CN114042087A - Application of human umbilical cord mesenchymal stem cell exosome in preparation of medicine for treating scleroderma - Google Patents

Application of human umbilical cord mesenchymal stem cell exosome in preparation of medicine for treating scleroderma Download PDF

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Publication number
CN114042087A
CN114042087A CN202111159905.XA CN202111159905A CN114042087A CN 114042087 A CN114042087 A CN 114042087A CN 202111159905 A CN202111159905 A CN 202111159905A CN 114042087 A CN114042087 A CN 114042087A
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umbilical cord
mesenchymal stem
human umbilical
cord mesenchymal
stem cell
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姜淼
俞悦
赵敬军
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Shanghai Tongji Hospital
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Shanghai Tongji Hospital
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    • 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
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • 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
    • 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
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • 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
    • 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

Abstract

The invention relates to application of human umbilical cord mesenchymal stem cell exosome in preparation of a medicament for treating scleroderma. According to the invention, the human umbilical cord mesenchymal stem cell exosome stem cells are adopted, so that the dermis layer of a scleroderma mouse is thinned, the subcutaneous fat layer is recovered to a certain degree, the deposition of extracellular matrix is reduced, and the epithelial-mesenchymal transition and M1/M2 macrophage balance (both of which participate in the regulation and control of scleroderma) of the scleroderma mouse are regulated.

