CN115354022A - Mesenchymal stem cells obtained by treating at least two cytokines of IL4, IL21 and IL27, exosome and application thereof - Google Patents
Mesenchymal stem cells obtained by treating at least two cytokines of IL4, IL21 and IL27, exosome and application thereof Download PDFInfo
- Publication number
- CN115354022A CN115354022A CN202211018225.0A CN202211018225A CN115354022A CN 115354022 A CN115354022 A CN 115354022A CN 202211018225 A CN202211018225 A CN 202211018225A CN 115354022 A CN115354022 A CN 115354022A
- Authority
- CN
- China
- Prior art keywords
- mesenchymal stem
- stem cells
- exosomes
- cytokines
- cytokine composition
- 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.)
- Withdrawn
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
- C12N5/0668—Mesenchymal stem cells from other natural sources
-
- 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
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2304—Interleukin-4 (IL-4)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2321—Interleukin-21 (IL-21)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2327—Interleukin-27 (IL-27)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Developmental Biology & Embryology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Biotechnology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Rheumatology (AREA)
- General Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Hematology (AREA)
- General Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Pain & Pain Management (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a mesenchymal stem cell obtained by processing at least two cytokines of IL4, IL21 and IL27, an exosome thereof and application thereof, relating to the technical field of biomedicine, wherein the mesenchymal stem cell and the exosome thereof are obtained by pretreating the mesenchymal stem cell by a cytokine composition, and the mesenchymal stem cell is obtained by the following steps: performing staged treatment stimulation on the mesenchymal stem cells by using a complete culture medium containing a cytokine composition to obtain energized mesenchymal stem cells; the mode of the exosome is as follows: performing staged treatment stimulation on the mesenchymal stem cells by using a complete culture medium containing a cytokine composition, and then replacing the treatment with starvation of a basic culture medium; the mesenchymal stem cells and the exosomes thereof obtained by using the cytokine composition provided by the invention for pretreatment can play a stronger immunoregulation function and can be better used for treating immune diseases.
Description
Technical Field
The invention relates to the technology in the field of biomedicine, in particular to a mesenchymal stem cell obtained by processing at least two cytokines of IL4, IL21 and IL27, and a exosome and application thereof.
Background
Refractory Systemic Lupus Erythematosus (SLE) is an autoimmune disease caused by multiple factors, which is developed among women of 20 to 40 weeks old, the onset of the disease is slow, and the clinical manifestations are various. Among them, autoantibodies produced by the dysfunction of patient's own cells and humoral immunity may affect multiple organs of the whole body, such as: skin, joints, kidneys, nervous system, etc.; common symptoms include arthritis, fever, chest pain, hair loss, lymphadenectasis, and facial erythema, among others. The cause of SLE is not clear, but it is thought that SLE may interact with genetic factors, environmental factors, endocrine factors, and the like. In China, the average incidence of SLE population is 30-70/10 ten thousand, wherein the incidence of diseases of men and women is 1.9, the average age of the diseases is 30.7 years old, the fatality rate and disability rate of patients after the diseases are high, and once the diseases are caused, certain economic and psychological burden can be caused to the patients and families. In clinical treatment of intractable systemic lupus erythematosus, the conventional therapy has no case of complete cure, the existing traditional therapy gradually loses the required curative effect along with the progress of diseases, and the body function is gradually disintegrated under long-term chronic inflammatory reaction, so that the life quality of patients is greatly influenced.
The mesenchymal stem cells are multipotent stem cells, widely exist in various tissues and organs in a body and play roles in tissue repair and immunoregulation. In vitro, mesenchymal stem cells have the characteristics of multidirectional differentiation potential, self-renewal and high proliferation speed. The biological characteristics of the compound which are widely concerned mainly include lower immunogenicity and stronger immunoregulation capability, so the compound is used in a large amount in a plurality of clinical trials and is favored by a plurality of experts and scholars in the field. In clinical trials for mesenchymal stem cell applications, a large part is involved in the treatment of autoimmune diseases, such as: systemic lupus erythematosus, rheumatoid arthritis, psoriasis, crohn's disease, etc. shows that mesenchymal stem cell has excellent application scene in treating autoimmune diseases and certain curative effect. However, not all clinical trial patients who participate in the treatment using mesenchymal stem cells can obtain better treatment effect, and common mesenchymal stem cells have certain individual difference in the treatment of autoimmune diseases, and not all subjects can obtain better expected curative effect, which is mainly caused by the heterogeneity of common mesenchymal stem cells. Although the common mesenchymal stem cells prepared for conventional clinical treatment are subjected to passage and amplification for several times, various cell subsets with different functional properties still exist in the cell population, the mesenchymal stem cells mainly play an immune regulation function by a paracrine pathway aiming at autoimmune diseases, and the common mesenchymal stem cells used in clinical treatment can gradually form the cell subsets with uniform functions and similar properties after being injected into a human body and stimulated by an in vivo microenvironment due to the cell heterogeneity, and then can play a treatment effect. The metabolic cycle of the cells is relatively fixed after the cells are injected into a body, a large part of the cells are trapped in the lung and cannot swim to the whole body, and the cells can exert functions after being stimulated in the body, so that the time for exerting the treatment effect is greatly reduced after the cells occupy the time in the large part of the metabolic cycle, and the treatment effect of different patients is uneven.
