CN106267161A - A kind of stem cell medicine and its preparation method and application - Google Patents
A kind of stem cell medicine and its preparation method and application Download PDFInfo
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- CN106267161A CN106267161A CN201610873067.5A CN201610873067A CN106267161A CN 106267161 A CN106267161 A CN 106267161A CN 201610873067 A CN201610873067 A CN 201610873067A CN 106267161 A CN106267161 A CN 106267161A
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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
- A61K35/48—Reproductive organs
- A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
- A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
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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
- A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/195—Chemokines, e.g. RANTES
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
Abstract
The present invention relates to regenerative medicine and biology techniques field, particularly to a kind of stem cell medicine and its preparation method and application.This stem cell medicine includes embryonic stem cell, BMNC, translation growth factor-β and solvent.The present invention uses embryonic stem cell and BMNC therapeutic alliance osteonecrosis, can effectively facilitate the healing of osteonecrosis, significantly improve the growth of area of new bone, and its therapeutic effect is considerably better than simple employing embryonic stem cell or the therapeutic effect of BMNC.
Description
Technical field
The present invention relates to regenerative medicine and biology techniques field, particularly to a kind of stem cell medicine and preparation method thereof
And application.
Background technology
Osteonecrosis refers to that skeleton living tissue composition is downright bad.Chinese medicine is referred to as osteomyelitis disease osteonecrosis.And for
Osteonecrosis, human body is likely at any position occur, only has been found that more than 40 locate with regard to ischemic necrosis, and femur head necrosis is sent out
Raw rate is the highest, and this is mainly determined by the feature of biomechanics and anatomical terms.
The cause of disease of femur head necrosis is not quite similar, and owing to lacking longitudinal comparison research and preferable animal model, it is true
The pathogenesis cut not yet obtains unified final conclusion, and current research focuses primarily upon capital blood vessel microcirculation and blood capillary lacks
Blood aspect, although the cause of disease of femur head necrosis exists the biggest difference, but has a common basic pathology change: femoral head
Blood supply reduces or interrupts causing osteocyte downright bad, and reaction occurs repairing in necrotic area subsequently, and process that is downright bad and that repair is interleaved into
OK, downright bad whole latter stage may occur in which femoral head at weight loading region articular step-off and the generation of Secondary cases osteoarthritis.
Myeloid-lymphoid stem cell has the following characteristics that and can be divided into osteoblast, chondrocyte, adipose cell and sarcoplast,
Several genes can also be expressed simultaneously, secrete the multiple osteogenic activity factor, create advantage for creeping substitution, union of fracture,
Research and internal bone demonstrate good application prospect with soft tissue healing aspect the most in vitro.Zoopery simultaneously is demonstrate,proved
Real whole body or be locally implanted myeloid-lymphoid stem cell or mescenchymal stem cell seldom occurs graft-rejection, for treatment osteonecrosis tool
There is certain curative effect.
At present, the seed cell for the treatment of osteonecrosis is mainly autologous bone marrow mesenchymal stem cells and umbilical cord mesenchyma is dry thin
Born of the same parents, but both are adult stem cell type, have had certain differentiation degree, and when carrying out amplification cultivation in vitro, these are two years old
Plant adult stem cell and be easy to that differentiating phenomenon occurs, cause therapeutic effect poor, therefore between mesenchymal stem cells MSCs and umbilical cord
Mesenchymal stem cells has certain limitation for treatment osteonecrosis, needs to research and develop and a kind of novel can effectively treat osteonecrosis
Stem cell medicine.
Summary of the invention
In view of this, the invention provides a kind of stem cell medicine and its preparation method and application.This stem cell medicine is adopted
By embryonic stem cell and BMNC therapeutic alliance osteonecrosis, the healing of osteonecrosis can be effectively facilitated, significantly improve new
The growth of bone growth promoting, its therapeutic effect is considerably better than simple employing embryonic stem cell or the therapeutic effect of BMNC.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides a kind of stem cell medicine, including embryonic stem cell, BMNC, monocyte chemotactic
The factor-1 (MCP-1) and solvent.
The present invention uses embryonic stem cell and BMNC therapeutic alliance osteonecrosis, substitutes bone marrow stem cell or umbilicus
With stem cell as the main cell repairing bone injury.Embryonic stem cell remains the differentiation potential that stem cell is the most original, and
Cellular degeneration apoptosis phenomenon do not occur, state is excellent, and abundance, and differentiation capability is strong in vitro, and therapeutic effect is notable.This
Outward, with the addition of BMNC, both are used in combination.Research shows that embryonic stem cell and BMNC are combined and controls
Treating, can effectively facilitate the healing of osteonecrosis, significantly improve the growth of area of new bone, its therapeutic effect is considerably better than and uses merely embryo
Stem cell or the therapeutic effect of BMNC.
