose-derived stem cell exosome capable of stably overexpressing IL-10 provided by the invention is obtained by selecting human adipose-derived stem cells, and performing specific cell culture, centrifugation, filtration and other steps to obtain the adipose-derived stem cell exosome capable of stably overexpressing IL-10 from human adipose-derived stem cells, which can overcome the defects, wherein the adipose-derived stem cell exosome capable of stably overexpressing IL-10 from human adipose-derived stem cells contains a plurality of beneficial factors for repairing myocardial cells after myocardial infarction, most notably IL-10, and due to exogenous overexpression, IL-10 is much higher than IL-10 in a common exosome, and in addition, other inflammation-inhibiting factors such as IL-7, HGF and TGF-beta are included, and active growth factors such as soma-145, VEGF, EGFR, PDGF and miR-146b cooperate to protect damaged cells, the specific action mechanism is as follows, and the intercellular information transfer and biological functions are mainly exerted through 4 modes:
(1) the adipose-derived stem cell exosome stably overexpressing IL-10 is used as a signal complex, can directly stimulate receptor cells through the action of ligands on the cell surface, and excites the signal cascade in the receptor cells to initiate a series of biochemical and physiological processes;
(2) the adipose-derived stem cell exosome stably overexpressing IL-10 transfers receptors among cells, transfers the receptors to target cells, promotes the effects of inflammation-inhibiting factors and active growth-promoting factors, and accelerates the recovery of damaged cardiac muscle cells;
(3) the adipose-derived stem cell exosome stably overexpressing IL-10 conveys functional proteins or infectious particles to receptor cells, and further promotes the recovery of the receptor cells;
(4) the adipose-derived stem cell exosome stably overexpressing the IL-10 transmits genetic information to a receptor cell through mRNA, microRNA or transcription factors. After the adipose-derived stem cell exosome stably overexpressing IL-10 is absorbed by receptor cells, the loaded lipid, protein, mRNA, microRNA and other inflammation-inhibiting and life activity factors can influence the cell phenotype and functions of the receptor cells by changing the transcription and translation programs of damaged cardiac muscle cells to influence the protein modification and positioning, and regulating the signal cascade pathway, key enzyme reaction, automatic regulation of cells and other modes, thereby playing roles in repairing and protecting the cardiac muscle cells;
(5) the repair function of the adipose-derived stem cell exosome stably overexpressing the IL-10 is further enhanced by stably overexpressing the IL-10. Myocardial infarction is one of the important causes of coronary atherosclerosis, and early and timely recovery of infarcted arteries can prevent further expansion of infarct range. The inflammatory reaction, the release and the regulation of inflammatory factors play an important role in the occurrence and the development process of myocardial infarction. Normal myocardial cells do not produce inflammatory factors such as TNF-alpha and the like, the concentration of TNF-alpha is increased along with the myocardial infarction degree after myocardial damage, and myocardial apoptosis, myocardial necrosis and the like are caused. The exosome from the human adipose mesenchymal stem cell stably overexpressing IL-10 is gathered at the myocardial infarction part, so that the IL-10 is gathered in a large amount in the myocardial damaged area, the retention rate of the IL-10 in the damaged area can be increased due to the structural characteristics of the exosome body, the high-concentration IL-10 in the damaged area is ensured for a long time, the activity of mononuclear/macrophage in the damaged area is inhibited, the generation of inflammatory factors such as TNF-alpha and the like is inhibited, the level of the inflammatory factors is reduced, the pathological process of the myocardial infarction is relieved, the myocardial protection effect is exerted, the angiogenin is promoted to be secreted by the heart, the myocardial infarction cell is repaired, and the blood ejection function is improved.
The invention has the advantages that: the human body fat can be obtained from subcutaneous fat, the material taking method is simple, when the human body fat is used for preparing the autologous adipose tissue-derived mesenchymal stem cell exosome, the human body fat is easier to be accepted by a patient psychologically, and meanwhile, due to the fact that the human body fat is the autologous adipose tissue-derived exosome, rejection reaction can not occur. Meanwhile, the adipose-derived mesenchymal stem cells have strong proliferation capacity and are less influenced by age, and even if the patients with the older age are, the adipose-derived mesenchymal stem cell exosomes with better activity can still be obtained. Therefore, the preparation method of the exosome derived from the human adipose-derived mesenchymal stem cells provided by the invention can simply and efficiently prepare a large amount of exosomes with high purity and good effect, and can be further applied to the treatment of myocardial infarction.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (10)
1. An IL-10 high-expression human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction, which is secreted by IL-10 over-expression human adipose-derived mesenchymal stem cells.