Exosomes are a class of small membrane vesicles containing complex RNAs, proteins, or carbohydrates, usually ranging in diameter between 40-100 nm. Exosomes are secreted outside cells in various cell states, play important roles in participating in cell communication and intercellular interaction as a specifically secreted membrane vesicle, and have the characteristic of difficulty in generating safety problems due to the fact that exosomes only have a membrane structure and no nuclear structure, and are widely concerned by clinical research and academic research. The exosome plays a paracrine function as the mesenchymal stem cell and has important significance in regulating the immune function of an organism, but the exosome with the immune function is very limited because common mesenchymal stem cells are difficult to react at the first time after entering the organism. Therefore, a technical way is needed to make mesenchymal stem cells receive the stimulation of the corresponding inflammatory environment signal, and rapidly produce the corresponding exosomes with the corresponding inflammatory environment regulation function in a large amount.
In conclusion, the problem of mesenchymal stem cell activation is solved, and the key problem of improving/enhancing the treatment effect of mesenchymal stem cells, and researches have proved that the mesenchymal stem cells can change corresponding biological characteristics after being stimulated by corresponding cytokines in vitro and can generate different effects along with the types, concentrations and stimulation degrees of the cytokines, so that the mesenchymal stem cells and exosomes thereof for treating SLE have important research significance.
Disclosure of Invention
In view of the above, the present invention provides a mesenchymal stem cell obtained by treating at least two cytokines IL4, IL21 and IL27, exosome thereof and application thereof, wherein the mesenchymal stem cell and exosome thereof obtained by pretreatment with the cytokine composition provided by the present invention can exert stronger immunomodulatory functions and can be better used for treating immune diseases.
In order to achieve the purpose, the invention adopts the following technical scheme:
the mesenchymal stem cell and the exosome obtained by treating at least two cytokines of IL4, IL21 and IL27 are obtained by pretreating the mesenchymal stem cell by using a cytokine composition, wherein the cytokine composition comprises at least two cytokines of IL4, IL21 and IL27; the manner of obtaining the mesenchymal stem cells by the pretreatment is as follows: performing staged treatment stimulation on the mesenchymal stem cells by using a complete culture medium containing a cytokine composition by adopting a large-scale bioreactor to obtain energized mesenchymal stem cells; the mode for obtaining the exosome by the pretreatment is as follows: and (3) performing staged treatment stimulation on the mesenchymal stem cells by using a complete culture medium containing a cytokine composition by adopting a large-scale bioreactor, replacing the treatment with starvation of a basic culture medium, collecting culture supernatant, and performing gradient ultracentrifugation to obtain exosomes.
As a preferred embodiment: the method for treating the stimulation in stages comprises the following steps: adding complete culture medium containing different cytokine compositions with corresponding concentrations into a large-scale bioreactor in stages to perform energized stimulation on the mesenchymal stem cells; the complete culture medium is any basic culture medium mixed serum substitute for culturing the mammalian cells.
As a preferred embodiment: the concentration ranges of the three cytokines of IL4, IL21 and IL27 are respectively as follows: IL4:25-150ng/ml, IL21:100-300ng/ml, IL27:50-250ng/ml.
As a preferred embodiment: the cytokine composition comprises IL4 and IL21, or the cytokine composition comprises IL21 and IL27, or the cytokine composition comprises IL4 and IL27.
As a preferred embodiment: the cytokine composition comprises IL4 at a concentration of 30ng/ml and IL21 at a concentration of 200 ng/ml; alternatively, the cytokine composition comprises IL21 at a concentration of 150ng/ml and IL27 at a concentration of 200 ng/ml; alternatively, the cytokine composition comprises IL-4 at a concentration of 25ng/ml and IL-27 at a concentration of 75ng/ml.
As a preferred embodiment: the contact culture of the cytokine composition for 16-36 hours is needed in the process of pretreating the mesenchymal stem cells; the cytokine composition comprises IL4 and IL21, wherein the IL4 pretreatment time is 10 hours, and the IL21 pretreatment time is 12 hours; the cytokine composition comprises IL21 and IL27, wherein the IL21 pretreatment time is 10 hours, and the IL27 pretreatment time is 12 hours; the cytokine composition comprises IL4, IL27, wherein; IL4 pretreatment time is 6 hours, IL27 pretreatment time is 10 hours.
As a preferred scheme: the treatment time for starvation using the basal medium was 24 hours.
As a preferred embodiment: the non-pretreated mesenchymal stem cells are derived from at least one of bone marrow, fat, placenta, umbilical cord and dental pulp in human tissue; the exosome is secreted by a mesenchymal stem cell.
As a preferred embodiment: the mesenchymal stem cells and exosomes thereof obtained by treating at least two cytokines of IL4, IL21 and IL27 have the effect of enhancing the anti-SLE therapeutic activity.