In bone repair process, need the participation of multiple somatomedin, and now Endogenous Growth Factors is often difficult to full
Foot needs, therefore exogenous growth factor the most gradually comes into one's own in the treatment of head necrosis.Therapeutic process at head necrosis
In, use certain method to introduce specific somatomedin timely and appropriately, contribute to reparation and the reconstruction of bone defect, these
Specific somatomedin is broadly divided into rush vascular endothelial cell growth factor and regulation and control bone, chondrocyte proliferation differentiation factor.?
In the present invention, stem cell medicine with the addition of monocyte chemotactic factor-1, beneficially embryonic stem cell and BMNC
Form osseous tissue.
As preferably, in every 1mL stem cell medicine, the consumption of each component is:
Embryonic stem cell: (1～10) × 106Individual；
BMNC: (1～10) × 106Individual；
Monocyte chemotactic factor-1:1～20ng；
Solvent: supply.
Preferably, in every 1mL stem cell medicine, the consumption of each component is:
Embryonic stem cell: (1～5) × 106Individual；
BMNC: (1～5) × 106Individual；
Monocyte chemotactic factor-1:10ng；
Solvent: supply.
In the embodiment that the present invention provides, in every 1mL stem cell medicine, the consumption of each component is:
Embryonic stem cell: (1～2.5) × 106Individual；
BMNC: (1～2.5) × 106Individual；
Monocyte chemotactic factor-1:10ng；
Solvent: supply.
In the embodiment that the present invention provides, solvent is normal saline.In preparation select normal saline as solvent, its with
The osmotic pressure of tissue is consistent, osseous tissue will not be produced damage.But the kind of solvent is not limited to this, art technology
The solvent of personnel's accreditation is all within protection scope of the present invention.
As preferably, storage temperature≤4 DEG C of stem cell medicine.Cryopreservation preparation, on the one hand can avoid MCP-1 to lose
Live, on the other hand the most well maintain the biological activity of stem cell, improve therapeutic effect.
As preferably, the dosage form of stem cell medicine is injection.
Present invention also offers the application in preparation treatment osteonecrosis medicine of this stem cell medicine.
In the embodiment that the present invention provides, osteonecrosis is femur head necrosis.
Present invention also offers the preparation method of this stem cell medicine, including: monocyte chemotactic factor-1 is dissolved in molten
In matchmaker, obtain monocyte chemotactic factor-1 solution, use monocyte chemotactic factor-1 resuspended embryonic stem cell of solution and bone
Marrow mononuclearcell, prepares stem cell medicine.
The invention provides a kind of stem cell medicine and its preparation method and application.This stem cell medicine includes that embryo is dry thin
Born of the same parents, BMNC, monocyte chemotactic factor-1 (MCP-1) and solvent.The present invention at least has one of following advantage:
1, the present invention uses embryonic stem cell and BMNC therapeutic alliance osteonecrosis, can effectively facilitate osteonecrosis
Healing, significantly improve the growth of area of new bone, its therapeutic effect is considerably better than and simple uses embryonic stem cell or bone marrow single core
The therapeutic effect of cell；
2, in the present invention, stem cell medicine with the addition of monocyte chemotactic factor-1, beneficially embryonic stem cell and bone
Marrow mononuclearcell forms osseous tissue, can improve curative effect；
3, the stem cell medicine preparation time that the present invention provides is short, and preparation efficiency is high, is suitable for industrialized production.
Detailed description of the invention
The invention discloses a kind of stem cell medicine and its preparation method and application, those skilled in the art can use for reference this
Literary composition content, is suitably modified technological parameter and realizes.Special needs to be pointed out is, all similar replacements and change are to art technology
Being apparent from for personnel, they are considered as being included in the present invention.Method and the application of the present invention have been passed through preferably
Embodiment is described, and related personnel substantially can be to side as herein described in without departing from present invention, spirit and scope
Method and application are modified or suitably change and combine, and realize and apply the technology of the present invention.
Term is explained:
BMNC refers to that the nucleus in bone marrow is single general name, include hematopoietic stem cell (HSC),
MSCs, endothelial progenitor cells (EPC) and stromal cell etc., be the mixed cellularity group containing various kinds of cell composition.
Monocyte chemotactic factor-1 (MCP-1) is the one-tenth of the CC class chemotactic factor family finding the earliest and being widely studied
One of member, it has chemotaxis to mononuclear cell, natural killer cell, T lymphocyte, basophil and dendritic cell.
