CN115957240A - Application of hematopoietic stem cells, pharmaceutical composition and kit - Google Patents
Application of hematopoietic stem cells, pharmaceutical composition and kit Download PDFInfo
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- CN115957240A CN115957240A CN202111195299.7A CN202111195299A CN115957240A CN 115957240 A CN115957240 A CN 115957240A CN 202111195299 A CN202111195299 A CN 202111195299A CN 115957240 A CN115957240 A CN 115957240A
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Abstract
The embodiment of the application discloses application, a pharmaceutical composition and a kit of hematopoietic stem cells. The application reports the new application of the hematopoietic stem cells in preparing the medicines for treating or preventing the joint diseases for the first time, and finds that the hematopoietic stem cells and the mesenchymal stem cells have synergistic effect in combination and have wide application prospect in treating or preventing the joint diseases.
Description
Technical Field
The present application relates generally to the field of medical technology, and more particularly, to an application of hematopoietic stem cells, a pharmaceutical composition and a kit thereof, and more particularly, to an application of hematopoietic stem cells in the preparation of a medicament for treating or preventing joint diseases, a pharmaceutical composition for treating or preventing joint diseases and a kit thereof.
Background
Joint diseases such as arthritis, which is a disease characterized by articular cartilage damage, osteophyte proliferation and subchondral bone change, can cause severe pain, stiffness, deformity, restricted movement of joints, and severe people can cause disability whose onset is associated with age, genetics, excessive exercise, trauma, obesity, etc.; among them, articular cartilage is highly vulnerable to damage and pathological degeneration, and has a limited self-repair ability due to its lack of blood vessels and nerves, and once damaged, it can only partially heal under certain physiological conditions.
At present, the clinical methods for treating joint diseases mainly comprise medicines, surgical treatment, physical treatment and the like, and reports show that mesenchymal stem cells can be used for treating joint diseases, but the treatment effect of the method is limited, and medicines suitable for joint diseases still need to be developed.
Disclosure of Invention
The present application is directed to solving the above technical problems in the prior art, and in one aspect, the present application relates to a method for treating or preventing joint diseases, comprising administering hematopoietic stem cells to a patient in need thereof, for example, by means of joint cavity injection or implantation. The application discovers that when the hematopoietic stem cells act on the cartilage injury part, the cartilage injury part is obviously repaired and healed, and the joint disease can be effectively treated.
In another aspect of the present application, the method for treating or preventing joint diseases of the present application further comprises administering the mesenchymal stem cells to a patient in need thereof, for example, sequentially or simultaneously administering the hematopoietic stem cells and the mesenchymal stem cells to the patient by means of joint cavity injection or implantation. The application unexpectedly finds that when the hematopoietic stem cells and the mesenchymal stem cells act on the cartilage injury part together, the repair and healing efficiency of the cartilage injury part is obviously higher than that of the hematopoietic stem cells or the mesenchymal stem cells which act alone, and the combination of the hematopoietic stem cells and the mesenchymal stem cells can generate a synergistic effect to promote the cartilage injury part to be repaired and healed more quickly and effectively.
In one aspect of the application, the amount of hematopoietic stem cells and/or mesenchymal stem cells is (0.01-100) x l0 per joint cavity 5 A plurality of; preferably, each joint cavity is (0.2-20) multiplied by l0 5 A plurality of; more preferably 10 × l0 per joint cavity 5 And (4) respectively. Experiments show that the cartilage injury part can be repaired and healed more quickly and effectively under the dosage.
In another aspect of the present application, there is provided a pharmaceutical composition for treating or preventing a joint disease, the pharmaceutical composition including hematopoietic stem cells. In another aspect of the present application, the pharmaceutical composition of the present application further comprises mesenchymal stem cells.
In another aspect of the present application, a kit comprising a pharmaceutical composition of the present application is provided.
Drawings
Other features, objects and advantages of the present application will become more apparent upon reading of the following detailed description of non-limiting embodiments thereof, made with reference to the accompanying drawings in which:
FIG. 1 is a graph showing the results of antigen expression and isotype control assay of hematopoietic stem cells according to one embodiment of the present application;
FIG. 2 is a graph showing the results of observation under a stereomicroscope of repair of cartilage damage of hematopoietic stem cells according to an embodiment of the present application;
fig. 3 is a graph showing the result of detecting surface markers of mesenchymal stem cells according to an embodiment of the present application;
FIG. 4 is a graph showing the result of observation under a stereomicroscope in the repair of cartilage damage according to an embodiment of the present application;
FIG. 5 is a graph of the results of a stereomicroscope examination of cartilage wear damage repair in accordance with one embodiment of the present application;
FIG. 6 is a graph showing the observation results of safranin fast green staining sections;
FIG. 7 is a graph showing the results of observation of toluidine blue-stained sections.