The mesenchymal stem cells obtained by treating at least two cytokines of IL4, IL21 and IL27 and the application of the exosome thereof are used for preparing the medicine for treating systemic and immune inflammation caused by SLE.
Compared with the prior art, the cytokine composition has obvious advantages and beneficial effects, and particularly, the cytokine composition provided by the invention is screened according to the expression characteristics of the inflammatory microenvironment cytokines in an organism in the disease attack process of an SLE patient, and can stimulate human mesenchymal stem cells to have better anti-inflammatory activity on SLE; compared with the human mesenchymal stem cells or exosomes obtained by the conventional culture method, the mesenchymal stem cells and exosomes provided by the invention have better anti-inflammatory environmental activity, so that the mesenchymal stem cells and exosomes can be used for treating immune inflammatory response caused by SLE, reducing the damage of the inflammatory response to the tissues of the whole body and relieving the disease progression speed; the concrete points are as follows: reducing the proliferation ratio of T/B lymphocytes, reducing the proliferation ratio of T helper lymphocytes and up-regulating the proliferation ratio of T regulatory lymphocytes; the protection degree of the human mesenchymal stem cells and the exosomes thereof obtained by the culture method of the invention on the disease model is superior to the protection degree of the human mesenchymal stem cells and the exosomes thereof obtained by the conventional method on the disease model.
To more clearly illustrate the structural features and effects of the present invention, the following detailed description is given with reference to the accompanying drawings and specific embodiments.
Drawings
FIG. 1 is a graph showing the changes in serum IL4, IL21 and IL27 during the disease progression in SLE model mice of the present invention;
FIG. 2 is a graph showing the therapeutic effects of human umbilical cord mesenchymal stem cells and exosomes in serum dsDNA and ANA of SLE mice before and after pretreatment according to the present invention;
FIG. 3 shows the effect of the biological properties of human umbilical cord mesenchymal stem cells and exosomes before and after pretreatment according to the present invention (A is the expression of IDO of different groups, B is the expression of PGE2 of different groups, and C is the expression of VEGF of different groups);
FIG. 4 is a graph showing the effect of different cytokine concentrations on the expression of cytokines by human umbilical cord mesenchymal stem cells and their exosomes according to the present invention (A is the expression of IDO of different groups, B is the expression of PGE2 of different groups);
FIG. 5 is a graph of the effect of in vitro immunosuppressive functions of human umbilical cord mesenchymal stem cells and exosomes after pretreatment with different cytokine compositions of the present invention;
FIG. 6 is a graph of the effect of factor combinations on the in vitro immunosuppressive function of human umbilical cord mesenchymal stem cells and exosomes at different treatment times of the present invention;
FIG. 7 is a graph of the effect of cytokine combinations of the present invention on the in vitro immunosuppressive function of human umbilical cord mesenchymal stem cells and exosomes at different concentrations;
FIG. 8 is a graph of the therapeutic effect of different cytokine compositions on human umbilical cord mesenchymal stem cells and exosomes in the SLE model of the present invention after pretreatment.
Detailed Description
As shown in fig. 1 to 8, a mesenchymal stem cell and an exosome thereof obtained by treating at least two cytokines of IL4, IL21 and IL27 are obtained by pretreating a mesenchymal stem cell with a cytokine composition, wherein the cytokine composition comprises at least two cytokines of IL4, IL21 and IL27; the way of obtaining the mesenchymal stem cells by the pretreatment is as follows: performing staged treatment stimulation on the mesenchymal stem cells by using a complete culture medium containing a cytokine composition by adopting a large-scale bioreactor to obtain energized mesenchymal stem cells; the way of obtaining the exosome by the pretreatment is as follows: and (2) performing staged treatment stimulation on the mesenchymal stem cells by using a complete culture medium containing a cytokine composition by adopting a large-scale bioreactor, replacing the treatment with starvation of a basic culture medium, collecting culture supernatant, and performing gradient ultracentrifugation to obtain exosomes.
The method for treating the stimulation in stages comprises the following steps: adding complete culture medium containing different cytokine compositions with corresponding concentrations to a large-scale bioreactor in stages to perform energized stimulation on the mesenchymal stem cells; the complete culture medium is any basic culture medium mixed with serum substitute for culturing the mammalian cells.
The basic medium may be Dulbecco's Modified Eagle's Medium (DMEM), a mixture of Dulbecco's modified eagle's medium and F12 (DMEM/F12), RPMI 1640.
The concentration ranges of the three cytokines of IL4, IL21 and IL27 are respectively as follows: IL4:25-150ng/ml, IL21:100-300ng/ml, IL27:50-250ng/ml.
The cytokine composition comprises IL4 and IL21, or the cytokine composition comprises IL21 and IL27, or the cytokine composition comprises IL4 and IL27.
