CN112294844A - Medicine for treating osteoarthritis by combining pluripotent cell active matter and platelet-rich plasma, preparation method and application - Google Patents
Medicine for treating osteoarthritis by combining pluripotent cell active matter and platelet-rich plasma, preparation method and application Download PDFInfo
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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
- A61K35/14—Blood; Artificial blood
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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
- A61K35/14—Blood; Artificial blood
- A61K35/16—Blood plasma; Blood serum
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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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
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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Abstract
The invention relates to the technical field of stem cell preparations, in particular to a preparation method of a medicine for treating osteoarthritis by combining a peripheral blood multipotential cell active substance and platelet-rich plasma, and a preparation method and application thereof. Obtaining peripheral blood multipotential cell active matter freeze-dried powder, adding platelet-rich plasma to obtain peripheral blood multipotential cell active matter suspension, and then dropwise adding NSADs into the suspension while uniformly mixing. The invention can prevent the release of inflammatory mediators and reduce inflammatory reaction by adopting the combination of peripheral blood multipotential cell active substances and platelet-rich plasma and NSADs, thereby reducing tissue damage; in addition, the pain of the patient is relieved, the treatment effect of the rheumatoid arthritis is improved, the treatment time is shortened, and the rheumatoid arthritis is not easy to relapse.
Description
Technical Field
The invention relates to the technical field of stem cell preparations, in particular to a preparation method of a medicine for treating osteoarthritis by combining a peripheral blood pluripotent cell active substance and platelet-rich plasma, and a preparation method and application thereof.
Background
Rheumatoid arthritis is a chronic inflammatory immune disease, vasculitis and synovitis are taken as basic pathological characteristics, and synovial membrane proliferation and neoplastic, articular cartilage and subchondral bone erosion are taken as main pathological changes along with the development of the disease, so that ankylosing deformity and function loss are finally caused; autoantigen-mediated hypersensitivity as well as non-antigen specific inflammatory reactions are currently thought to be the major cause of destructive pathology.
For the treatment of RA, non-steroidal anti-inflammatory drugs and antirheumatic drugs are mainly used conventionally, but the relapse rate of the disease is high, the side effect is obvious, and the progress of the disease cannot be changed. Joint replacement is more serious and has serious side effects, and the artificial joint has a limited life span, which results in the failure to repair damaged articular cartilage and the failure to control the further development of the diseases.
Disclosure of Invention
Aiming at the technical problems, the invention provides a medicament prepared by combining peripheral blood multipotential cell active matter and platelet-rich plasma for treating osteoarthritis, a preparation method and application thereof, and solves the defects of poor treatment efficiency of rheumatoid arthritis and the like in the prior art.
In order to solve the technical problems, the specific technical scheme is as follows:
a medicine for treating osteoarthritis is prepared from the peripheral blood multipotential cell active matter freeze-dried powder, platelet rich plasma and NSADs.
The content of the peripheral blood multipotential cell active matter freeze-dried powder in the medicine is 10-15 ng/g; the mass fraction content of human serum albumin in the platelet-rich plasma in the medicine is 0.5-2%; the content concentration of the NSADs in the medicine is 25-40 mg/ml.
The preparation method of the medicine for treating osteoarthritis comprises the following steps: obtaining peripheral blood multipotential cell active matter freeze-dried powder, adding platelet-rich plasma to obtain peripheral blood multipotential cell active matter suspension, and then dropwise adding NSADs into the suspension while uniformly mixing.
The preparation method of the peripheral blood pluripotent cell active substance freeze-dried powder comprises the following steps:
(1) detecting exogenous factors; adopting a diagnostic kit to detect the exogenous factors of the absorbed peripheral blood supernatant so as to ensure that the peripheral blood does not contain the exogenous factors;
(2) primary separation, culture and passage of peripheral blood stem cells; the specific processes of primary separation, culture and passage of the peripheral blood stem cells comprise:
firstly, diluting peripheral blood, blowing off, centrifuging, carefully extracting a bottom precipitate, and then digesting the extracted bottom precipitate by using a digestive juice, wherein the digestive juice is trypsin with the concentration of 0.1-0.3% or collagenase with the concentration of 0.5-2%;
centrifuging again, placing the aspirated cells into a disposable culture dish, adding a proper amount of mesenchymal stem cell serum-free culture medium, placing in an environment of 37 ℃, and filling with 5% CO2Culturing for 10-15 days in the incubator, when the cell density is not less than 80%, cleaning the cell surface twice by using normal saline, then adding pancreatin for digestion, centrifuging and inoculating, and continuing subculturing;
(3) identifying the mesenchymal stem cells;
(4) collecting the supernatant; the supernatant is P3-P5 generation cell supernatant, when the cell grows to reach 70-80%, the supernatant is collected, then the original volume is supplemented, the culture is carried out, and the supernatant is collected again when the cell grows to reach 70-80%;
(5) preparing freeze-dried powder; the preparation process of the freeze-dried powder comprises the following steps:
firstly, adding a freeze-dried powder protective agent into the collected supernatant, filtering and sterilizing through a 0.22-micron filter membrane, subpackaging the filtrate into penicillin bottles, half plugging, placing into a freeze dryer, freeze-drying the filtrate according to a freeze-drying program, and taking out after plugging; the freeze-drying protective agent is one or a mixture of mannitol, dextran and sucrose.