In the embodiment that the present invention provides, the isolation and purification method list of references of BMNC is: Sun Lin, week
The rising sun, Zhang Tiling. the extracting and developing of BMNC, labelling and In vitro culture. China cardiovascular diseases's research [J], 2008,6
(8):777-779。
Biomaterial used, reagent or instrument in stem cell medicine that the present invention provides and its preparation method and application
Buied by market.Wherein embryonic stem cell is bought in the Chinese Academy of Sciences.
Below in conjunction with embodiment, the present invention it is expanded on further:
The preparation of embodiment 1 stem cell medicine
1. with physiological saline solution MCP-1 (monocyte chemotactic factor-1), the final concentration of 10ng/mL of MCP-1.
2. with the resuspended BMNC of the normal saline containing MCP-1 and embryonic stem cell, BMNC
Final concentration of 5.0 × 105Individual/mL, final concentration of the 5.0 × 10 of embryonic stem cell5Individual/mL.
3. preparation is divided equally to 1-2 syringe.
4. preparation preserves transport: preparation is put in aseptic sylphon, the sylphon equipped with preparation is transferred to be placed with
In the storage box of ice bag, low temperature (0～4 DEG C) transports.
5. cell and quality of the pharmaceutical preparations evaluation
A, cell quality evaluation (form, vigor, surface antigen):
The motility rate of the cell in table 1 preparation
| Project | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 |
| Survival rate (%) | 97.89 | 96.47 | 98.76 | 99.43 |
BMNC is a group cell, is to be combined by polytype cell, therefore can not be resisted by surface
Former detect, according to the method for document report, BMNC can be obtained.
B, quality of the pharmaceutical preparations evaluation (antibacterial, fungus, endotoxin, five viroids):
Rear testing result made by table 2 preparation
Note: owing to embryonic stem cell is bought in the Chinese Academy of Sciences, its quality need not detect again.
The preparation of embodiment 2 stem cell medicine
1. with physiological saline solution MCP-1 (monocyte chemotactic factor-1), the final concentration of 10ng/mL of MCP-1.
2. with the resuspended BMNC of the normal saline containing MCP-1 and embryonic stem cell, BMNC
Final concentration of 1.0 × 106Individual/mL, final concentration of the 2.5 × 10 of embryonic stem cell6Individual/mL.
3. preparation is divided equally to 1-2 syringe.
4. preparation preserves transport: preparation is put in aseptic sylphon, the sylphon equipped with preparation is transferred to be placed with
In the storage box of ice bag, low temperature (0～4 DEG C) transports.
5. cell and quality of the pharmaceutical preparations evaluation: result of the test approximates with embodiment 1.
The preparation of embodiment 3 stem cell medicine
1. with physiological saline solution MCP-1 (monocyte chemotactic factor-1), the final concentration of 10ng/mL of MCP-1.
2. with the resuspended BMNC of the normal saline containing MCP-1 and embryonic stem cell, BMNC
Final concentration of 2.5 × 106Individual/mL, final concentration of the 1.0 × 10 of embryonic stem cell6Individual/mL.
3. preparation is divided equally to 1-2 syringe.
4. preparation preserves transport: preparation is put in aseptic sylphon, the sylphon equipped with preparation is transferred to be placed with
In the storage box of ice bag, low temperature (0～4 DEG C) transports.
5. cell and quality of the pharmaceutical preparations evaluation: result of the test approximates with embodiment 1.
The preparation of embodiment 4 stem cell medicine
1. with physiological saline solution MCP-1 (monocyte chemotactic factor-1), the final concentration of 10ng/mL of MCP-1.
2. with the resuspended BMNC of the normal saline containing MCP-1 and embryonic stem cell, BMNC
Final concentration of 5.0 × 106Individual/mL, final concentration of the 5.0 × 10 of embryonic stem cell6Individual/mL.
3. preparation is divided equally to 1-2 syringe.
4. preparation preserves transport: preparation is put in aseptic sylphon, the sylphon equipped with preparation is transferred to be placed with
In the storage box of ice bag, low temperature (0～4 DEG C) transports.
5. cell and quality of the pharmaceutical preparations evaluation: result of the test approximates with embodiment 1.
Embodiment 5 zoopery
1, animal model:
Taking 30 new zealand white rabbits, 3% pentobarbital sodium auricular vein injecting anesthetic (30mg/kg), before taking hip joint
Outside otch, appears femoral head and neck of femur front upper part, and row Light B μ Lb performs the operation.At neck of femur cartilage and bone intersection, with
3.5mm electric drill head drilling on inner side before femoral head, the most about 4.0mm.Curet moves under water to inner side and strikes off part cancellous bone of femoral head extremely
Subchondral bone, accounts for the 50%～60% of femoral head cumulative volume, and analog bone necrosis row focus cleaning art, dry gauze protects femoral head
Organize in addition, at once clog with the cotton balls speckling with liquid nitrogen freezing in defective region, the most freezing 25 times, the most about 8s, the most about
3min, makes osteocyte and medullary cell downright bad, and normal saline rewarming prepares ANFH animal model.
2, femur head necrosis (ANFH) animal model restorative procedure:
Test packet:
Matched group: at once sew up the incision after simple normal saline rewarming；
Embodiment 1 group: the stem cell medicine drawing 50 μ L embodiments 1 implants defective region, uses bone wax to close bone hole, sews up
Otch；
Embodiment 2 groups: the stem cell medicine drawing 50 μ L embodiments 2 implants defective region, uses bone wax to close bone hole.
Embodiment 3 groups: the stem cell medicine drawing 50 μ L embodiments 3 implants defective region, uses bone wax to close bone hole.
Embodiment 4 groups: the stem cell medicine drawing 50 μ L embodiments 4 implants defective region, uses bone wax to close bone hole.
Sewing up the incision for each group, postoperative gluteus maximus for three days on end injects gentamicin injection liquid, 2ml/, once a day.
3, evaluation methodology:
A, imaging observation (x-ray scoring)
Postoperative 2,4,8 weeks, laboratory animal took after using 3% pentobarbital sodium auricular vein injecting anesthetic (30mg/kg) and faces upward
Clinostatism, takes the photograph hip joint normotopia X-ray film, observes hip joint and the change of Cranial defect stove bone density, and parallel Lane-Sandhu x-ray is marked
Evaluate skeletonization situation.
B, histology (different time points new bone area percent)
4, x-ray evaluation comparison result
Table 3: the postoperative different time points x-ray scoring of each group is compared
| Group | Preparation | Number of elements | 2 weeks | 4 weeks | 8 weeks |
| Matched group | Normal saline | 3 | 0.34±0.16 | 1.05±0.04 | 1.48±0.03 |
| Embodiment 1 group | Embodiment 1 preparation | 3 | 0.46±0.19 | 1.24±0.55 | 1.64±0.49 |
| Embodiment 2 groups | Embodiment 2 preparation | 3 | 1.75±0.42* | 2.28±0.37* | 3.81±0.56* |
| Embodiment 3 groups | Embodiment 3 preparation | 3 | 1.89±0.37* | 2.33±0.51* | 3.96±0.43* |
| Embodiment 4 groups | Embodiment 4 preparation | 3 | 2.59±0.27* | 4.57±0.16* | 6.47±0.38* |
* represent that compared with matched group P < 0.05 has statistical significance.
Lane-Sandhu x-ray scoring display: postoperative 2,4,8 weeks, embodiment 1 contrasted with matched group 1, does not has statistics to anticipate
Justice, P > 0.05.Embodiment 2～4 composition bone be substantially better than matched group 1 and embodiment 1 group, difference statistically significant (P <
0.01), and embodiment 4 skeletonization is better than embodiment 2,3 groups, difference statistically significant (P < 0.05).
Embodiment 6 zoopery (contrast experiment)
The present embodiment animal model, restorative procedure and evaluation methodology are with embodiment 5, and each group preparation composition is as follows:
Table 4: preparation used in the present embodiment
| Group | Particular make-up |
| Matched group 1 | Normal saline |
| Matched group 2 | 3.5×106/ mL BMNC+10ng/mL MCP-1 |
| Matched group 3 | 3.5×106/ mL embryonic stem cell+10ng/mL MCP-1 |
| Embodiment 2 | 1.0×106/ mL embryonic stem cell+2.5 × 106/ mL BMNC+10ng/mL MCP-1 |
| Embodiment 3 | 2.5×106/ mL embryonic stem cell+1.0 × 106/ mL BMNC+10ng/mL MCP-1 |
1, x-ray evaluation result is as follows:
Table 5: the postoperative different time points x-ray scoring of each group is compared
| Group | Number of elements | 2 weeks | 4 weeks | 8 weeks |
| Matched group 1 | 6 | 0.29±0.16 | 0.89±0.35 | 1.27±0.51 |
| Matched group 2 | 6 | 1.24±0.19* | 1.67±0.26* | 1.98±0.33* |
| Matched group 3 | 6 | 1.38±0.17* | 1.85±0.19* | 2.24±0.28* |
| Embodiment 2 groups | 6 | 1.75±0.13# | 2.28±0.17# | 3.81±0.16# |
| Embodiment 3 groups | 6 | 1.80±0.20# | 2.32±0.28# | 3.96±0.23# |
* represent compared with matched group 1, P < 0.05, there is statistical significance；
