CN112870229A - Mesenchymal stem cell preparation for treating knee osteoarthritis and research method thereof - Google Patents
Mesenchymal stem cell preparation for treating knee osteoarthritis and research method thereof 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
- A61K35/28—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
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
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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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- 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
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
Abstract
The invention discloses a mesenchymal stem cell preparation for treating knee osteoarthritis and a research method thereof. The invention has the beneficial effects that: the activity of prepared cells is ensured, the survival number of the cells is ensured to be used for experimental research, partial cells are prevented from remaining in a culture bottle, the accuracy of cell counting is improved, the fact that the pain of patients suffering from the knee osteoarthritis can be obviously improved by MSCs treatment is confirmed, the effect is superior to that of a traditional hyaluronic acid treatment method, the optimal treatment frequency can be selected according to experimental data, and scientific basis is provided for treating the knee osteoarthritis by the human umbilical cord mesenchymal stem cells in the future.
Description
Technical Field
The invention relates to a research method of a mesenchymal stem cell preparation, in particular to a mesenchymal stem cell preparation for treating knee osteoarthritis and a research method thereof, and belongs to the technical field of chemical medical treatment.
Background
The mesenchymal stem cells from umbilical cord have the advantages of easy expansion and differentiation of mesenchymal stem cells from other sources, anti-inflammation and recruitment functions, and the like, and also has the characteristics of cell rejuvenation, large acquisition amount, no ethical problem, no pain in the material taking process, high proliferation property, multipotential differentiation property, low immunogenicity, no tumorigenicity and the like, and is the most commonly used seed cell for clinical cell therapy at present.
Mesenchymal stem cells have the advantages of wide tissue source, easy amplification and differentiation, tissue specificity of differentiated cells, anti-inflammation and recruitment, low tumor formation risk, low immunogenicity and the like, but the number of the current clinical random control tests is limited, and further clinical research is needed to confirm the curative effect of the cells.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide a mesenchymal stem cell preparation for treating knee osteoarthritis and a method for studying the same.
The invention realizes the purpose through the following technical scheme: a mesenchymal stem cell preparation for treating knee osteoarthritis and a research method thereof comprise the following steps:
step A: preparing platelet lysate, taking 300ml of venous blood of a patient, centrifuging to obtain the platelet lysate, and storing at the low temperature of-80 ℃ for later use;
and B: separating and culturing human umbilical cord MSCs, and using umbilical cord in super clean benchWashing with sterile physiological saline, and cutting into pieces smaller than 1mm3Placing the crushed tissue blocks into a centrifuge tube, paving the tissue blocks at the bottoms of 10T 75 culture bottles, adding a serum-free culture medium, placing the culture medium in a constant-temperature incubator at 37 ℃ and with 5% carbon dioxide saturation humidity, changing the culture medium once after 6 days, observing whether cells climb out from the periphery of the tissue blocks under a mirror, and changing the culture medium once every 3 days later until the visible cells climb out and the density reaches 70-80%, wherein the subculture operation can be considered;
and C: human umbilical cord MSCs are transplanted, subjects are randomly divided into 10 cases of treatment groups and control groups, the two groups have no significant difference in sex constitution, age, disease course and the like, and the treatment groups are injected with 2 x 10 medicines every month7cells/4mL, continuously injecting for 3 months, and performing sodium hyaluronate 25mg knee joint cavity injection treatment on a control group, wherein joint fluid is obtained;
step D: and evaluating the curative effect, and performing WOMAC total score and knee joint lequesne index score.
As a still further scheme of the invention: in the step B, when the growth is observed under a cell mirror and the density reaches 70-80%, passage operation is started, the side wall of the culture bottle is lightly tapped to enable the tissue block to completely fall off, the culture supernatant containing the tissue block is discarded, the bottom of the culture bottle is washed by PBS (phosphate buffer solution) for 2 times, 1mL of preheated 0.05% pancreatin is added, after the cells under a microscope become round, digestion is stopped by old culture solution, the cells are lightly tapped, cell suspension is sucked into a centrifuge tube, the centrifuge tube is centrifuged at 1500rpm/min and 4 ℃ for 5min, the cells are collected and counted, and the number is 5 multiplied by 103The seeding density was passaged at/cm 2.
As a still further scheme of the invention: and in the step C, 4ml of platelet lysate which is activated after unfreezing is injected into the joint cavity 1 week before transplantation every time, cells which have the survival rate of not less than 90% and are free from EBV, HBV, HCV and HIV pollution are selected for resuscitation before transplantation, and the cells are uniformly mixed and packaged after trypsin collection and activated platelet lysate heavy suspension treatment are performed before transplantation.