2. The human adipose-derived mesenchymal stem cell exosome for highly expressing IL-10 according to claim 1, which is prepared by the following steps: culturing human adipose mesenchymal stem cells over expressing IL-10, and collecting cell culture solution; and (3) centrifuging the cell culture solution at a differential speed, collecting supernatant, ultracentrifuging the supernatant to obtain target microspheres, and filtering, purifying and sterilizing the target microspheres to obtain the exosome.
3. The IL-10 highly expressing human adipose-derived mesenchymal stem cell exosome according to claim 2, wherein the IL-10 overexpressing human adipose-derived mesenchymal stem cell is prepared by the following method: preparing an IL-10 overexpression vector, transfecting the IL-10 overexpression vector into the human adipose-derived mesenchymal stem cells, and screening and identifying to obtain the human adipose-derived mesenchymal stem cells overexpressing IL-10.
4. The exosome for human adipose-derived mesenchymal stem cells highly expressing IL-10 according to claim 3, wherein the means for transfecting an IL-10 overexpression vector into the human adipose-derived mesenchymal stem cells comprises adenovirus, lentivirus, Lip2000 and Lip 3000.
5. The IL-10 highly expressing human adipose-derived mesenchymal stem cell exosome according to claim 4, wherein the identification and screening comprises adding 700 μ G/mL G418 to the human adipose-derived mesenchymal stem cells, and culturing for 10-14 days.
6. The human adipose-derived mesenchymal stem cell exosome according to claim 5, wherein the human adipose-derived mesenchymal stem cells are prepared by digesting human adipose tissues, inoculating and subculturing, and the enzymes for digesting the human adipose tissues comprise 0.06-0.16% by volume of collagenase type IV and 0.015-0.02% by mass of BSA.
7. The human adipose-derived mesenchymal stem cell exosome according to claim 6, wherein the human adipose-derived mesenchymal stem cells are from P2 generation to P8 generation.
8. The human adipose-derived mesenchymal stem cell exosome according to claim 7, wherein the enzymes for digesting the human adipose tissues are 0.1% type IV collagenase and 0.02% BSA (bovine serum albumin) by volume percentage, and the digestion time is 30-35 min.
9. The exosome for high-expression IL-10 human adipose-derived mesenchymal stem cells according to any one of claims 2 to 8, wherein the culture solution for culturing the human adipose-derived mesenchymal stem cells is 10% by volume of exosome-free fetal bovine serum culture medium; the density of inoculation is 2-6 multiplied by 104cells/cm2(ii) a And when the supernate is subjected to ultracentrifugation, performing differential centrifugation, wherein the differential centrifugation comprises centrifugation for 10min at 1500g and then centrifugation for 10min at 10000 g.