The cytokine composition comprises IL4 at a concentration of 30ng/ml and IL21 at a concentration of 200 ng/ml; alternatively, the cytokine composition comprises IL21 at a concentration of 150ng/ml and IL27 at a concentration of 200 ng/ml; alternatively, the cytokine composition comprises IL-4 at a concentration of 25ng/ml and IL-27 at a concentration of 75ng/ml.
The contact culture of the cytokine composition for mesenchymal stem cells is required for 16-36 hours in the pretreatment process; the cytokine composition comprises IL4 and IL21, wherein the IL4 pretreatment time is 10 hours, and the IL21 pretreatment time is 12 hours; the cytokine composition comprises IL21 and IL27, wherein the IL21 pretreatment time is 10 hours, and the IL27 pretreatment time is 12 hours; the cytokine composition comprises IL4, IL27, wherein; IL4 pretreatment time is 6 hours, IL27 pretreatment time is 10 hours.
The treatment time for starvation using the basal medium was 24 hours.
The non-pretreated mesenchymal stem cells are derived from at least one of bone marrow, fat, placenta, umbilical cord and dental pulp in human tissue; the exosome is secreted by a mesenchymal stem cell.
The mesenchymal stem cells and exosomes thereof obtained by treating at least two cytokines of IL4, IL21 and IL27 have the effect of enhancing the anti-SLE therapeutic activity.
The application of the mesenchymal stem cells obtained by treating at least two cytokines of IL4, IL21 and IL27 and exosomes thereof in preparing the medicine for treating systemic and immune inflammation caused by SLE.
In the present invention, the reagents and test materials are commercially available, and the sources of the biological materials are as follows:
IL4, IL21, and IL27 cytokines were purchased from R & D Systems, inc.
The first embodiment is as follows:
the preparation method of the human umbilical cord mesenchymal stem cells and the exosomes thereof pretreated by the cytokine composition comprises the following steps:
firstly: the preparation method of the human umbilical cord mesenchymal stem cells comprises the following steps:
separating and culturing human umbilical cord mesenchymal stem cells:
under the supervision of the medical ethics committee, fresh umbilical cords are obtained from healthy donors to separate the Wharton jelly, the Wharton jelly is sheared into pieces, the Wharton jelly is cultured by a wall attaching method, and primary cells are obtained and amplified in a serum-free culture system, wherein the serum-free culture system comprises the following steps: MSC
XF Medium (BI, israel) +2% platelet lysate + MSC
XF Supplement Mix (BI, israel).
The primary cells were expanded and cryopreserved at "P2 passage" as seed cells:
"P2 generation" means: performing amplification culture on primary cells in a cell factory according to 3000-5000cells/m & lt 2 & gt, transferring the cells into a cylindrical cell amplification bioreactor after accumulating a certain amount of the cells, performing mixed culture on the cells and microcarriers, performing cell amplification with the culture concentration of 10000-12000cells/ml, and timely supplementing a culture solution through a perfusion system; after the culture 24, the stage of treatment with the cytokine composition is entered.
Secondly, the method comprises the following steps: cell factor composition pretreatment of human umbilical cord mesenchymal stem cells:
different cell factors are added into a cell culture medium in stages at corresponding concentrations according to a combination mode, the original culture medium is gradually replaced along with a perfusion system, and after each factor in the combination stimulates the human umbilical cord mesenchymal stem cells for corresponding time according to the corresponding sequence, the next cell factor is replaced or the basic culture medium is replaced for starvation treatment.
"pretreatment" refers to a process of contacting and culturing a cell culture medium supplemented with a cytokine composition comprising at least 2 cytokines among IL4, IL21 and IL27 with human umbilical cord-derived mesenchymal stem cells in the process of culturing human umbilical cord-derived mesenchymal stem cells.
The "basal medium" may be any commercially available mammalian cell culture medium, such as: DMEM, MEM, DMEM/F12; in this embodiment, MSC is used
XF Medium (BI, israel).
In the specification, aiming at the pathological characteristics of the SLE disease, the cytokine composition comprises IL4 and IL21, and is called as a cytokine composition 1; the cytokine composition comprises IL21 and IL27, and is called cytokine composition 2; the cytokine composition comprises IL4, IL27, and is called cytokine composition 3; cytokine concentrations ranged from IL4:25-150ng/ml, IL21:100-300ng/ml, IL27:50-250ng/ml.
In the present specification, the pretreatment medium containing the cytokine composition is contacted with the cells for the culture time:
the pretreatment time of the cytokine composition 1 is 10 hours for IL4, and 12 hours for IL21; the pretreatment time of the cytokine composition 2 for IL21 is 10 hours, and the pretreatment time of IL27 is 12 hours; the pretreatment time for IL4 was 6 hours and the pretreatment time for IL27 was 10 hours for cytokine composition 3.
In the present specification, the mesenchymal stem cells treated by the pretreatment medium of the cytokine composition may be digested after 24 hours of starvation treatment, and prepared into a cell preparation to be stored and transported to a patient for use.