The freeze drying procedure is as follows: precooling at-50 ℃, then vacuumizing, and keeping at-50 ℃ for 6 h; then heating to-35 ℃ and keeping for 20 h; gradually heating to-20 deg.C, and maintaining for 4 hr; then heating to 0-10 ℃ and keeping for 4-6 h; then heating to 10-25 ℃ and keeping for 4 h; finally, drying for 6h at 25 ℃.
(6) Detecting stem cell active matters of the freeze-dried powder; when the stem cell active material is detected, 2ml of supernatant freeze-dried powder is diluted by 4ml of solvent, and then the kit is used for determining the content of the stem cell active factor. The solvent comprises glycerol and hyaluronic acid.
The medicine for treating osteoarthritis is applied to treating rheumatoid arthritis.
The invention provides a medicine for treating osteoarthritis prepared by combining a peripheral blood multipotential cell active substance and platelet-rich plasma, a preparation method and application thereof, wherein the peripheral blood multipotential cell active substance is combined with the platelet-rich plasma and NSADs, so that the release of inflammatory mediators can be prevented, the inflammatory reaction is reduced, and the tissue injury is reduced; in addition, the pain of the patient is relieved, the treatment effect of the rheumatoid arthritis is improved, the treatment time is shortened, and the rheumatoid arthritis is not easy to relapse.
Detailed Description
In order to make those skilled in the art better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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.
The invention provides a medicine prepared by combining a peripheral blood multipotential cell active matter and platelet rich plasma and used for treating osteoarthritis.
There are a small number of multipotent mesenchymal stem cells in peripheral blood. They have biological characteristics similar to bone marrow mesenchymal stem cells, can self-proliferate and can differentiate in multiple directions. Can be induced in vitro and differentiated in vivo to form mesodermal and other germ layer tissues. The ecto-multipotential mesenchymal stem cells can be differentiated into cells such as osteogenesis, cartilage, fat, tendon, muscle and the like in vitro and can participate in the regeneration and repair of damaged tissues; the joint lesion can promote the accumulation of mesenchymal stem cells to the lesion synovium and promote the differentiation of the mesenchymal stem cells into chondrocytes and the like, thereby repairing the damaged joint tissues. In addition, the multipotential mesenchymal stem cells can secrete various cell factors, have a nutritional effect and can promote the proliferation and differentiation of the stem cells; the multipotential mesenchymal stem cells have unique immunoregulation function, and autologous or allogeneic mesenchymal stem cells are added into a mitogen-stimulated peripheral blood lymphocyte culture system, can obviously inhibit the proliferation and activation of T lymphocytes, play an important role in regulating an immune network, and can fundamentally regulate a patient's pathologic immune system. In addition, the multipotent mesenchymal stem cells differentiate into other types of cells, and the immunoregulation function of the multipotent mesenchymal stem cells is also continuously maintained; in addition, the multipotential mesenchymal stem cells have chemotactic effect, can prevent the release of inflammatory mediators and relieve inflammatory reaction, thereby relieving tissue injury and being beneficial to the progress of tissue disease.
NSADs are used in combination with human serum albumin and peripheral blood multipotential cells for alleviating inflammatory reactions and regulating immune reactions to improve treatment efficiency; in addition, the human blood albumin in the platelet-rich plasma can provide certain nutritional support for the multipotential mesenchymal stem cells simultaneously, so that the multipotential mesenchymal stem cells can keep the cell activity thereof, the survival time of the multipotential mesenchymal stem cells is prolonged, the biocompatibility of the medicine is improved, and the immune exclusion reaction is reduced.
Wherein the content of the peripheral blood multipotential cell active matter freeze-dried powder in the medicine is 10-15 ng/g; the mass fraction content of human serum albumin in the platelet-rich plasma in the medicine is 0.5-2%; the content concentration of the NSADs in the medicine is 25-40 mg/ml.
Obtaining peripheral blood multipotential cell active matter freeze-dried powder, adding platelet-rich plasma to obtain peripheral blood multipotential cell active matter suspension, and then dropwise adding NSADs into the suspension while uniformly mixing.