Description

Application of human umbilical cord mesenchymal stem cell exosome in preparation of medicine for treating scleroderma
Technical Field
The invention relates to the technical field of medicament treatment, in particular to application of human umbilical cord mesenchymal stem cell exosome in preparation of a medicament for treating scleroderma.
Background
Scleroderma, an autoimmune disease characterized by fibrosis, vascular destruction and immune abnormalities, is a serious threat to the life of patients and the prognosis of scleroderma is therefore of widespread concern. At present, no exact effective treatment method exists, and clinically, glucocorticoid and immunosuppressant are combined as a main treatment scheme, but the slow release rate is low, and the safety and drug resistance are uncertain. In addition, biologies have also become a new star for treating scleroderma, but have not yet been able to reverse the progression of the disease.
Stem cells are cells with the potential for self-renewal and multipotentiality, and can repair damaged tissues autonomously. Human umbilical cord mesenchymal stem cells (HUMSCs) have the characteristics of lower immunogenicity, stronger differentiation capacity and easier acquisition. Stem cells function primarily through paracrine, while human umbilical cord mesenchymal stem cell exosomes (HUMSCs-Ex) are paracrine vesicles. By cell-free transplantation, the active substances of the exosome can be released, and the effect of treating scleroderma is achieved.
Disclosure of Invention
The invention aims to provide an application of human umbilical cord mesenchymal stem cell exosome in preparation of a medicament for treating scleroderma, aiming at the defects in the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that:
provides the application of human umbilical cord mesenchymal stem cell exosome in preparing a medicament for treating scleroderma.
Preferably, the preparation method of the human umbilical cord mesenchymal stem cell exosome comprises the following steps:
s1, taking the frozen human umbilical cord mesenchymal stem cells, carrying out constant-temperature water bath at 35-40 ℃ for 1-5 min, centrifuging, adding a fresh culture medium, and carrying out conventional culture;
s2, taking 3 rd-5 th generation human umbilical cord mesenchymal stem cells, when the human umbilical cord mesenchymal stem cells are fused to 70% -80%, updating a fresh serum-free culture medium, and collecting supernatant after culturing for 24-72 h;
s3, high-speed centrifugation is carried out to obtain concentrated solution, and the concentrated solution is refrigerated at the temperature of-100 ℃ to-60 ℃ after PBS heavy suspension.
Preferably, the amount of the human umbilical cord mesenchymal stem cell exosome protein in the medicament for treating scleroderma is 2-6mg/kg body weight.
Preferably, the human umbilical cord mesenchymal stem cell exosomes thin the dermal layer.
Preferably, the human umbilical cord mesenchymal stem cell exosome reduces extracellular matrix deposition and inhibits epithelial-mesenchymal transition.
Preferably, the human umbilical cord mesenchymal stem cell exosomes inhibit M2 macrophage polarization, and modulate M1/M2 macrophage balance.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
according to the invention, the human umbilical cord mesenchymal stem cell exosome stem cells are adopted, so that the dermis layer of a scleroderma mouse is thinned, the subcutaneous fat layer is recovered to a certain degree, the deposition of extracellular matrix is reduced, and the epithelial-mesenchymal transition and M1/M2 macrophage balance (both of which participate in the regulation and control of scleroderma) of the scleroderma mouse are regulated.
Drawings
FIG. 1 is a graph showing the therapeutic effect of human umbilical cord mesenchymal stem cell exosome on scleroderma.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be 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.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.
Examples
The embodiment provides application of human umbilical cord mesenchymal stem cell exosome in preparation of a medicament for treating scleroderma, wherein the preparation method of the human umbilical cord mesenchymal stem cell exosome comprises the following steps:
s1, taking out the cell cryopreservation tube of the human umbilical cord mesenchymal stem cells from the liquid nitrogen tank, carrying out water bath in a constant-temperature water bath kettle at 37 ℃ for 3min, centrifuging, adding a fresh culture medium, and carrying out conventional culture;
s2, taking 3 rd-5 th-generation human umbilical cord mesenchymal stem cells in a good state, when the human umbilical cord mesenchymal stem cells are fused to 75%, updating a fresh serum-free culture medium, and collecting a supernatant after culturing for 48 hours;
s3, obtaining a concentrated solution of the human umbilical cord mesenchymal stem cell exosome by adopting a high-speed centrifugation method, and refrigerating at-80 ℃ after PBS (phosphate buffer solution) heavy suspension;
after a scleroderma mouse animal model is successfully constructed, the identified human umbilical cord mesenchymal stem cell exosome is injected subcutaneously at a skin damage part, and the total amount of the human umbilical cord mesenchymal stem cell exosome is 100 mu g.
The boundary among the epidermal layer, the dermal layer and the subcutaneous fat layer of the scleroderma mouse is disordered, the dermal layer is thickened, collagen fibers are increased, and extracellular matrix is deposited; after the human umbilical cord mesenchymal stem cell exosome stem cells are predicted, the dermis layer is thinned, the subcutaneous fat layer is restored to a certain degree, the extracellular matrix deposition is reduced, and the epithelial-mesenchymal transition and M1/M2 macrophage balance (both of which are involved in the regulation and control of scleroderma) of scleroderma mice are regulated.
Detection examples
The detection embodiment is used for judging the treatment effect of the human umbilical cord mesenchymal stem cell exosome on scleroderma:
fixing mouse skin tissue with 4% paraformaldehyde, and then carrying out conventional dehydration, paraffin embedding and slicing; after slicing, dewaxing by xylene and hydrating by gradient ethanol, then staining by hematoxylin and eosin in sequence, and finally dehydrating and sealing; as shown in panel a, the disordered skin structure was significantly corrected;
each HE stained section was randomly selected 3 fields under x 100 fields, dermis thickness was calculated using Image J software, and averaged. The thickened dermal layer was thinned as shown in panel D.
Fixing mouse skin tissue with 4% paraformaldehyde, and then performing conventional dehydration, paraffin embedding and slicing; after slicing, dewaxing by xylene and hydrating by gradient ethanol, then dyeing by hematoxylin and aniline blue in sequence, and finally dehydrating and sealing; as shown in figure B, replacement of the dermal layer by collagen fibers was alleviated after the human umbilical cord mesenchymal stem cell exosome stem cells were pretreated;
randomly selecting 3 visual fields from each Masson stained section under the visual field of multiplied by 100, calculating the percentage of extracellular matrix deposition area to the total tissue area by using Image J software, and taking an average value; as shown in panel E, human umbilical cord mesenchymal stem cell exosomes decreased the area of extracellular matrix deposition;
fixing mouse skin tissue with 4% paraformaldehyde, and then performing conventional dehydration, paraffin embedding and slicing; dropwise adding 3% H2O2Blocking endogenous peroxidase, and dropwise adding goat serum for sealing; then primary antibody (TGF-beta 1) is dripped, and the temperature is kept overnight at 4 ℃; after being washed by PBS, secondary antibody is dripped, and then diaminobenzidine is used for color development, hematoxylin counterstain and mounting;
each TGF- β immunohistochemically stained section was randomly selected 5 fields at x 400 field and the mean optical density value (IOD) was calculated using Image Pro Plus software;
as shown in panels C and F, the expression level of scleroderma critical pathway molecule TGF- β was corrected;
extracting total RNA of the tissue by using a TRIzol method, carrying out cDNA reverse transcription, and carrying out qPCR detection on the designed and synthesized primer; each sample was replicated three times, and the relative expression levels of the respective genes were calculated as 2(Ct reference gene-Ct target gene)Carrying out statistical analysis; as shown in panels G and H, markers of extracellular matrix, Col1 α 1 and Col3 α 1, were also similarly altered as above.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