Thirdly, the steps of: the preparation method of the exosome comprises the following steps:
collecting culture supernatant after starvation treatment, and centrifuging for 10min under a centrifugal force of 200g to remove most cell debris; thereafter, tangential flow concentration using a hollow fiber column as the filter support was performed, adjusting the pressure between 40-100psi, removing impurities above 250kD and concentrating the liquid by a factor of 50 to 100, in this example, a factor of 50.
And (3) carrying out ultragradient centrifugation on the concentrated solution, wherein the specific method comprises the following steps:
centrifuging at 10,000g for 30min at 4 deg.C, and collecting supernatant; centrifuging the supernatant at 10,0000g for 90min at 4 deg.C, and keeping precipitate; adding 20ml of D-PBS into the precipitate, centrifuging for 90min at the temperature of 4 ℃ at 100,000g, and reserving the centrifugal precipitate to obtain the separated exosome; dissolving with 1ml D-PBS, mixing, centrifuging, precipitating, and storing at-80 deg.C.
First effect embodiment:
comparison of biological characteristics of human umbilical cord mesenchymal stem cells and exosomes before and after pretreatment
In the present specification, in order to further elucidate the influence of the cytokine composition on the biological properties of human umbilical cord mesenchymal stem cells, an in vitro cytokine secretion test was performed. The in vitro cytokine secretion detection mainly examines the concentration of cytokines in culture supernatants of common human umbilical cord mesenchymal stem cells, human umbilical cord mesenchymal stem cells pretreated by different cytokine concentrations and human umbilical cord mesenchymal stem cells pretreated by different cytokine compositions (the combination types comprise a cytokine composition 1, a cytokine composition 2 and a cytokine composition 3) under the condition of in vitro culture and in the same culture time, and examines the influence of different cytokine concentrations or different cytokine compositions on the expression cytokines of the human umbilical cord mesenchymal stem cells.
Specifically, when the cell fusion degree reaches more than 80%, adding complete culture media containing cytokines with corresponding concentrations or different combinations to stimulate the mesenchymal stem cells for 10h-24h, then collecting culture supernatant to detect the cytokine expression condition of the mesenchymal stem cells, and simultaneously replacing the culture supernatant with a basic culture medium and then carrying out starvation treatment for 24h to detect the cytokine expression condition in exosomes.
The method for obtaining the exosomes of the human umbilical cord mesenchymal stem cells before and after pretreatment comprises the steps of starving the pretreated mesenchymal stem cells after replacing a basic culture medium, collecting culture supernatant, carrying out gradient centrifugation to obtain the concentrated exosomes of the mesenchymal stem cells with enhanced SLE (systemic lupus erythematosus) resistance activity, and detecting and identifying the exosomes through a nano-particle size analyzer and a western blot; "pretreatment time" refers to the contact incubation time.
The results of fig. 3 and 4 show that the concentrations of IDO, PGE2 and VEGF in the culture supernatant and exosomes of the human umbilical cord mesenchymal stem cells after pretreatment are obviously increased by detecting the expression amounts of IDO, PGE2 and VEGF by using an ELISA method.
FIG. 4 shows that when different cytokine concentrations of individual components are used for expression of human umbilical cord mesenchymal stem cells and exosomes, IL4 is preferably at a concentration of 45ng/ml, IL21 is preferably at a concentration of 200ng/ml, and IL27 is preferably at a concentration of 75ng/ml.
Effect example two:
in vitro immunosuppression test of human umbilical cord mesenchymal stem cells and exosomes thereof before and after pretreatment
The mesenchymal stem cells are co-cultured with human peripheral blood mononuclear cells in vitro, and the method is an important experimental detection method for detecting the immunosuppressive capacity of the mesenchymal stem cells in vitro. In the invention, after the CFSE is marked by the peripheral blood mononuclear cells and activated by the CD3 and CD28 monoclonal antibodies, the CFSE is respectively cultured with common human umbilical cord mesenchymal stem cells or exosomes thereof and the pretreated human umbilical cord mesenchymal stem cells or exosomes thereof (the combination types comprise cytokine composition 1, cytokine composition 2 and cytokine composition 3) for 72 to 80 hours, and the proliferation condition of the PBMC is detected in a flow mode.
The results of fig. 5, 6 and 7 show that: after 80 hours of co-culture, the immunosuppressive effect of the human umbilical cord mesenchymal stem cells or the exosomes thereof pretreated by the cell factor composition is obviously superior to that of the common human umbilical cord mesenchymal stem cells or the exosomes thereof. From the flow detection result, the proliferation proportion of the CD3+ total T cells co-cultured by the human umbilical cord mesenchymal stem cells or the exosomes after the cytokine composition 1, the cytokine composition 2 and the cytokine composition 3 are pretreated is obviously reduced, and the proliferation reduction proportion is obviously higher than that of the CD3+ total T cells co-cultured with the common human umbilical cord mesenchymal stem cells or the exosomes. According to the statistics of the inhibition percentage, the inhibition effect of the CD3+ total T cells co-cultured with the human umbilical cord mesenchymal stem cells or the exosomes after the pretreatment by combining the factors is good, and the inhibition ratio is obviously higher than that of the CD3+ total T cells co-cultured with the common human umbilical cord mesenchymal stem cells or the exosomes.