Detailed Description
The present application will be described in further detail with reference to examples. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant invention and not restrictive of the invention.
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict.
It is noted that the endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and that such ranges or values are understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The inventor firstly discovers and proves that the hematopoietic stem cells have the effect of promoting the repair and regeneration of the articular cartilage, and can quickly and effectively repair the damage when acting on the articular cartilage damaged part, thereby being used for preparing the medicine for treating or preventing the joint diseases.
In the embodiments of the present application, the joint disease refers to osteoarthritis characterized by joint bone damage, which causes bone damage including, but not limited to, aging, traumatic injury, etc., and joints including, but not limited to, joints in the hand, wrist, toe, neck, back, knee, hip, etc.
In the embodiments of the present application, "treatment" refers to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects resulting from the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including inhibition of disease, e.g., retardation of disease progression; or relieving the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce, or inhibit a disease in the individual, including, but not limited to, administering a drug containing a compound described herein to an individual in need thereof.
In the embodiments of the present application, "prevention" refers to preventing a disease (e.g., preventing arthritis) or delaying or preventing the onset of a condition in an individual who is susceptible to the disease but has not yet been diagnosed.
Among them, hematopoietic Stem Cells (HSCs) are adult stem cells in the blood system that have long-term self-renewal ability and the potential to differentiate into various types of mature blood cells, and express CD34 marker antigens.
In some embodiments of the present application, the hematopoietic stem cells are previously screened, for example by flow cytometry, such that the hematopoietic stem cells are CD 34-positively expressed hematopoietic stem cells; wherein the positive expression rate of CD34 in the hematopoietic stem cells is more than 80%, such as more than 85%, such as more than 90%, such as more than 95%, such as more than 99%, to obtain hematopoietic stem cells with stronger capability of repairing articular bone injury.
In some embodiments of the present application, the hematopoietic stem cells are hematopoietic stem cells isolated from at least one tissue of cord blood, bone marrow, and mobilized peripheral blood: and/or, the hematopoietic stem cells are expanded hematopoietic stem cells.
It will be understood by those skilled in the art that the hematopoietic stem cells are separately cultured and purified from, for example, umbilical cord blood, bone marrow and/or mobilized peripheral blood of a patient to be administered or/and treated, respectively, or isolated and purified from the above-mentioned tissues of other normal donors, or obtained by expanding hematopoietic stem cells, or commercially available. In some embodiments of the application, the hematopoietic stem cells may also be genetically engineered hematopoietic stem cells.
In some embodiments of the present application, the joint disease is cartilage damage, i.e. the medicament is for the treatment or prevention of cartilage damage.
In the present application, cartilage damage specifically refers to the occurrence of characteristic changes in articular cartilage such as inflammation, deformation, hardening and loss of elasticity, regional wear or loss, and more specifically to the occurrence of regional wear or loss of articular cartilage; among them, the main factors inducing the damage of the articular cartilage include, but are not limited to, degenerative changes, inflammation, trauma, sports injury, etc. caused by aging.
In some embodiments of the present application, the hematopoietic stem cells are administered by joint cavity injection or implantation.
For example, the hematopoietic stem cells can be prepared into a suspension or a gel liquid or a liquid tissue engineering material, and injected into the joint cavity to be repaired, or the hematopoietic stem cells can be embedded in a suitable biological material for tissue engineering to prepare a hard tissue engineering material, and implanted into the joint cavity to be repaired by means of surgery.
In some embodiments of the present application, the hematopoietic stem cells are administered in an amount of (0.01 to 100) x l0 per joint space 5 And (4) respectively.
Experiments show that the cartilage injury part can be repaired and healed more quickly and effectively under the dosage.
In some embodiments of the present application, the hematopoietic stem cells are administered in an amount of (0.2 to 20) x l0 per joint space 5 And (4) respectively.
In some embodiments of the present application, the hematopoietic stem cells are administered in an amount of 10 × l0 per joint space at a time 5 And (4) respectively.
In some embodiments of the present application, the hematopoietic stem cells are administered in an amount of 3 × l0 per joint space at a time 5 And (4) respectively.
In some embodiments of the present application, the medicament further comprises mesenchymal stem cells.
The inventor surprisingly finds that the combination of the hematopoietic stem cells and the mesenchymal stem cells has a synergistic effect, and compared with the single administration, the curative effect is obviously improved when the hematopoietic stem cells and the mesenchymal stem cells jointly act on a cartilage injury part, and the injury can be quickly and effectively repaired, so that the hematopoietic stem cells and the mesenchymal stem cells can be used for treating or preventing diseases related to or generated by cartilage injury.