As a still further scheme of the invention: and in the step C, when the knee joint is injected, disinfecting and paving a hole towel conventionally, holding a 5ml sterile injector to enter a joint cavity, then drawing out a proper amount of joint fluid, keeping a needle head, drawing out a needle cylinder, connecting the needle cylinder with uniformly mixed MSCs, slowly injecting 4ml, pressing and covering a needle eye with sterile gauze after the operation, lying on the back for 20min, discontinuously bending and stretching the knee joint for dozens of times during the period, and injecting 4ml of platelet lysate activated after unfreezing again after the MSCs are transplanted for 1 night.
As a still further scheme of the invention: in the step D, data analysis is carried out by using SPSS17.0 statistical software, VAS24 WOMAC scale scores of 10mm are completed under the guidance of doctors before and after treatment, but the scores can not be substituted for patients to be scored, each score is recorded in detail, and knee joint Lequesne index scores are respectively carried out before and after treatment, so that the method is used for judging the severity of knee joint osteoarthritis, disease activity and knee joint function.
The invention has the beneficial effects that: the mesenchymal stem cell preparation for treating knee osteoarthritis and the research method thereof are reasonably designed, and in the step B, passage operation is started when the growth is observed under a cell mirror and the density reaches 70-80%. Gently patting the side wall of the culture flask to completely remove the tissue block, discarding the culture supernatant containing the tissue block, washing the bottom of the culture flask with PBS, washing with PBS for 2 times, adding 1mL of preheated 0.05% pancreatin, stopping digestion with old culture solution after cell rounding under microscope, gently blowing, sucking cell suspension into a centrifuge tube, centrifuging at 4 ℃ for 5min at 1500rpm/min, collecting cells, counting, and centrifuging at 5 × 103The inoculation density of/cm 2 is used for passage, the activity of prepared cells is ensured, the survival number of the cells can be used for experimental research, partial cells are prevented from remaining in a culture bottle, the accuracy of cell counting is improved, the foundation is laid for subsequent experiments, in the step C, 4ml of platelet lysate which is activated after unfreezing is injected into a joint cavity 1 week before transplantation, the survival rate is not lower than 90% before transplantation, cells without EBV, HBV, HCV and HIV pollution are recovered, trypsin collection is performed before use, the activated platelet lysate is subjected to heavy suspension treatment and then is uniformly mixed and subpackaged, the subpackaged activated platelet lysate is conveniently distinguished, the activity of the transplanted cells is ensured, the accuracy of experimental research data is improved, in the step C, when knee joint injection is performed, a proper amount of joint fluid is extracted after a 5ml sterile syringe enters the joint cavity, retaining needleThe head is pulled out of the syringe, the syringe which is provided with uniformly mixed MSCs is connected, 4ml is slowly injected, sterile gauze is pressed to cover a needle eye after operation, the patient lies on the back for 20min, the knee joint is intermittently bent and stretched for dozens of times during the period, 4ml of platelet lysate which is activated after unfreezing is injected again after the MSCs are transplanted for 1 evening, impurity interference in the whole processes of extracting the joint fluid and injecting the platelet lysate is reduced, sterile operation is ensured, accidental errors are avoided, in the step D, data analysis is carried out by SPSS17.0 statistical software, VAS24 WOMAC scale scoring of 10mm is completed under the guidance of doctors before and after treatment, scoring of patients is not required, each score is recorded in detail, knee joint Lequesne index scoring is respectively carried out before and after treatment, the mesenchymal stem cell preparation is used for judging the severity of knee joint osteoarthritis, disease activity and knee joint function, and is convenient to judge whether the mesenchymal stem cell preparation is effective for treating knee joint osteoarthritis according to the, making the entire data more convincing.
Drawings
FIG. 1 is a schematic view of the structure of the present invention.
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.
Referring to fig. 1, a mesenchymal stem cell preparation for treating knee osteoarthritis and a research method thereof include the following steps:
step A: preparing platelet lysate, taking 300ml of venous blood of a patient, centrifuging to obtain the platelet lysate, and storing at the low temperature of-80 ℃ for later use;
and B: separating and culturing human umbilical cord MSCs, washing umbilical cord with sterile normal saline in super clean bench, and cutting into pieces smaller than 1mm3Placing the crushed tissue blocks into a centrifuge tube, spreading the tissue blocks at the bottom of 10T 75 culture bottles, adding serum-free culture medium, placing at 37 deg.C and 5 deg.CIn a constant-temperature incubator with% carbon dioxide saturation humidity, after 6 days, the whole culture solution is changed once, whether cells climb out from the periphery of the tissue block is observed under a microscope, and then the solution is changed once every 3 days until the visible cells climb out and the density reaches 70-80%, and the subculture operation can be considered;
and C: human umbilical cord MSCs are transplanted, subjects are randomly divided into 10 cases of treatment groups and control groups, the two groups have no significant difference in sex constitution, age, disease course and the like, and the treatment groups are injected with 2 x 10 medicines every month7cells/4mL, continuously injecting for 3 months, and performing sodium hyaluronate 25mg knee joint cavity injection treatment on a control group, wherein joint fluid is obtained;
step D: and evaluating the curative effect, and performing WOMAC total score and knee joint lequesne index score.
Further, in the embodiment of the present invention, in the step B, when the growth is observed under the cytoscope and the density reaches 70-80%, the passage operation is started, the side wall of the culture flask is lightly tapped to completely drop the tissue block, the culture supernatant containing the tissue block is discarded, the bottom of the culture flask is washed with PBS, PBS is washed for 2 times, 1mL of preheated 0.05% pancreatin is added, after the cells under the microscope become round, digestion is stopped with old culture solution, the cell suspension is gently tapped, sucked into a centrifuge tube, 1500rpm/min and centrifuged at 4 ℃ for 5min, the cells are collected and counted, and the number is 5 × 103The inoculation density of/cm 2 is used for passage, the activity of the prepared cells is ensured, the survival number of the cells can be used for experimental study, partial cells are prevented from remaining in a culture bottle, the accuracy of cell counting is improved, and a foundation is laid for subsequent experiments.
Further, in the embodiment of the present invention, in step C, 4ml of platelet lysate activated after thawing is injected into the joint cavity 1 week before transplantation every time, cells with a survival rate not lower than 90% and without EBV, HBV, HCV, and HIV contamination are selected before transplantation, are resuscitated, trypsin is collected before use, and the activated platelet lysate is subjected to heavy suspension treatment, then are uniformly mixed and packaged, so that the packaged activated platelet lysate can be conveniently distinguished, the activity of the transplanted cells is ensured, and the accuracy of experimental research data is improved.
Further, in the embodiment of the invention, in the step C, when injecting the knee joint, conventionally sterilizing and paving a hole towel, holding a 5ml sterile injector to enter the joint cavity, then drawing out a proper amount of joint fluid, retaining the needle head, pulling out the needle cylinder, connecting the needle cylinder with uniformly mixed MSCs, slowly injecting 4ml, covering the needle hole with sterile gauze by pressing after the operation is finished, lying on the back for 20min, intermittently extending the knee joint for tens of times, injecting 4ml of platelet lysate activated after thawing again after transplanting the MSCs for 1 night, reducing impurity interference in the whole processes of drawing the joint fluid and injecting the platelet lysate, ensuring sterile operation, and avoiding accidental errors.
Further, in the embodiment of the present invention, in the step D, data analysis is performed by using SPSS17.0 statistical software, scoring of VAS24 WOMAC scales of 10mm is completed under the guidance of a doctor before and after treatment, but scoring of a patient cannot be replaced, each score is recorded in detail, knee joint Lequesne index scoring is performed before and after treatment, and the method is used for determining the severity of knee joint osteoarthritis, disease activity and knee joint function, and is convenient for determining whether the mesenchymal stem cell preparation is effective for treating knee osteoarthritis according to comparison of the scores before and after treatment, so that the whole data is more convincing.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (5)
1. A mesenchymal stem cell preparation for treating knee osteoarthritis and a research method thereof are characterized in that: the method comprises the following steps:
step A: preparing platelet lysate, taking 300ml of venous blood of a patient, centrifuging to obtain the platelet lysate, and storing at the low temperature of-80 ℃ for later use;
and B: separating and culturing human umbilical cord MSCs, washing umbilical cord with sterile normal saline in super clean bench, and cutting into pieces smaller than 1mm3Placing the crushed tissue blocks into a centrifuge tube, paving the tissue blocks at the bottoms of 10T 75 culture bottles, adding a serum-free culture medium, placing the culture medium in a constant-temperature incubator at 37 ℃ and with 5% carbon dioxide saturation humidity, changing the culture medium once after 6 days, observing whether cells climb out from the periphery of the tissue blocks under a mirror, and changing the culture medium once every 3 days later until the visible cells climb out and the density reaches 70-80%, wherein the subculture operation can be considered;
and C: human umbilical cord MSCs are transplanted, subjects are randomly divided into 10 cases of treatment groups and control groups, the two groups have no significant difference in sex constitution, age, disease course and the like, and the treatment groups are injected with 2 x 10 medicines every month7cells/4mL, continuously injecting for 3 months, and performing sodium hyaluronate 25mg knee joint cavity injection treatment on a control group, wherein joint fluid is obtained;
step D: and evaluating the curative effect, and performing WOMAC total score and knee joint lequesne index score.
2. The preparation of mesenchymal stem cells for treating knee osteoarthritis and the research method thereof according to claim 1, wherein the mesenchymal stem cells are selected from the group consisting of: in the step B, when the growth is observed under a cell mirror and the density reaches 70-80%, passage operation is started, the side wall of the culture bottle is lightly tapped to enable the tissue block to completely fall off, the culture supernatant containing the tissue block is discarded, the bottom of the culture bottle is washed by PBS (phosphate buffer solution) for 2 times, 1mL of preheated 0.05% pancreatin is added, after the cells under a microscope become round, digestion is stopped by old culture solution, the cells are lightly tapped, cell suspension is sucked into a centrifuge tube, the rpm is 1500/min, and the temperature is 4 DEG CCentrifuging for 5min, collecting cells, counting at 5 × 103The seeding density was passaged at/cm 2.
3. The preparation of mesenchymal stem cells for treating knee osteoarthritis and the research method thereof according to claim 1, wherein the mesenchymal stem cells are selected from the group consisting of: and in the step C, 4ml of platelet lysate which is activated after unfreezing is injected into the joint cavity 1 week before transplantation every time, cells which have the survival rate of not less than 90% and are free from EBV, HBV, HCV and HIV pollution are selected for resuscitation before transplantation, and the cells are uniformly mixed and packaged after trypsin collection and activated platelet lysate heavy suspension treatment are performed before transplantation.
4. The preparation of mesenchymal stem cells for treating knee osteoarthritis and the research method thereof according to claim 1, wherein the mesenchymal stem cells are selected from the group consisting of: and in the step C, when the knee joint is injected, disinfecting and paving a hole towel conventionally, holding a 5ml sterile injector to enter a joint cavity, then drawing out a proper amount of joint fluid, keeping a needle head, drawing out a needle cylinder, connecting the needle cylinder with uniformly mixed MSCs, slowly injecting 4ml, pressing and covering a needle eye with sterile gauze after the operation, lying on the back for 20min, discontinuously bending and stretching the knee joint for dozens of times during the period, and injecting 4ml of platelet lysate activated after unfreezing again after the MSCs are transplanted for 1 night.
5. The preparation of mesenchymal stem cells for treating knee osteoarthritis and the research method thereof according to claim 1, wherein the mesenchymal stem cells are selected from the group consisting of: in the step D, data analysis is carried out by using SPSS17.0 statistical software, VAS24 WOMAC scale scores of 10mm are completed under the guidance of doctors before and after treatment, but the scores can not be substituted for patients to be scored, each score is recorded in detail, and knee joint Lequesne index scores are respectively carried out before and after treatment, so that the method is used for judging the severity of knee joint osteoarthritis, disease activity and knee joint function.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112007049A (en) * | 2020-09-21 | 2020-12-01 | 济南磐升生物技术有限公司 | Stem cell exosome composition for treating knee osteoarthritis |
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| CN108865986A (en) * | 2018-06-29 | 2018-11-23 | 马琳 | For repairing articular cartilage damage/defect mescenchymal stem cell preparation and its preparation method and application |
| CN110237095A (en) * | 2019-05-28 | 2019-09-17 | 武汉汉密顿生物科技股份有限公司 | For treating stem cell injection liquid and its preparation and the application of cartilage defect of osteoarthritis |
| CN112007049A (en) * | 2020-09-21 | 2020-12-01 | 济南磐升生物技术有限公司 | Stem cell exosome composition for treating knee osteoarthritis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108865986A (en) * | 2018-06-29 | 2018-11-23 | 马琳 | For repairing articular cartilage damage/defect mescenchymal stem cell preparation and its preparation method and application |
| CN110237095A (en) * | 2019-05-28 | 2019-09-17 | 武汉汉密顿生物科技股份有限公司 | For treating stem cell injection liquid and its preparation and the application of cartilage defect of osteoarthritis |
| CN112007049A (en) * | 2020-09-21 | 2020-12-01 | 济南磐升生物技术有限公司 | Stem cell exosome composition for treating knee osteoarthritis |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112007049A (en) * | 2020-09-21 | 2020-12-01 | 济南磐升生物技术有限公司 | Stem cell exosome composition for treating knee osteoarthritis |
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