FIG. 6 shows that in the pretreatment, the pretreatment time for IL4 and IL21 in the cytokine composition 1 are 10 hours and 12 hours, respectively; the pretreatment time of the cytokine composition 2 for IL21 is 10 hours, and the pretreatment time of IL27 is 12 hours; the pretreatment time for IL4 was 6 hours and the pretreatment time for IL27 was 10 hours for cytokine composition 3.
The results in FIGS. 7 and 8 show that the preferred concentration of 30ng/ml IL4 and 200ng/ml IL21 for cytokine composition 1 was obtained by in vitro immunosuppression experiments and SLE model treatment experiments; preferred concentrations of cytokine composition 2 are 150ng/ml IL21, 200ng/ml IL27; preferred concentrations of cytokine composition 3 are 25ng/ml IL4, 75ng/ml IL27.
Effect example three:
mouse model for treating SLE (systemic lupus erythematosus) by human umbilical cord mesenchymal stem cells or exosomes pretreated by different cytokine compositions
MRL/lpr mice were used as SLE mouse models, and after the mice were raised in the laboratory to 12 weeks of age, they started to develop spontaneous morbidity, and the serum cytokine (IL 4, IL-18, IL 27) changes in SLE mice were measured from week 10, and the serum antinuclear antibody (ANA) and anti-dsDNA antibody (dsDNA) concentrations in mice were measured. In the invention, the immunosuppressive function characteristics of common human umbilical cord mesenchymal stem cells or exosomes thereof and pretreated human umbilical cord mesenchymal stem cells or exosomes (combination types comprise a cytokine composition 1, a cytokine composition 2 and a cytokine composition 3) are utilized to treat SLE.
The SLE model treatment experiment refers to that when MRL/lpr gene mice (the gene mice can generate disease characteristics similar to clinical systemic lupus erythematosus when being aged for 12 weeks, and the gene mice are a good SLE disease research model), 1 x 106 human umbilical cord mesenchymal stem cells before and after pretreatment are injected into tail veins for treatment, or human umbilical cord mesenchymal stem cell exosomes before and after pretreatment with total protein content of 200 mug are injected into tail veins for treatment, mice are killed on day 14 after model creation, the internal organ tissues of the mice are taken for pathological section, and the pathological section and a control group are subjected to H & E staining together to judge the treatment effect.
As shown in fig. 2 and fig. 5 to fig. 8, the results show that the common human umbilical cord mesenchymal stem cells or exosomes have a certain therapeutic effect on SLE, which indicates that the common human umbilical cord mesenchymal stem cells or exosomes can exert corresponding immunosuppressive effects after being stimulated by in-vivo inflammatory factors, but the common human umbilical cord mesenchymal stem cells are not pretreated by specific cytokines and cannot exert effects immediately after entering into the body, so that the therapeutic effect is limited, and exosomes released by the untreated human umbilical cord mesenchymal stem cells also show the same biological characteristics, which indicates that the mesenchymal stem cells which are not pretreated can exert immunomodulatory functions; however, the human umbilical cord mesenchymal stem cells or exosomes pretreated by the cytokine composition 1, the cytokine composition 2 and the cytokine composition 3 in the SLE disease model show the immunosuppressive capability superior to that of the common human umbilical cord mesenchymal stem cells or exosomes.
Therefore, this result is consistent with the conclusion drawn in the in vitro experiments in this specification, and the cytokine composition provided by the present invention has an enhancing effect on human umbilical cord mesenchymal stem cells or exosomes for the treatment of SLE.
As shown in fig. 2, 5 to 8, the present invention provides mesenchymal stem cells cultured with a cytokine-containing composition and exosomes thereof, which have good anti-inflammatory activity against SLE. Compared with the common existing mesenchymal stem cells and exosomes thereof, the mesenchymal stem cells and exosomes thereof treated by the cytokine composition containing at least 2 cytokines of IL4, IL21 and IL27 have obviously improved treatment effect on SLE and can effectively reduce the secretion level of dsDNA and ANA on an animal model.
Wherein: the term "anti-SLE activity" refers to that the mesenchymal stem cells and exosomes thereof have better immunosuppressive function on T/B lymphocytes after being treated by a cytokine composition containing at least 2 cytokines of IL4, IL21 and IL27.
In the present invention, the main mechanism of realization of said "anti-SLE activity" is: the mesenchymal stem cells are pretreated in advance in vitro by using the corresponding cytokine composition, and the cytokines which mainly play a role in contacting a disease microenvironment in advance are activated in vitro culture, so that the activation time of the cytokines entering a body is shortened, and the functionality of the cytokines aiming at a special microenvironment is enhanced, thereby improving the antagonistic activity and the treatment effect of the corresponding diseases.
The results in fig. 8 show that after the mesenchymal stem cells or exosomes pretreated in the specification are used for treatment, obvious treatment effects are achieved.
The design key point of the invention is that the mesenchymal stem cells and exosomes thereof obtained after the pretreatment by using the cytokine composition provided by the invention can exert stronger immunoregulation function, can be better used for treating immune diseases, and lay a foundation for providing personalized cell treatment schemes aiming at different states of the diseases; the invention provides a set of feasible clinical stem cell treatment scheme for accurate treatment, can better perform targeted accurate treatment on different degrees of disease severity of different SLE patients, effectively improves the treatment effect and reduces the clinical symptoms of the disease.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the technical scope of the present invention, so that any minor modifications, equivalent changes and modifications made to the above embodiments according to the technical spirit of the present invention are within the technical scope of the present invention.
Claims (10)
1. Mesenchymal stem cells obtained by treatment with at least two cytokines IL4, IL21, IL27 and exosomes thereof; the mesenchymal stem cell and an exosome thereof are obtained after the mesenchymal stem cell is pretreated by a cytokine composition, wherein the cytokine composition comprises at least two cytokines of IL4, IL21 and IL27; the manner of obtaining the mesenchymal stem cells by the pretreatment is as follows: performing staged treatment stimulation on the mesenchymal stem cells by using a complete culture medium containing a cytokine composition by adopting a large-scale bioreactor to obtain energized mesenchymal stem cells; the mode for obtaining the exosome by the pretreatment is as follows: and (3) performing staged treatment stimulation on the mesenchymal stem cells by using a complete culture medium containing a cytokine composition by adopting a large-scale bioreactor, replacing the treatment with starvation of a basic culture medium, collecting culture supernatant, and performing gradient ultracentrifugation to obtain exosomes.
2. Mesenchymal stem cells and exosomes of at least two cytokines obtained from IL4, IL21, IL27 treatment according to claim 1 characterized in that; the method for treating the stimulation in stages comprises the following steps: adding complete culture medium containing different cytokine compositions with corresponding concentrations to a large-scale bioreactor in stages to perform energized stimulation on the mesenchymal stem cells; the complete culture medium is any basic culture medium mixed serum substitute for culturing the mammalian cells.
3. Mesenchymal stem cells and exosomes obtained from treatment of at least two cytokines IL4, IL21, IL27 according to claim 1 characterized in that; the concentration ranges of the three cytokines of IL4, IL21 and IL27 are respectively as follows: IL4:25-150ng/ml, IL21:100-300ng/ml, IL27:50-250ng/ml.
4. Mesenchymal stem cells and exosomes obtained from treatment of at least two cytokines IL4, IL21, IL27 according to claim 1 characterized in that; the cytokine composition comprises IL4 and IL21, or the cytokine composition comprises IL21 and IL27, or the cytokine composition comprises IL4 and IL27.
5. Mesenchymal stem cells and exosomes of at least two cytokines obtained from IL4, IL21, IL27 according to claim 4 characterized in that; the cytokine composition comprises IL4 at a concentration of 30ng/ml and IL21 at a concentration of 200 ng/ml; alternatively, the cytokine composition comprises IL21 at a concentration of 150ng/ml and IL27 at a concentration of 200 ng/ml; alternatively, the cytokine composition comprises IL4 at a concentration of 25ng/ml and IL27 at a concentration of 75ng/ml.
6. Mesenchymal stem cells obtained by treatment with at least two cytokines IL4, IL21, IL27 and exosomes according to claim 4 characterized in that; the contact culture of the cytokine composition for 16-36 hours is needed in the process of pretreating the mesenchymal stem cells; the cytokine composition comprises IL4 and IL21, wherein the IL4 pretreatment time is 10 hours, and the IL21 pretreatment time is 12 hours; the cytokine composition comprises IL21 and IL27, wherein the IL21 pretreatment time is 10 hours, and the IL27 pretreatment time is 12 hours; the cytokine composition comprises IL4, IL27, wherein; IL4 pretreatment time is 6 hours, IL27 pretreatment time is 10 hours.
7. Mesenchymal stem cells and exosomes of at least two cytokines obtained from IL4, IL21, IL27 treatment according to claim 1 characterized in that; the treatment time for starvation using basal medium was 24 hours.
8. Mesenchymal stem cells and exosomes obtained from treatment of at least two cytokines IL4, IL21, IL27 according to claim 1 characterized in that; the non-pretreated mesenchymal stem cells are derived from at least one of bone marrow, fat, placenta, umbilical cord and dental pulp in human tissue; the exosome is secreted by a mesenchymal stem cell.
9. Mesenchymal stem cells and exosomes of at least two cytokines obtained from IL4, IL21, IL27 treatment according to claim 1 characterized in that; has effect in enhancing therapeutic activity of resisting SLE.