The preparation method of the peripheral blood pluripotent cell active substance freeze-dried powder comprises the following steps:
(1) detecting exogenous factors; adopting a diagnostic kit to detect the exogenous factors of the absorbed peripheral blood supernatant so as to ensure that the peripheral blood does not contain the exogenous factors;
(2) primary separation, culture and passage of peripheral blood stem cells; the specific processes of primary separation, culture and passage of the peripheral blood stem cells comprise:
firstly, diluting peripheral blood, blowing off, centrifuging, carefully extracting a bottom precipitate, and then digesting the extracted bottom precipitate by using a digestive juice, wherein the digestive juice is trypsin with the concentration of 0.1-0.3% or collagenase with the concentration of 0.5-2%;
centrifuging again, placing the aspirated cells into a disposable culture dish, adding a proper amount of mesenchymal stem cell serum-free culture medium, placing in an environment of 37 ℃, and filling with 5% CO2Culturing for 10-15 days in the incubator, when the cell density is not less than 80%, cleaning the cell surface twice by using normal saline, then adding pancreatin for digestion, centrifuging and inoculating, and continuing subculturing;
(3) identifying the mesenchymal stem cells;
(4) collecting the supernatant; the supernatant is P3-P5 generation cell supernatant, when the cell grows to reach 70-80%, the supernatant is collected, then the original volume is supplemented, the culture is carried out, and the supernatant is collected again when the cell grows to reach 70-80%;
(5) preparing freeze-dried powder; firstly, adding a freeze-dried powder protective agent into the collected supernatant, filtering and sterilizing through a 0.22-micron filter membrane, subpackaging the filtrate into penicillin bottles, half plugging, placing into a freeze dryer, freeze-drying the filtrate according to a freeze-drying program, and taking out after plugging; the freeze-drying protective agent is one or a mixture of mannitol, dextran and sucrose.
The freeze-drying procedure was: precooling at-50 ℃, then vacuumizing, and keeping at-50 ℃ for 6 h; then heating to-35 ℃ and keeping for 20 h; gradually heating to-20 deg.C, and maintaining for 4 hr; then heating to 0-10 ℃ and keeping for 4-6 h; then heating to 10-25 ℃ and keeping for 4 h; finally, drying for 6h at 25 ℃.
(6) Detecting stem cell active matters of the freeze-dried powder; when the stem cell active material is detected, 2ml of supernatant freeze-dried powder is diluted by 4ml of solvent, and then the kit is used for determining the content of the stem cell active factor. The solvent comprises glycerol and hyaluronic acid.
In order to verify the beneficial effects of the medicament for treating osteoarthritis provided by the invention, the following experiments are set up:
30 patients were selected according to the diagnostic criteria revised by the american college of rheumatology (ARA) in 1987, specifically:
(1) morning stiffness of at least 1 hour (more than or equal to 6 weeks); (2) swelling of the metacarpophalangeal joints or proximal interphalangeal joints (more than or equal to 6 weeks); (3)3 or more than 3 arthrodema (more than or equal to 6 weeks); (4) symmetric arthrocele (more than or equal to 6 weeks); (5) subcutaneous rheumatoid nodules; (6) a hand line change; (7) rheumatoid factor positive;
the patient with rheumatoid arthritis can be diagnosed by the four items.
According to three groups of random bits:
(1) experimental groups: 30ml of the medicine provided by the invention is infused intravenously for 10 patients, and meanwhile, the medicine is diluted by normal saline according to the proportion of 1: 4 and is injected in a punctate way along the spinal erector spinae muscles and sacroiliac joint areas on two sides of the spinal column;
(2) first control group: 10 patients, mesenchymal stem cell preparation infused only.
(3) Second control group: 10 patients, only the NSADs preparation alone was infused.
The disease activity score criteria for the joints were: checking 28 joints in total of proximal interphalangeal joints, metacarpophalangeal joints, wrist joints, elbow joints, shoulder joints and knee joints at two sides; the joint tenderness number (T28) and swollen joint number (SW28) are obtained and are calculated by combining the Erythrocyte Sedimentation Rate (ESR) according to the following formula:
DAS28＝[0.56×T28+0.28×SW28+0.70×Ln(ESR)]×1.08+0.16；
DAS28 is less than or equal to 2.6 points to indicate the disease is in remission;
the disease condition is indicated to be in low activity degree within 2.6-3.2 minutes;
3.2-5.1 points out the disease condition as middle activity;
a score of > 5.1 indicates a high activity.
TABLE 1 average DAS28 score of each group of patients before treatment
| Group of | Experimental group | First control group | Second control group |
| Pre-treatment scoring | 5.3±0.37 | 5.1±0.38 | 5.4±0.43 |
The following data are follow-up records for patients within 4-14 months of treatment, as shown in table 2;
TABLE 2 mean score of patients in each group
The results show that the patients in the experimental group are in remission stage when the DAS28 is less than or equal to 2.6 points in the 10 th month of treatment; and the patients of the first control group and the second control group are not relieved at the 14 th month of treatment, so that the stem cell preparation provided by the invention shortens the treatment period of rheumatoid arthritis and improves the treatment efficiency.
The above description is only intended to illustrate one embodiment of the present invention, and the description is more specific and detailed, but not to be construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (8)
1. A medicine prepared by combining peripheral blood multipotential cell active matter and platelet rich plasma for treating osteoarthritis is characterized by comprising peripheral blood multipotential cell active matter freeze-dried powder, platelet rich plasma and NSADs.
2. The preparation method of the drug for treating osteoarthritis by combining the peripheral blood multipotential cell active substance and the platelet rich plasma according to claim 1, wherein the content of the peripheral blood multipotential cell active substance freeze-dried powder in the drug is 10-15 ng/g; the mass fraction content of human serum albumin in the platelet-rich plasma in the medicine is 0.5-2%; the content concentration of the NSADs in the medicine is 25-40 mg/ml.
3. A method according to claim 1 or 2 for the preparation of a medicament for the treatment of osteoarthritis from a combination of peripheral blood multipotential cell active substance and platelet rich plasma, comprising the steps of: obtaining peripheral blood multipotential cell active matter freeze-dried powder, adding platelet-rich plasma to obtain peripheral blood multipotential cell active matter suspension, and then dropwise adding NSADs into the suspension while uniformly mixing.
4. The method for preparing a medicament for treating osteoarthritis by using the combination of peripheral blood multipotential cell active substance and platelet rich plasma according to claim 3, wherein the method for preparing the peripheral blood multipotential cell active substance freeze-dried powder comprises the following steps:
(1) detecting exogenous factors; adopting a diagnostic kit to detect the exogenous factors of the absorbed peripheral blood supernatant so as to ensure that the peripheral blood does not contain the exogenous factors;
(2) primary separation, culture and passage of peripheral blood stem cells; the specific processes of primary separation, culture and passage of the peripheral blood stem cells comprise:
firstly, diluting peripheral blood, blowing off, centrifuging, carefully extracting a bottom precipitate, and then digesting the extracted bottom precipitate by using a digestive juice, wherein the digestive juice is trypsin with the concentration of 0.1-0.3% or collagenase with the concentration of 0.5-2%;
centrifuging again, placing the aspirated cells into a disposable culture dish, adding a proper amount of mesenchymal stem cell serum-free culture medium, placing in an environment of 37 ℃, and filling with 5% CO2Culturing for 10-15 days in the incubator, when the cell density is not less than 80%, cleaning the cell surface twice by using normal saline, then adding pancreatin for digestion, centrifuging and inoculating, and continuing subculturing;
(3) identifying the mesenchymal stem cells;
(4) collecting the supernatant; the supernatant is P3-P5 generation cell supernatant, when the cell grows to reach 70-80%, the supernatant is collected, then the original volume is supplemented, the culture is carried out, and the supernatant is collected again when the cell grows to reach 70-80%;
(5) preparing freeze-dried powder;
(6) detecting stem cell active matters of the freeze-dried powder; when the stem cell active material is detected, 2ml of supernatant freeze-dried powder is diluted by 4ml of solvent, and then the kit is used for determining the content of the stem cell active factor.
5. The method for preparing a medicament for treating osteoarthritis by using peripheral blood multipotential cell active substances and platelet rich plasma according to claim 4, wherein the process for preparing the lyophilized powder in the step (5) comprises the following steps:
firstly, adding a freeze-dried powder protective agent into the collected supernatant, filtering and sterilizing through a 0.22-micron filter membrane, subpackaging the filtrate into penicillin bottles, half plugging, placing into a freeze dryer, freeze-drying the filtrate according to a freeze-drying program, and taking out after plugging; the freeze-drying protective agent is one or a mixture of mannitol, dextran and sucrose.
6. The method of claim 5, wherein the lyophilization process comprises: precooling at-50 ℃, then vacuumizing, and keeping at-50 ℃ for 6 h; then heating to-35 ℃ and keeping for 20 h; gradually heating to-20 deg.C, and maintaining for 4 hr; then heating to 0-10 ℃ and keeping for 4-6 h; then heating to 10-25 ℃ and keeping for 4 h; finally, drying for 6h at 25 ℃.
7. The method for preparing a medicament for treating osteoarthritis according to claim 4, wherein the vehicle in step (6) comprises glycerol and hyaluronic acid.
8. Use of a peripheral blood multipotential cell active substance in combination with platelet rich plasma according to claim 1 or 2 for the preparation of a medicament for the treatment of osteoarthritis, in a medicament for the treatment of rheumatoid arthritis.
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