10. The use of the human adipose-derived mesenchymal stem cell exosome highly expressing IL-10 according to any one of claims 1-9 in the preparation of a medicament for treating myocardial infarction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010820947.2A CN111944748A (en) | 2020-08-14 | 2020-08-14 | high-IL-10-expression human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010820947.2A CN111944748A (en) | 2020-08-14 | 2020-08-14 | high-IL-10-expression human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111944748A true CN111944748A (en) | 2020-11-17 |
Family
ID=73342990
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010820947.2A Pending CN111944748A (en) | 2020-08-14 | 2020-08-14 | high-IL-10-expression human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111944748A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877294A (en) * | 2021-02-23 | 2021-06-01 | 赛浦生物科技(长春)有限公司 | Preparation and application of gene-modified mesenchymal stem cell exosome |
| CN116656617A (en) * | 2023-01-03 | 2023-08-29 | 生物岛实验室 | Exosome and preparation method and application thereof |
| CN116688101A (en) * | 2023-06-26 | 2023-09-05 | 南通大学附属医院 | Application of high-expression IL-10 neutrophil extracellular vesicles in treatment of temporomandibular arthritis |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130302273A1 (en) * | 2012-05-08 | 2013-11-14 | Bioregenerative Sciences | Compositions derived from stem cell released molecules & methods for formulation thereof |
| CN104974984A (en) * | 2015-04-13 | 2015-10-14 | 深圳市默赛尔生物医学科技发展有限公司 | Adipose tissue-derived mesenchymal stem cell amplification culture method |
| CN105395571A (en) * | 2015-12-31 | 2016-03-16 | 苏州大学 | New application of small exosome secreted by mesenchymal stem cells in treatment of myocardial infarction |
| CN106222134A (en) * | 2016-07-29 | 2016-12-14 | 中卫华医（北京）生物科技有限公司 | The cultural method of effective acquisition fat mesenchymal stem cell |
| CN108728410A (en) * | 2018-06-19 | 2018-11-02 | 中国医学科学院阜外医院 | The preparation method of source for mesenchymal stem cells excretion body based on medical preconditioning |
| CN108743620A (en) * | 2018-06-21 | 2018-11-06 | 南开大学 | Bioactive materials promote source of human stem cell excretion body to treat corneal injury |
| CN110564682A (en) * | 2019-09-30 | 2019-12-13 | 陕西中鸿科瑞再生医学研究院有限公司 | Method for large-scale production of human adipose-derived mesenchymal stem cell exosomes |
| CN110577931A (en) * | 2019-10-29 | 2019-12-17 | 上海交通大学医学院附属第九人民医院 | Intermittent hypoxia treatment stem cell source exosome and application thereof in myocardial tissues |
| CN110846275A (en) * | 2019-12-12 | 2020-02-28 | 赛瑞诺(北京)生物科技有限公司 | Serum-free culture method of human autologous adipose-derived mesenchymal stem cells |
| CN111304164A (en) * | 2019-11-30 | 2020-06-19 | 杭州拜欧津生物科技有限公司 | Preparation method and application of adipose-derived stem cell exosomes |
| CN111514166A (en) * | 2020-05-14 | 2020-08-11 | 天津医科大学眼科医院 | Application of mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 in drugs for autoimmune diseases |
-
2020
- 2020-08-14 CN CN202010820947.2A patent/CN111944748A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130302273A1 (en) * | 2012-05-08 | 2013-11-14 | Bioregenerative Sciences | Compositions derived from stem cell released molecules & methods for formulation thereof |
| CN104974984A (en) * | 2015-04-13 | 2015-10-14 | 深圳市默赛尔生物医学科技发展有限公司 | Adipose tissue-derived mesenchymal stem cell amplification culture method |
| CN105395571A (en) * | 2015-12-31 | 2016-03-16 | 苏州大学 | New application of small exosome secreted by mesenchymal stem cells in treatment of myocardial infarction |
| CN106222134A (en) * | 2016-07-29 | 2016-12-14 | 中卫华医（北京）生物科技有限公司 | The cultural method of effective acquisition fat mesenchymal stem cell |
| CN108728410A (en) * | 2018-06-19 | 2018-11-02 | 中国医学科学院阜外医院 | The preparation method of source for mesenchymal stem cells excretion body based on medical preconditioning |
| CN108743620A (en) * | 2018-06-21 | 2018-11-06 | 南开大学 | Bioactive materials promote source of human stem cell excretion body to treat corneal injury |
| CN110564682A (en) * | 2019-09-30 | 2019-12-13 | 陕西中鸿科瑞再生医学研究院有限公司 | Method for large-scale production of human adipose-derived mesenchymal stem cell exosomes |
| CN110577931A (en) * | 2019-10-29 | 2019-12-17 | 上海交通大学医学院附属第九人民医院 | Intermittent hypoxia treatment stem cell source exosome and application thereof in myocardial tissues |