10. Use of mesenchymal stem cells obtained by treatment with at least two cytokines IL4, IL21, IL27 and exosomes according to any of claims 1 to 9, characterized in that: can be used for preparing medicine for treating systemic and immune inflammation caused by SLE.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211018225.0A CN115354022A (en) | 2022-08-24 | 2022-08-24 | Mesenchymal stem cells obtained by treating at least two cytokines of IL4, IL21 and IL27, exosome and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211018225.0A CN115354022A (en) | 2022-08-24 | 2022-08-24 | Mesenchymal stem cells obtained by treating at least two cytokines of IL4, IL21 and IL27, exosome and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115354022A true CN115354022A (en) | 2022-11-18 |
Family
ID=84005231
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211018225.0A Withdrawn CN115354022A (en) | 2022-08-24 | 2022-08-24 | Mesenchymal stem cells obtained by treating at least two cytokines of IL4, IL21 and IL27, exosome and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115354022A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014093948A1 (en) * | 2012-12-14 | 2014-06-19 | Rutgers, The State University Of New Jersey | Methods modulating immunoregulatory effect of stem cells |
| US20210052641A1 (en) * | 2019-08-23 | 2021-02-25 | United Therapeutics Corporation | Pre-conditioned extracellular vesicles and methods of production |
| WO2022169049A1 (en) * | 2021-02-03 | 2022-08-11 | 서울대학교산학협력단 | Method for obtaining mesenchymal stem cell-derived exosome |
-
2022
- 2022-08-24 CN CN202211018225.0A patent/CN115354022A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014093948A1 (en) * | 2012-12-14 | 2014-06-19 | Rutgers, The State University Of New Jersey | Methods modulating immunoregulatory effect of stem cells |
| US20210052641A1 (en) * | 2019-08-23 | 2021-02-25 | United Therapeutics Corporation | Pre-conditioned extracellular vesicles and methods of production |
| WO2022169049A1 (en) * | 2021-02-03 | 2022-08-11 | 서울대학교산학협력단 | Method for obtaining mesenchymal stem cell-derived exosome |
Non-Patent Citations (4)
| Title |
|---|
| AIFEN LI等: "Mesenchymal Stem Cell Therapy: Hope for Patients With Systemic Lupus Erythematosus", 《FRONT. IMMUNOL.》, vol. 12, pages 728190 * |
| JICUI LI等: "Immunomodulatory Activity of Mesenchymal Stem Cells in Lupus Nephritis: Advances and Applications", 《FRONT. IMMUNOL.》, vol. 13, pages 843192 * |
| 王婕等: "炎症因子干预影响间充质干细胞的免疫调控特性", 《中国组织工程研究》, vol. 24, no. 13, pages 2108 - 2113 * |
| 王颖翠等: "间充质干细胞对系统性红斑狼疮T、B细胞的免疫调节机制", 《中国组织工程研究》, vol. 21, no. 29, pages 4734 - 4741 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115161276B (en) | Mesenchymal stem cells obtained by treatment of at least two cytokines, namely IL4, IL21 and IL27, and exosomes and application thereof | |
| JP7455165B2 (en) | Methods and devices for production and delivery of beneficial factors from stem cells | |
| Liu et al. | Mesenchymal stem cells prevent hypertrophic scar formation via inflammatory regulation when undergoing apoptosis | |
| Li et al. | Potential pre-activation strategies for improving therapeutic efficacy of mesenchymal stem cells: current status and future prospects | |
| KR101993027B1 (en) | Stem cell microparticles | |
| CN105120879A (en) | Stem cell microparticles and mirna | |
| KR20090065478A (en) | Mesenchymal stem cells and uses therefor | |
| CN113197919B (en) | Application of pilose antler stem cell exosome in preparing product for improving or treating osteoarthritis and delaying cell senescence | |
| JP2021531288A (en) | Mitochondrial enhancement therapy for eye diseases | |
| US20230077870A1 (en) | Method for enhancing secretory function of mesenchymal stem cells and application thereof | |
| JP2021532093A (en) | Mitochondrial enhancement therapy for brain diseases | |
| JP2021532095A (en) | Mitochondrial enhancement therapy for liver disease | |
| JP2021532091A (en) | Mitochondrial enhancement therapy for pancreatic disease | |
| US20180221411A1 (en) | Improved Cell Therapies | |
| JP2021532094A (en) | Mitochondrial enhancement therapy for muscle diseases | |
| CN111973632A (en) | Stem cell preparation for treating diabetes and preparation method thereof | |
| CN115354022A (en) | Mesenchymal stem cells obtained by treating at least two cytokines of IL4, IL21 and IL27, exosome and application thereof | |
| CN114561349B (en) | Exosomes produced by IL18, IL12, IL15 treated mesenchymal stem cells and antiviral use | |
| CN113980896B (en) | Application of IRF1 in regulation and control of mesenchymal stem cell immunoregulation and product | |
| CN113789333B (en) | Application of Chi3l1 in regulating and controlling hUC-MSCs to inhibit Th17 differentiation-mediated immunoregulation | |
| JP2021531281A (en) | Mitochondrial enhancement therapy for kidney disease | |
| KR20150091519A (en) | A method of generating multilineage potential cells | |
| CN111087442B (en) | Polypeptide extraction method | |
| CN114517177A (en) | Composition for enhancing mesenchymal stem cell efficiency and application thereof | |
| KR101577553B1 (en) | Mesenchymal stem cells and uses therefor |
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 | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20221118 |