Wherein, mesenchymal Stem Cells (MSCs) are multipotent stromal cells defined by the mesenchymal and tissue stem cell committee of the international society for cell therapy, have the ability to adhere to the wall and differentiate into bone, cartilage, ligament, tendon, muscle, fat and other histiocyte cells in vitro under standard culture conditions, express membrane surface markers such as CD29, CD44, CD73, CD90, CD105 and CD106, and do not express hematopoietic stem cell marker antigens such as CD14, CD20 and CD 34;
in some embodiments of the present application, the mesenchymal stem cells are previously screened, for example by flow cytometry, such that the mesenchymal stem cells are CD73, CD90 and CD105 positively expressed mesenchymal stem cells; wherein the positive expression rate of CD73, CD90 and CD105 in the positively expressed mesenchymal stem cells is more than 80%, such as more than 85%, such as more than 90%, such as more than 95%, such as more than 99%, to obtain mesenchymal stem cells with stronger ability to repair bone damage.
In some embodiments of the present application, the mesenchymal stem cells are mesenchymal stem cells isolated from at least one of umbilical cord, adipose, dental pulp, and synovial tissue; and/or, the mesenchymal stem cell is a mesenchymal stem cell that induces differentiation of a pluripotent stem cell; and/or, the mesenchymal stem cell is an expanded mesenchymal stem cell.
It will be understood by those skilled in the art that the above mesenchymal stem cells are separately cultured and purified from, for example, umbilical cord, fat, dental pulp and/or synovial membrane of the patient himself/herself to be administered or/and treated, may be separated and purified from the above tissues of other normal donors, may be obtained by expanding mesenchymal stem cells, or may be commercially available. In some embodiments of the application, the mesenchymal stem cell may also be a genetically engineered mesenchymal stem cell.
In some embodiments of the present application, the mesenchymal stem cells are administered by joint cavity injection or implantation.
For example, the mesenchymal stem cells can be prepared into a suspension or a gel liquid or a liquid tissue engineering material and injected into the joint cavity to be repaired, or the mesenchymal stem cells are embedded in a proper biological material for tissue engineering to prepare a hard tissue engineering material and are implanted into the joint cavity to be repaired by means of a surgery.
In some embodiments of the present application, the mesenchymal stem cells are administered in an amount of (0.01-100) x l0 per joint cavity 5 And (4) respectively.
In some embodiments of the present application, the mesenchymal stem cell is a mesenchymal stem cellThe dosage of the cells is 0.2-20 multiplied by l0 for each joint cavity 5 And (4) respectively.
In some embodiments of the present application, the mesenchymal stem cells are administered in an amount of 10 × l0 per joint cavity at a time 5 And (4) respectively.
In some embodiments of the present application, the mesenchymal stem cells and the hematopoietic stem cells are administered sequentially or simultaneously.
By "sequentially" is meant administration of mesenchymal stem cells first, and then hematopoietic stem cells; or, hematopoietic stem cells are administered first, and then mesenchymal stem cells; the subsequent application time may be within 1-48 h.
By "simultaneously" is meant that the mesenchymal stem cells are administered simultaneously with the hematopoietic stem cells.
Further, the present application relates to a pharmaceutical composition for treating or preventing joint diseases, which comprises hematopoietic stem cells.
In some embodiments of the present application, the hematopoietic stem cells are hematopoietic stem cells isolated from at least one tissue of cord blood, bone marrow, and mobilized peripheral blood: and/or, the hematopoietic stem cells are expanded hematopoietic stem cells.
In some embodiments of the present application, the pharmaceutical composition further comprises mesenchymal stem cells.
In some embodiments of the present application, the mesenchymal stem cells are mesenchymal stem cells isolated from at least one of umbilical cord, adipose, dental pulp, and synovial tissue; and/or, the mesenchymal stem cell is a mesenchymal stem cell that induces differentiation of a pluripotent stem cell; and/or, the mesenchymal stem cell is an expanded mesenchymal stem cell.
In some embodiments of the present application, the hematopoietic stem cells and the mesenchymal stem cells are administered sequentially or simultaneously.
In some embodiments of the present application, the pharmaceutical composition is administered by joint cavity injection or implantation.
Illustratively, in some modes, the mesenchymal stem cells and the hematopoietic stem cells can be prepared into a suspension or a gel liquid or a tissue engineering material in a liquid state, and are injected into a position to be repaired step by step or simultaneously by means of injection; illustratively, in some specific embodiments, the hematopoietic stem cells and the mesenchymal stem cells are resuspended in, for example, PBS buffer solution or pharmaceutical saline solution to form a suspension, and the suspension is injected into the site to be repaired, wherein the mixing ratio (i.e. the ratio of cell number) of the hematopoietic stem cells and the mesenchymal stem cells in the suspension is 1:3-3:1, for example, the ratio of the hematopoietic stem cells and the mesenchymal stem cells is 1:3, 1:2, 1:1, 2:1, 3:1, etc.;
in other forms, the hematopoietic stem cells and mesenchymal stem cells can be embedded in a suitable tissue engineering biological material to form a hard tissue engineering material, and then implanted into a part to be repaired through a surgery;
it is understood that, for example, hematopoietic stem cells and mesenchymal stem cells may be prepared as a cell suspension or a gel solution, respectively, and applied to the site to be repaired sequentially at the time of administration, or pre-mixed before administration while being applied to the site to be repaired.
In some embodiments of the present application, the hematopoietic stem cells and the mesenchymal stem cells are used in an amount of (0.01-100) x l0 per joint space 5 And (4) respectively.
Experiments show that the medicine can repair and heal the damaged part of cartilage more quickly and effectively under the dosage.
According to the embodiment of the application, the dosage of the hematopoietic stem cells and the mesenchymal stem cells is (0.2-20) multiplied by l0 per joint cavity 5 And (4) respectively.
According to the embodiment of the application, the dosage of the hematopoietic stem cells and the mesenchymal stem cells is 10 xL 0 per joint cavity 5 And (4) respectively.
According to the embodiment of the application, the dosage of the hematopoietic stem cells is 3 xL 0 per joint cavity 5 The dosage of the mesenchymal stem cells is 10 xL 0 per joint cavity 5 And (4) respectively.
In some embodiments of the present application, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.
The excipients comprise one or more solvents, excipients or nutrients for maintaining cell viability/activity.
According to an embodiment of the present application, the pharmaceutical composition further comprises hyaluronic acid, a cytokine, a vitamin and/or a pharmaceutically acceptable salt.
Hyaluronic acid is the main component of joint synovial fluid, can protect articular cartilage, prevent or delay further degeneration, remove substances causing pain, has the function of obviously relieving pain and accelerates wound healing; cytokines can regulate a variety of inflammatory responses and have the ability to induce cartilage formation, such as interleukins, transforming growth factors, insulin-like growth factors, and the like; vitamins such as vitamin D and pharmaceutically acceptable salts may exert anti-inflammatory effects.
In some embodiments of the present application, the joint disease is cartilage damage, i.e. the pharmaceutical composition is for use in the treatment or prevention of cartilage damage.
Further, the present application relates to a kit comprising a pharmaceutical composition as described above.
In some embodiments of the present application, the kit comprises hematopoietic stem cells in individually sealed packages; or, the hematopoietic stem cells and the mesenchymal stem cells are separately and hermetically packaged.
In some embodiments of the present application, the kit comprises a hermetically sealed package of a mixture of hematopoietic stem cells and mesenchymal stem cells.
In some embodiments of the present application, the kit further comprises a device for delivering the pharmaceutical composition to a joint of a mammalian subject to be administered, and the device has a reservoir means for storing the pharmaceutical composition, a piston means movable along a longitudinal axis of the reservoir for dispensing the pharmaceutical composition, and a hollow needle secured to the reservoir means for delivering the pharmaceutical composition to the joint cavity of the mammalian subject.
It will be understood that the frequency and dose of administration of the drugs and/or pharmaceutical compositions and/or compositions of the present application can be determined by a variety of relevant factors, including the type of disease to be treated, the route of administration, the age, sex, weight and severity of the disease of the patient, and the type of drug as the active ingredient. According to some embodiments of the invention, the daily dose may be divided into 1, 2 or more doses in a suitable form for administration 1, 2 or more times over the entire period, as long as a therapeutically effective amount is achieved.
According to embodiments herein, the medicaments and/or pharmaceutical compositions and/or kits of the present application may be used in combination with conventional methods and/or therapies or may be used separately from conventional methods and/or therapies. When the agents of the present application are administered in combination therapy with other agents, they may be administered to the individual sequentially or simultaneously.
Additional aspects and advantages of the present application will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Example 1 isolated culture and identification of umbilical cord blood hematopoietic Stem cells
Uniformly mixing a blood sample and physiological saline according to the proportion of 2:1-1:2 to obtain a mixed solution, then respectively adding the mixed solution and lymphocyte separation solution into a centrifuge tube according to the proportion of 3:1-1:3, wherein the adding sequence is that the lymphocyte separation solution is firstly added into the centrifuge tube, then the mixed solution is slowly added, the liquid level of the lymphocyte separation solution is kept flat in the adding process, after the adding is finished, the lymphocyte separation solution is centrifuged for 20-25 minutes at 500-1500g, the slow acceleration and slow deceleration are carried out during centrifugation, after the centrifugation is finished, an intermediate leucocyte layer is collected into a new centrifuge tube, the physiological saline is added according to the proportion of 2:1-1:2, and the mixture is centrifuged for 10-15 minutes at 500-1000 g; removing supernatant after centrifugation, adding 10-20mL of normal saline, blowing, beating and precipitating, centrifuging for 10-15 minutes at 200-500g, removing supernatant after centrifugation, adding 2-10mL of normal saline, blowing, beating and precipitating, and counting cells;
adopting a CD34 magnetic bead sorting kit with gentle and gentle characteristics in the America, and carrying out CD34 magnetic bead sorting according to the steps of the specification + Sorting hematopoietic stem cells, or sorting the mononuclear cells labeled with a CD34 fluorescent antibody by using a flow cytometer;
and blowing and resuspending the sorted cells by using 1-2mL of physiological saline, culturing the cells and the mold, and detecting the positive rate of CD34 molecules, wherein the detection result is shown in figure 1, and the positive expression rate of CD34 reaches 96.82%.