| CN111304164A (en) * | 2019-11-30 | 2020-06-19 | 杭州拜欧津生物科技有限公司 | Preparation method and application of adipose-derived stem cell exosomes |
| CN110846275A (en) * | 2019-12-12 | 2020-02-28 | 赛瑞诺(北京)生物科技有限公司 | Serum-free culture method of human autologous adipose-derived mesenchymal stem cells |
| CN111514166A (en) * | 2020-05-14 | 2020-08-11 | 天津医科大学眼科医院 | Application of mesenchymal stem cell-derived small extracellular vesicles overexpressing interleukin 10 in drugs for autoimmune diseases |
Non-Patent Citations (3)
| Title |
|---|
| YUJIA YUE等: "Interleukin-10 Deficiency Alters Endothelial Progenitor Cell-Derived Exosome Reparative Effect on Myocardial Repair via Integrin-Linked Kinase Enrichment", 《CIRC RES.》 * |
| 洪梅等: "急性心肌梗死患者IL-6和IL-10水平测定及其临床意义", 《血栓与止血学》 * |
| 赵辉: "过表达IL-10的脂肪间充质干细胞促进糖尿病小鼠慢性创面愈合的研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877294A (en) * | 2021-02-23 | 2021-06-01 | 赛浦生物科技(长春)有限公司 | Preparation and application of gene-modified mesenchymal stem cell exosome |
| CN112877294B (en) * | 2021-02-23 | 2021-08-24 | 赛浦生物科技(长春)有限公司 | Preparation and application of gene-modified mesenchymal stem cell exosome |
| CN116656617A (en) * | 2023-01-03 | 2023-08-29 | 生物岛实验室 | Exosome and preparation method and application thereof |
| CN116688101A (en) * | 2023-06-26 | 2023-09-05 | 南通大学附属医院 | Application of high-expression IL-10 neutrophil extracellular vesicles in treatment of temporomandibular arthritis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105985985B (en) | Preparation method of allogeneic mesenchymal stem cells edited by CRISPR technology and optimized by IGF (insulin-like growth factor) and application of allogeneic mesenchymal stem cells in treatment of myocardial infarction | |
| KR101310578B1 (en) | Methods of using adipose tissue-derived cells in the treatment of cardiovascular conditions | |
| JP5450072B2 (en) | Kidney-derived cells and their use in tissue repair and regeneration | |
| CN111944748A (en) | high-IL-10-expression human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction and application thereof | |
| CN110577931B (en) | Intermittent hypoxia treatment stem cell source exosome and application thereof in myocardial tissues | |
| JP2016116523A (en) | Use of adipose tissue-derived stromal stem cells in treating fistula | |
| US20210032598A1 (en) | Activation-induced tissue-effector cells suitable for cell therapy and extracelluar vesicles derived therefrom | |
| CN111471650B (en) | Exosome derived from human umbilical cord mesenchymal stem cells, identification method and application | |
| CN112430567B (en) | Culture method and application of urine-derived renal stem cells | |
| CN113278591B (en) | Cardiac targeting genetic engineering exosome and preparation method and application thereof | |
| CN105769910B (en) | Application of human amniotic mesenchymal stem cells | |
| JP2020534344A (en) | Methods and compositions for treating epidermolysis bullosa | |
| CN108588026A (en) | The preparation method and its usage of the clinical grade mescenchymal stem cell of height expression IL10 | |
| CN113274411A (en) | Application of genetically modified bone marrow mesenchymal stem cell-derived microvesicles in preparation of medicines for treating renal injury | |
| CN109402175B (en) | Adipose-derived stem cells expressing chemokine receptor CCR2B, and preparation method and application thereof | |
| CN111925983A (en) | Preparation method of high-IL-10-expression human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction | |
| CN113234811A (en) | Application of CircRNA000338 and medicine thereof | |
| CN111944747A (en) | Human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction and application thereof | |
| US20240034997A1 (en) | Myogenin-expressing fibroblast-like cell (meflc) line and construction method and use thereof | |
| US9950016B2 (en) | Cell secreted proteins for the treatment of myocardial infarction | |
| CN110090227B (en) | Use of human amniotic epithelial cells in treatment of graft-versus-host disease | |
| CN101182493A (en) | Transgenic mesenchyma stem cell for curing radiation enteritis and method of producing the same | |
| CN114984219B (en) | Use of PD1 inhibitors in the preparation of inhibitors of cardiac fibroblast transdifferentiation | |
| CN115478048A (en) | Preparation of exosome by culturing adipose-derived mesenchymal stem cells | |
| CN115671137A (en) | Application of human deciduous tooth pulp stem cell exosome in preparation of tendon injury medicine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |