CN116115661A - Adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease and preparation method thereof - Google Patents
Adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease and preparation method thereof Download PDFInfo
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- 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
- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
- A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
- A61K36/258—Panax (ginseng)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- 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
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- 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
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- 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]
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- 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
- C12N2509/10—Mechanical dissociation
Abstract
The invention discloses a adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease and a preparation method thereof, wherein each 100ml of the preparation comprises the following components: adipose-derived mesenchymal stem cells (1-8). Times.10 8 20-50 mg of bone marrow mesenchymal stem cell exosome, 5-10 ml of pseudo-ginseng injection, 1-4 g of sodium hyaluronate, 0.2-0.5 g of vitamin C, 0.2-0.4 g of maltitol, 4-8 ml of compound amino acid injection and the balance of physiological saline; the adipose-derived mesenchymal stem cell preparation can improve the microenvironment of a lesion part, reduce inflammatory effusion, relieve pain and repair damaged tissues by being injected to the lesion position, and further treat joint degenerative disease.
Description
Technical Field
The invention relates to the technical field of biological preparations, in particular to a adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease and a preparation method thereof.
Background
Joint degenerative disease is also called hyperosteogeny, i.e. degenerative disease of bones, and is a manifestation of arthritis. The causative factor of this disease is mainly due to cartilage wear caused by imbalance in mechanical stress distribution or excessive loading. Joint degeneration is mainly caused by aging, occupational, genetic and physical factors, and comprises cervical degeneration, lumbar degeneration, knee degeneration and the like, wherein clinical manifestations mainly comprise: the neck has a strong feeling, limited movement, and rattle sounds when the neck moves, the pain is usually radiated to the shoulder and upper limbs, the hands and fingers have numbness and electric shock feeling, and the neck can be aggravated when the neck moves to a certain angle. Different lesions involve different parts and have different symptoms, and severe patients can press the cervical marrow to cause paralysis. The third and fourth waist are the most common. Clinically, pain, distending pain, stiffness and fatigue, and even bending limitation of lumbar and lumbar soft tissues often occur. If the adjacent nerve roots are pressed, corresponding symptoms can be caused, and local pain, stiff hair, rear root neuralgia, numbness and the like can occur. If the sciatica is pressed, the sciatica is caused, the pain of the affected limb is caused by radioactive tingling, burning, pain of the affected limb, and the affected limb is irradiated to the whole lower limb, so that intermittent claudication can occur when the vertebral canal is narrow. The knee joint pain of the patient suffering from the slow disease is not serious, the pain is sustained and hidden, the pain is aggravated when the temperature is reduced, the patient is related to climate change, the patient starts to move after the patient is in the morning and walks for a long time, the patient is in intense exercise or the patient starts to walk after sitting for a long time, the patient is improved after the patient is slightly moved, the patient is difficult to go upstairs and downstairs, and the knee joint is soft and easy to fall down when the patient goes downstairs. Pain and stiffness in squatting, pain and swelling pain in joints, lameness and limited joint functions in severe cases, the squatting is most obvious, the stretching and bending activities have bouncing sounds, part of patients can see joint effusion, obvious swelling and pain pressing phenomena are locally caused, and joint redness, swelling and deformity of rheumatics are combined.
At present, western medicine treatment, physical therapy and operation treatment are mainly adopted for the treatment of joint degenerative disease, however, western medicine treatment mainly aims at symptomatic medication, cannot radically treat the disease and is easy to relapse; the physical therapy has the advantages of durable efficacy and poor treatment effect; the operation treatment is mainly aimed at patients with serious illness, and can be considered when seriously affecting the life of the patients.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a adipose-derived mesenchymal stem cell preparation for treating joint degenerative diseases and a preparation method thereof, and the adipose-derived mesenchymal stem cell preparation can improve microenvironment and inflammatory effusion of a lesion part, relieve pain and repair damaged tissues by being injected to the lesion position, so as to treat the joint degenerative diseases.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
in a first aspect, the present invention provides a adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease, comprising the following components per 100ml of the preparation:
adipose-derived mesenchymal stem cells (1-8). Times.10 8 20-50 mg of bone marrow mesenchymal stem cell exosome, 5-10 ml of pseudo-ginseng injection, 1-4 g of sodium hyaluronate, 0.2-0.5 g of vitamin C, 0.2-0.4 g of maltitol, 4-8 ml of compound amino acid injection and the balance of physiological saline.
Preferably, the following components are included per 100ml of the formulation:
adipose-derived mesenchymal stem cells 5×10 8 30mg of mesenchymal stem cell exosome, 8ml of pseudo-ginseng injection, 3g of sodium hyaluronate, 0.3g of vitamin C, 0.3g of maltitol, 6ml of compound amino acid injection and the balance of physiological saline.
Preferably, the adipose tissue-derived mesenchymal stem cells are autologous adipose tissue-derived mesenchymal stem cells of generation 3 or generation 4.
Preferably, the bone marrow mesenchymal stem cell exosome is prepared by the following method:
selecting supernatant of the bone marrow mesenchymal stem cell culture, and extracting exosomes in the supernatant by adopting an ultracentrifugation method.
The second aspect of the invention provides a preparation method of the adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease, which comprises the following steps:
(a) Placing sodium hyaluronate, vitamin C and maltitol into normal saline, stirring, filtering and sterilizing to obtain a mixed solution;
(b) Placing adipose-derived mesenchymal stem cells and bone marrow mesenchymal stem cell exosomes in a mixed solution to obtain a suspension;
(c) And uniformly mixing the suspension, the pseudo-ginseng injection and the compound amino acid injection to obtain the adipose-derived mesenchymal stem cell preparation for treating the joint degenerative disease.
Preferably, the entire preparation process of the preparation method is carried out under aseptic conditions.
Preferably, in the step (a), the stirring speed is 50-100 r/min, and the stirring time is 5-10 min.
Preferably, in the step (b), the temperature of the mixed solution is 4 to 10 ℃.
Preferably, in the step (c), the mixing is carried out at a temperature of 4-10 ℃ and a speed of 50-100 r/min for 2-4 min.
Compared with the prior art, the invention has the beneficial effects that at least:
the adipose-derived mesenchymal stem cell preparation can improve the microenvironment of a lesion part, reduce inflammatory effusion, relieve pain and repair damaged tissues by being injected to the lesion position, and further treat joint degenerative disease.
Detailed Description
Embodiments of the technical scheme of the present invention will be described in detail below with reference to the embodiments. The following examples are only for more clearly illustrating the technical aspects of the present invention, and thus are merely examples, and are not intended to limit the scope of the present invention.
It is noted that unless otherwise indicated, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
Example 1
The embodiment is a adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease, wherein each 100ml of the preparation comprises the following components:
3 rd generation adipose mesenchymal stem cells 1×10 8 20mg of bone marrow mesenchymal stem cell exosome, 10ml of pseudo-ginseng injection, 1g of sodium hyaluronate, 0.5g of vitamin C, 0.2g of maltitol, 4ml of compound amino acid injection and the balance of physiological saline.
The preparation method of the adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease comprises the following steps:
(a) Placing sodium hyaluronate, vitamin C and maltitol into physiological saline, stirring for 10min at 50r/min, filtering, and sterilizing to obtain mixed solution;
(b) Placing adipose-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cell exosomes in the mixed solution at the temperature of 4 ℃ to obtain a suspension;
(c) Mixing the suspension, notoginseng radix injection and compound amino acid injection, and stirring at 4deg.C for 4min at 50r/min to obtain the final product.
Example 2
The embodiment is a adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease, wherein each 100ml of the preparation comprises the following components:
fourth generation adipose tissue-derived stem cells 8×10 8 50mg of bone marrow mesenchymal stem cell exosome, 5g of pseudo-ginseng injection, 4g of sodium hyaluronate, 0.2g of vitamin C, 0.4g of maltitol, 8ml of compound amino acid injection and the balance of physiological saline.
The preparation method of the adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease comprises the following steps:
(a) Placing sodium hyaluronate, vitamin C and maltitol into physiological saline, stirring for 5min at 100r/min, filtering, and sterilizing to obtain mixed solution;
(b) Placing adipose-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cell exosomes in the mixed solution at the temperature of 10 ℃ to obtain a suspension;
(c) Mixing the suspension, notoginseng radix injection and compound amino acid injection, and stirring at 10deg.C and 100r/min for 2min to obtain the final product.
Example 3
The embodiment is a adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease, wherein each 100ml of the preparation comprises the following components:
generation 3 adipose mesenchymal stem cells 5×10 8 30mg of mesenchymal stem cell exosome, 8ml of pseudo-ginseng injection, 3g of sodium hyaluronate, 0.3g of vitamin C, 0.3g of maltitol, 6ml of compound amino acid injection and the balance of physiological saline.
The preparation method of the adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease comprises the following steps:
(a) Placing sodium hyaluronate, vitamin C and maltitol into physiological saline, stirring for 10min at 50r/min, filtering, and sterilizing to obtain mixed solution;
(b) Placing adipose-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cell exosomes in the mixed solution at the temperature of 4 ℃ to obtain a suspension;
(c) Mixing the suspension, notoginseng radix injection and compound amino acid injection, and stirring at 4deg.C for 4min at 50r/min to obtain the final product.
Comparative example 1
The comparative example is a adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease, and each 100ml of the preparation comprises the following components:
3 rd generation fatMesenchymal stem cells 5×10 8 8ml of pseudo-ginseng injection, 3g of sodium hyaluronate, 0.3g of vitamin C, 0.3g of maltitol, 6ml of compound amino acid injection and the balance of physiological saline.
The preparation method of the adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease comprises the following steps:
(a) Placing sodium hyaluronate, vitamin C and maltitol into physiological saline, stirring for 10min at 50r/min, filtering, and sterilizing to obtain mixed solution;
(b) Placing adipose-derived mesenchymal stem cells into the mixed solution at the temperature of 4 ℃ to obtain a suspension;
(c) Mixing the suspension, notoginseng radix injection and compound amino acid injection, and stirring at 4deg.C for 4min at 50r/min to obtain the final product.
Comparative example 2
The comparative example is a adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease, and each 100ml of the preparation comprises the following components:
generation 3 adipose mesenchymal stem cells 5×10 8 30mg of bone marrow mesenchymal stem cell exosome, 3g of sodium hyaluronate, 0.3g of vitamin C, 0.3g of maltitol, 6ml of compound amino acid injection and the balance of physiological saline.
The preparation method of the adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease comprises the following steps:
(a) Placing sodium hyaluronate, vitamin C and maltitol into physiological saline, stirring for 10min at 50r/min, filtering, and sterilizing to obtain mixed solution;
(b) Placing adipose-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cell exosomes in the mixed solution at the temperature of 4 ℃ to obtain a suspension;
(c) Mixing the suspension and the compound amino acid injection, and stirring at 50r/min for 4min at 4deg.C to obtain the final product.
Experimental example
Rat knee joint cartilage injury model and repair experiment
Selecting a male rat, and manufacturing a cartilage defect model by drilling holes between the knee joint, the femur and the ankle through an operation, wherein the diameter of the drilled holes is about 1mm, and the depth of the drilled holes is about 1 mm;
the rats successfully molded are randomly divided into 4 groups, 5 rats are respectively injected into the groups 1 to 3, 30 mu l of the adipose-derived mesenchymal stem cell preparation for treating the joint degeneration of the example 3 and the comparative examples 1 to 2 are respectively injected into the groups 4, and 30 mu physiological saline is injected into the groups 4; once a week, 5 times after the injection, the experimental animals were sacrificed, and the cartilage surface regeneration condition of the rats was scored according to the Wakitani scoring standard, and the scoring results are shown in table 1;
TABLE 1
As can be seen from table 1:
the adipose-derived mesenchymal stem cell preparation for treating the joint degenerative disease, which is prepared by the embodiment of the application and the comparative example, can better repair the joint injury of the rat, and compared with the comparative example, the adipose-derived mesenchymal stem cell preparation for treating the joint degenerative disease, which is prepared by the embodiment of the application, has obviously improved repair effect.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention, and are intended to be included within the scope of the appended claims and description.
Claims (9)
1. A adipose-derived mesenchymal stem cell preparation for treating joint degenerative diseases, characterized in that the preparation comprises the following components per 100 ml:
adipose-derived mesenchymal stem cells (1-8). Times.10 8 20-50 mg of bone marrow mesenchymal stem cell exosome, 5-10 ml of pseudo-ginseng injection, 1-4 g of sodium hyaluronate, 0.2-0.5 g of vitamin C, 0.2-0.4 g of maltitol, 4-8 ml of compound amino acid injection and the balance of physiological saline.
2. The adipose mesenchymal stem cell preparation for treating joint degeneration according to claim 1, wherein the following components are included per 100ml of the preparation:
adipose-derived mesenchymal stem cells 5×10 8 30mg of mesenchymal stem cell exosome, 8ml of pseudo-ginseng injection, 3g of sodium hyaluronate, 0.3g of vitamin C, 0.3g of maltitol, 6ml of compound amino acid injection and the balance of physiological saline.
3. The adipose mesenchymal stem cell preparation for treating joint degenerative diseases according to claim 1 or 2, wherein the adipose mesenchymal stem cells are autologous adipose mesenchymal stem cells of 3 rd generation or 4 th generation.
4. The adipose mesenchymal stem cell preparation for treating joint degenerative diseases according to claim 1 or 2, wherein the bone marrow mesenchymal stem cell exosome is prepared by the following method:
selecting supernatant of the bone marrow mesenchymal stem cell culture, and extracting exosomes in the supernatant by adopting an ultracentrifugation method.
5. The method for preparing the adipose-derived mesenchymal stem cell preparation for treating joint degenerative disease according to any one of claims 1 to 4, comprising the following steps:
(a) Placing sodium hyaluronate, vitamin C and maltitol into normal saline, stirring, filtering and sterilizing to obtain a mixed solution;
(b) Placing adipose-derived mesenchymal stem cells and bone marrow mesenchymal stem cell exosomes in a mixed solution to obtain a suspension;
(c) And uniformly mixing the suspension, the pseudo-ginseng injection and the compound amino acid injection to obtain the adipose-derived mesenchymal stem cell preparation for treating the joint degenerative disease.
6. The preparation method according to claim 5, wherein the whole preparation process of the preparation method is performed under aseptic conditions.
7. The method according to claim 5, wherein in the step (a), the stirring speed is 50 to 100r/min and the stirring time is 5 to 10min.
8. The method according to claim 5, wherein in the step (b), the temperature of the mixed solution is 4 to 10 ℃.
9. The method according to claim 5, wherein in the step (c), the mixing is carried out at a temperature of 4 to 10 ℃ for 2 to 4 minutes at a speed of 50 to 100 r/min.
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