Example 2 experiment of repairing rat articular cartilage injury by hematopoietic stem cells
1. Establishing rat articular cartilage damage model
Adopting a healthy Wistar rat as a model, male, 3-month-old and 150-220g in weight;
anesthetizing a rat, and then placing the rat on a constant-temperature heating pad to keep the temperature of the rat; using a shaver to remove hair near the knee joint, using a scalpel to open the skin along the inner side of the knee joint, and cutting muscle tissue along the patellar ligament; firstly, straightening the leg of a rat, moving the patella to the outer side of the knee joint, and exposing the joint cavity; punching a rat femoral sliding groove by using a 1mm spherical drill to form an abrasion round hole with the diameter of about 2 mm; then flushing with normal saline, scrubbing with iodophor, straightening the leg of the rat, and resetting the patella; the wound is sutured by using an absorbable suture line with a round 19mm needle; injecting gentamicin sulfate into leg muscle of rat in the dosage of 40mg/kg, and heating the rat to restore temperature; three days after the operation, continuous intramuscular injection of gentamicin sulfate prevents infection; after one month of operation, inflammation caused by the operation is resolved, cartilage damage is stable, and model construction is completed.
2. Hematopoietic stem cell transplantation
Rats were randomly divided into four groups, two of which were locally administered in the joint cavity using the cord blood hematopoietic stem cells obtained as described in the above example, to transplant HSCs at cartilage injury sites, respectively; the HSCs are resuspended into cell suspension by adopting PBS, and two groups injected with PBS only are used as negative controls, and the administration method is as follows:
after a rat is anesthetized by ether inhalation, the hair of the rat near the knee joint is removed by using a shaver, cells are injected into the cavity of an insulin syringe, resistance is not felt when the joint cavity is penetrated, and a needle head is slowly drawn out after administration to avoid leakage of the cells; wherein the total volume of injected PBS or cell suspension is 50 μ L, and the number of injected cells in each group is 5 × 10 5 And (4) respectively.
3. Repair effect detection
After four weeks of hematopoietic stem cell transplantation treatment, rats were euthanized with carbon dioxide, rat knee joints were harvested, washed with physiological saline, and excess muscle and bone were removed, and after observation and photographing under a stereomicroscope to record the appearance of rat knee joints, they were immediately placed in a tissue fixative for fixation for 48 hours. After fixation is finished, the knee joint is washed twice by PBS, and is decalcified in decalcifying liquid; after the decalcification is finished, taking out the ddH for the knee joint 2 Cleaning for about 3 times with O, storing in 75% ethanol for a long time, and directly performing subsequent dehydrated paraffin embedding operation.
The observation result of the body type microscope is shown in fig. 2, and it can be seen that the worn round holes of the PBS treatment group are still obvious and have no repairing effect basically, and the cartilage layer of the HSCs treatment group is better repaired, which indicates that the HSCs have cartilage repairing capability.
Example 3 culture and identification of umbilical cord mesenchymal Stem cells
Adding umbilical cord mesenchymal stem cells to 10mL mesenchymal stem cell culture medium (DMEM/Fl 2+10% FBS +2mM L-Glutamine +50 μ g/mL Gentamycin); place the plate in CO 2 Culturing in a 5% culture box at 37 deg.C for 1 week, and changing the culture solution for 1 time 2-4 days; when the mesenchymal stem cell fusion rate reaches about 80%, marking as the 1 st generation, and carrying out passage amplification;
detaching adherent cells from the bottom of the plate by using digestive enzyme (such as TrypLE Express of Gibco) during passage amplification, removing supernatant after centrifugation, adding a fresh mesenchymal stem cell culture medium to re-suspend the cells, and inoculating the cells in a T25 cell culture bottle for passage amplification; then changing the liquid every 2 days until the fusion rate reaches 80%, and then freezing or subculturing again;
when the number of the cells reaches a certain number (generally not more than 10 th generation), performing cryopreservation or identification, and identifying the mesenchymal stem cells by flow cytometry; in this example, the human mesenchymal stem cell typing kit of Miltenyi was selected to identify the cells, and the specific process was as follows:
digesting the cells with trypsase/EDTA digestive juice when the cell fusion rate reaches 80-90 percent to prepare 1 multiplied by 10 6 Cell suspension per mL, adding antibodyThe result of the mixture specifically comprises CD73-APC, CD90-FITC, CD105-PE, CD14/CD20/CD34/CD45-PerCP, and is shown in figure 3, it can be seen that the positive rates of CD73, CD90 and CD105 of the mesenchymal stem cell reach more than 85%, and the expression of CD14/CD20/CD34/CD45 is negative, which indicates that the mesenchymal stem cell has better characteristics.