Claims (6)

1. Application of human umbilical cord mesenchymal stem cell exosome in preparation of medicines for treating scleroderma.
2. The use according to claim 1, wherein the preparation method of the human umbilical cord mesenchymal stem cell exosome comprises the following steps:
s1, taking the frozen human umbilical cord mesenchymal stem cells, carrying out constant-temperature water bath at 35-40 ℃ for 1-5 min, centrifuging, adding a fresh culture medium, and carrying out conventional culture;
s2, taking 3 rd-5 th generation human umbilical cord mesenchymal stem cells, when the human umbilical cord mesenchymal stem cells are fused to 70% -80%, updating a fresh serum-free culture medium, and collecting supernatant after culturing for 24-72 h;
s3, high-speed centrifugation is carried out to obtain concentrated solution, and the concentrated solution is refrigerated at the temperature of-100 ℃ to-60 ℃ after PBS heavy suspension.
3. The use according to claim 1, wherein the amount of human umbilical cord mesenchymal stem cell exosome protein in the medicament for treating scleroderma is 2-6mg/kg body weight.
4. The use of claim 1, wherein the human umbilical cord mesenchymal stem cell exosomes thin the dermal layer.
5. The use according to claim 1, wherein the human umbilical cord mesenchymal stem cell exosomes reduce extracellular matrix deposition and inhibit epithelial-to-mesenchymal transition.
6. The use of claim 1, wherein the human umbilical cord mesenchymal stem cell exosomes inhibit M2 macrophage polarization, modulating M1/M2 macrophage balance.
CN202111159905.XA 2021-09-30 2021-09-30 Application of human umbilical cord mesenchymal stem cell exosome in preparation of medicine for treating scleroderma Pending CN114042087A (en)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
CN111565732A (en) * 2018-01-12 2020-08-21 国立大学法人大阪大学 Agent for promoting normal differentiation-maturation of stratified squamous epithelial cell, agent for treating epithelial disease, and method for promoting normal differentiation-maturation of stratified squamous epithelial cell
CN112538456A (en) * 2019-09-20 2021-03-23 北京干细胞与再生医学研究院 Pluripotent stem cells, pharmaceutical composition, preparation method and application thereof
CN112553149A (en) * 2019-09-25 2021-03-26 深圳光彩生命工程技术有限公司 Preparation method of exosome
CN112831464A (en) * 2019-11-23 2021-05-25 路春光 Industrial preparation method, quality research and application of human cell exosomes
JP2021122205A (en) * 2020-02-03 2021-08-30 株式会社フルステム Exosome recovery method

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111565732A (en) * 2018-01-12 2020-08-21 国立大学法人大阪大学 Agent for promoting normal differentiation-maturation of stratified squamous epithelial cell, agent for treating epithelial disease, and method for promoting normal differentiation-maturation of stratified squamous epithelial cell
CN112538456A (en) * 2019-09-20 2021-03-23 北京干细胞与再生医学研究院 Pluripotent stem cells, pharmaceutical composition, preparation method and application thereof
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JP2021122205A (en) * 2020-02-03 2021-08-30 株式会社フルステム Exosome recovery method

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MAN LI ET AL: "Mesenchymal Stem Cell-Derived Exosomes Ameliorate Dermal Fibrosis in a Murine Model of Bleomycin-Induced Scleroderma", pages 1 - 2, Retrieved from the Internet <URL:https://www.researchgate.net/publication/354121077_Mesenchymal_Stem_Cell-Derived_Exosomes_Ameliorate_Dermal_Fibrosis_in_a_Murine_Model_of_Bleomycin-Induced_Scleroderma> *
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