Example 4 experiment of repairing rat articular cartilage injury by hematopoietic stem cell in combination with mesenchymal stem cell
1. Establishing rat articular cartilage damage model
Adopting a healthy Wistar rat as a model, male, 3-month-old and 150-220g in weight;
anesthetizing a rat, and then placing the rat on a constant-temperature heating pad to keep the temperature of the rat; removing hair near the knee joint by using a shaver, opening the skin along the inner side of the knee joint by using a scalpel, and cutting muscle tissues along patellar ligaments; firstly, straightening the leg of a rat, then moving the patella to the outer side of the knee joint, and exposing the joint cavity; punching a femoral trochlear groove of a rat by using a 1mm spherical drill to form an abrasion round hole with the diameter of about 2 mm; then, the rat is washed by normal saline and scrubbed by iodophor, and then the leg of the rat is straightened to reset the patella; the wound is sutured by using an absorbable suture line with a round needle of 19 mm; injecting gentamicin sulfate into leg muscle of rat in 40mg/kg dosage, and heating the rat to recover; three days after the operation, continuous intramuscular injection of gentamicin sulfate prevents infection; after one month of operation, inflammation caused by the operation is resolved, cartilage damage is stable, and model construction is completed.
2. Stem cell transplantation
Rats are randomly divided into four groups, wherein one group adopts the suspension (marked as the composition of MSCs and HSCs) of umbilical cord mesenchymal stem cells and umbilical cord blood hematopoietic stem cells obtained in the above embodiment to carry out intra-articular local administration, and MSCs and HSCs are transplanted at cartilage injury parts respectively to serve as positive controls; the MSCs, the HSCs and the MSCs and HSCs composition adopt PBS to resuspend cells to form cell suspension, only PBS is injected to serve as a negative control, and the administration specific method is as follows:
after the rats were anesthetized by ether inhalation, the rat hairs near the knee joint were removed by a shaverInjecting cells in the cavity of an insulin syringe, wherein the joint cavity is penetrated without resistance, and the needle head is slowly drawn out after administration to avoid leakage of the cells; wherein the total volume of injected PBS or cell suspension is 50 μ L, and the number of injected cells in each group is 5 × 10 5 And in the MSCs + HSCs cell suspension, the mixing ratio of the MSCs and the HSCs is 1:1.
3. Repair effect detection
After four weeks of stem cell transplantation treatment, rats were euthanized with carbon dioxide, the knee joints of the rats were harvested, rinsed with normal saline, and excess muscle and bone were removed, and after observation under a stereomicroscope and photographing recording of the appearance of the knee joints of the rats, they were immediately placed in a tissue fixative for fixation for 48 hours. After fixation is finished, the knee joint is washed twice by PBS, and is decalcified in decalcifying liquid; after the decalcification is finished, taking out the ddH for the knee joint 2 Cleaning for about 3 times with O, storing in 75% ethanol for a long time, and directly performing subsequent dehydrated paraffin embedding operation.
The observation result of the body type microscope is shown in fig. 4, and it can be seen that the worn round holes of the PBS treatment group are still obvious and have no repair effect basically, the MSCs and HSCs treatment groups have good repair effect, but the repair effect is incomplete, while the MSCs + HSCs treatment group has no damage seen completely, and the cartilage layer is well repaired, which indicates that the MSCs and HSCs combination has better cartilage repair capability compared with the MSCs and HSCs alone.
Example 5 experiment of repairing abraded rat articular cartilage injury by hematopoietic stem cell in combination with mesenchymal stem cell
1. Establishing rat articular cartilage abrasion damage model
Adopting a healthy Wistar rat as a model, male, 3-month-old and 150-220g in weight;
anesthetizing a rat, and then placing the rat on a constant-temperature heating pad to keep the temperature of the rat; removing hair near the knee joint by using a shaver, opening the skin along the inner side of the knee joint by using a scalpel, and cutting muscle tissues along patellar ligaments; firstly, straightening the leg of a rat, then moving the patella to the outer side of the knee joint, and exposing the joint cavity; grinding cartilage at the bulges at the two sides of the rat femur chute by using a 1mm spherical drill; then, the rat is washed by normal saline and scrubbed by iodophor, the leg of the rat is straightened, and the patella is reset; the wound is sutured by using an absorbable suture line with a round 19mm needle; gentamicin sulfate was injected intramuscularly in the legs of rats at a dose of 40mg/kg and was rewarmed in a heating pad. Three days after surgery, continuous intramuscular injection of gentamicin sulfate prevented infection. After one month of operation, the inflammation caused by the operation is removed, the cartilage damage is stable, and the model construction is completed.
In other embodiments, the model may be constructed by injecting a drug into the joint cavity, such as sodium iodoacetate (MIA). The specific method comprises the following steps: a rat is anesthetized by 60mg/kg of 0.6% sodium pentobarbital solution, the rat hair at the knee joint is shaved off, a small opening is cut at the knee joint skin after iodophor wiping sterilization, the patella and the patella ligament are exposed, an insulin needle syringe needle is inserted into a cavity between the patella and the femur from the inner side of the knee joint, and 0.1-1mg MIA is injected into each joint cavity with the volume of 50 mu L. There was significant damage to the knee cartilage after 4 weeks.
2. Stem cell transplantation
Rats were randomly divided into three groups, one of which was administered locally in the joint cavity using the suspension of umbilical cord mesenchymal stem cells and umbilical cord blood hematopoietic stem cells (denoted as MSCs + HSCs composition) obtained in the above example, and MSCs were transplanted at the cartilage injury site as a positive control; the MSCs and the MSCs + HSCs composition adopt PBS to resuspend cells to form cell suspension, only PBS is injected to serve as a negative control, and the administration method is as follows:
after a rat is anesthetized by ether inhalation, the hair of the rat near the knee joint is removed by using a shaver, cells are injected into the cavity of an insulin syringe, resistance is not felt when the joint cavity is penetrated, and a needle head is slowly drawn out after administration to avoid leakage of the cells; wherein the total volume of injected PBS or cell suspension is 50 μ L, and the number of injected cells in each group is 5 × 10 5 And in the MSCs + HSCs cell suspension, the mixing ratio of the MSCs and the HSCs is 1:1.
3. Repair effect detection
Four weeks after stem cell transplantation therapy, rats were euthanized with carbon dioxide,harvesting the knee joint of the rat, flushing the knee joint with normal saline, removing redundant muscle and bone, observing the knee joint of the rat under a body type microscope, taking a picture, recording the appearance of the knee joint of the rat, and immediately placing the rat into tissue fixing liquid for fixing for 48 hours; after fixation is finished, the knee joint is washed twice by PBS, and is decalcified in decalcifying liquid; after the decalcification is finished, taking out the ddH for the knee joint 2 Cleaning for about 3 times by using an O cleaning machine, and performing paraffin embedding;
the paraffin embedding operation process is as follows:
firstly, melting the paraffin of the section used for embedding in a 65 ℃ oven, preheating a tissue embedding instrument to melt the paraffin, and precooling a cold platform and a cold spot;
tissue dehydration embedding: 75% ethanol for 2 hours, 85% ethanol for 2 hours, 90% ethanol for 2 hours, 95% ethanol for 1 hour, absolute ethanol I for 0.5 hour, absolute ethanol II for 0.5 hour, xylene I for 1 hour, xylene II for 0.5 hour, paraffin I for 1 hour, paraffin II for 1 hour, and paraffin III for 1 hour; placing the tissue fixing frame at a hot point, injecting paraffin into the tissue fixing frame, then placing the tissue, immediately placing the tissue in an embedding frame, and injecting paraffin to enable the paraffin to be parallel to the tissue fixing frame; placing the tissue fixing frame on a cold platform, taking down the paraffin block after the paraffin block is solidified and molded, and placing the paraffin block in a refrigerator at the temperature of minus 20 ℃ for overnight freezing;
slicing: firstly, the developing machine is replaced by clean ddH 2 O, setting the temperature to 42 ℃, setting the temperature of a chip baking machine to 72 ℃, setting the thickness of the slices to 5 μm, placing the cut Dan Lapian in the chip unfolding machine for unfolding, fishing out the unfolded glass slide, adhering the unfolded glass slide to a glass slide (an anti-falling glass slide is required for immunohistochemistry), baking the cut glass slide on the chip baking machine for 2 hours, and dyeing;
and (3) safranin fast green staining:
prior to the staining procedure, the sections were dewaxed to water as follows: xylene I20 min, xylene II 20 min, anhydrous ethanol I10 min, anhydrous ethanol II 10 min, 95% ethanol 5 min, 85% ethanol 5 min, 75% ethanol 5 min, ddH 2 O3 minutes;
the dyeing operation is then carried out: fixing green and dyeing for 5 minutes, washing with tap water for 1 minute, washing off excessive dye, performing microscopic examination until the target part is light green and the rest parts are emerald green, 1% hydrochloric acid ethanol for 15 seconds, and safranine for 3 minutes (the green and red can be differentiated by tap water and hydrochloric acid ethanol respectively to control the dyeing degree);
dehydrating and sealing after dyeing, wherein 75%, 85% and 95% ethanol are respectively used for 3 minutes, absolute ethyl alcohol I, absolute ethyl alcohol II and absolute ethyl alcohol III are respectively used for slightly washing, xylene I, II is respectively used for 3 minutes, and the xylene and neutral resin 1:1 are proportionally sealed;
wherein, acidophilic bone can be combined with acid dye fast green to present green or blue, basophilic cartilage can be combined with basic dye safranin O to present red, and the red green is in sharp contrast, so that cartilage tissue and bone tissue can be easily distinguished, and the method can be used for evaluating the cartilage repair capability of stem cells;
toluidine blue staining:
prior to the staining procedure, the sections were dewaxed to water as follows: xylene I20 min, xylene II 20 min, anhydrous ethanol I10 min, anhydrous ethanol II 10 min, 95% ethanol 5 min, 85% ethanol 5 min, 75% ethanol 5 min, ddH 2 O3 minutes;
the dyeing operation is then carried out: 100 mu L of toluidine blue staining solution, adding 900 mu L of diluent to prepare 1mL of toluidine blue staining solution, carrying out drop dyeing for 10 minutes, washing for about 2 minutes by tap water, soaking for 2 minutes twice by deionized water, dehydrating and sealing according to the operation;
wherein, cations in toluidine blue have dyeing effect, can be combined with acidic substances in cells and tissues to form blue, and can be combined with cartilage to form blue-purple, so that the method can be used for further evaluating the cartilage repair capacity of stem cells.
The observation result of the body type microscope is shown in fig. 5, and it can be seen that the cartilage abrasion of the MSCs treatment group is still obvious, the repair degree is not good, while the MSCs + HSCs treatment group has no damage seen, and is completely covered by cartilage-like tissues, and the new tissue has no obvious boundary with the surrounding normal cartilage tissues, and the surface is smooth.
The results of safranin fast green staining are shown in fig. 6, and it can be seen that the cartilage layer (layer shown in box) of the MSCs + HSCs treated group is obviously thickened, chondrocytes are increased, and the chondrocytes are arranged in order, indicating that the treatment has better cartilage regeneration effect compared with the treatment of MSCs alone.
The toluidine blue staining results are shown in fig. 7, and it can be seen that the cartilage layer of the PBS treated group is thinnest, cartilage damage is obvious, the cartilage layer of the MSCs treated group is thicker than the PBS treated group, but the staining is lighter, which indicates that cartilage damage repair is not obvious, while the blue-purple region (the layer shown in the box) of the MSCs + HSCs treated group is obviously enlarged, the color is darker, the cartilage layer is thick, and the cells are arranged regularly, which indicates that the good cartilage regeneration effect is achieved.
In the description of the specification, reference to the description of "one embodiment," "some embodiments," "an example," "a specific example," or "some examples" or the like means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and not to be construed as limiting the present invention and that modifications, substitutions and alterations may be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (11)
1. Use of hematopoietic stem cells in the manufacture of a medicament for the treatment or prevention of a joint disease.
2. The use of claim 1, wherein the medicament further comprises mesenchymal stem cells.
3. Use according to claim 1 or 2, wherein the joint disease is cartilage damage.
4. The use according to claim 1 or 2, wherein the hematopoietic and/or mesenchymal stem cells are administered by joint cavity injection or implantation.
5. Use according to claim 1 or 2, wherein the hematopoietic and/or mesenchymal stem cells are used in an amount of (0.01-100) x l0 per joint space 5 A plurality of; preferably, each joint cavity is (0.2-20) multiplied by l0 each time 5 A plurality of; more preferably 10 × l0 per joint cavity 5 And (4) respectively.
6. The use of claim 2, wherein the mesenchymal stem cells and the hematopoietic stem cells are administered sequentially or simultaneously.
7. A pharmaceutical composition for treating or preventing a joint disease, comprising hematopoietic stem cells.
8. The pharmaceutical composition of claim 7, further comprising mesenchymal stem cells.
9. The pharmaceutical composition of claim 7, further comprising one or more pharmaceutically acceptable excipients.
10. The pharmaceutical composition according to any one of claims 7 to 9, wherein the joint disease is cartilage damage.
11. A kit comprising a pharmaceutical composition according to any one of claims 7 to 10.
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