# represents compared with matched group 2,3, P < 0.01, has extremely significantly statistical significance.
Lane-Sandhu x-ray scoring display: postoperative 2,4,8 weeks, matched group 2,3 contrasted with matched group 1, and difference has statistics
Learn meaning (P < 0.05)；And embodiment 2,3 contrasts with matched group 1, difference has extremely significantly statistical significance (P < 0.05).Real
Executing example 2,3 to contrast with matched group 2,3, difference is respectively provided with extremely significantly statistical significance (P < 0.05), show embryonic stem cell with
BMNC can produce more preferable therapeutic effect after being applied in combination.
2, new bone area percent
Table 6: the postoperative different time points new bone area percent of each group compares
| Group | Number of elements | 2 weeks | 4 weeks | 8 weeks |
| Matched group 1 | 6 | 6.7±1.7 | 17.5±2.4 | 24.8±3.6 |
| Matched group 2 | 6 | 12.6±2.1* | 26.7±2.1* | 45.0±4.1* |
| Matched group 3 | 6 | 14.2±1.8* | 28.9±2.5* | 54.6±5.4* |
| Embodiment 2 groups | 6 | 18.4±1.3# | 42.5±3.4# | 72.6±3.2# |
| Embodiment 3 groups | 6 | 19.3±1.8# | 44.1±2.9# | 74.5±3.7# |
* represent compared with matched group 1, P < 0.05, there is statistical significance；
# represents compared with matched group 2,3, P < 0.01, has extremely significantly statistical significance.
New bone area percent testing result shows: postoperative 2,4,8 weeks, matched group 2,3 contrasted with matched group 1, difference
Statistically significant (P < 0.05)；And embodiment 2,3 contrasts with matched group 1, difference have extremely significantly statistical significance (P <
0.05).Embodiment 2,3 contrasts with matched group 2,3, and difference is respectively provided with extremely significantly statistical significance (P < 0.05), shows embryo
Stem cell and BMNC can produce more preferable therapeutic effect after being applied in combination.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a stem cell medicine, it is characterised in that include embryonic stem cell, BMNC, monocyte chemotactic because of
Son-1 and solvent.
Stem cell medicine the most according to claim 1, it is characterised in that the consumption of each component in every 1mL stem cell medicine
For:
Embryonic stem cell: (1～10) × 106Individual；
BMNC: (1～10) × 106Individual；
Monocyte chemotactic factor-1:1～20ng；
Solvent: supply.
Stem cell medicine the most according to claim 1 and 2, it is characterised in that the use of each component in every 1mL stem cell medicine
Amount is:
Embryonic stem cell: (1～5) × 106Individual；
BMNC: (1～5) × 106Individual；
Monocyte chemotactic factor-1:10ng；
Solvent: supply.
Stem cell medicine the most according to any one of claim 1 to 3, it is characterised in that each in every 1mL stem cell medicine
The consumption of component is:
Embryonic stem cell: (1～2.5) × 106Individual；
BMNC: (1～2.5) × 106Individual；
Monocyte chemotactic factor-1:10ng；
Solvent: supply.
Stem cell medicine the most according to any one of claim 1 to 4, it is characterised in that described solvent is normal saline.
Stem cell medicine the most according to any one of claim 1 to 5, it is characterised in that the guarantor of described stem cell medicine
Deposit temperature≤4 DEG C.
Stem cell medicine the most according to any one of claim 1 to 6, it is characterised in that the agent of described stem cell medicine
Type is injection.
8. stem cell medicine application in preparation treatment osteonecrosis medicine as according to any one of claim 1 to 7.
Application the most according to claim 8, it is characterised in that described osteonecrosis is femur head necrosis.
10. the preparation method of stem cell medicine as according to any one of claim 1 to 7, it is characterised in that including: by monokaryon
Cell chemotactic factor-1 is dissolved in solvent, obtains monocyte chemotactic factor-1 solution, use described monocyte chemotactic factor-
The 1 resuspended embryonic stem cell of solution and BMNC, prepares described stem cell medicine.
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| CN107412744A (en) * | 2017-04-27 | 2017-12-01 | 广州资生生物科技有限公司 | A kind of Endometrial stem cell preparation and its application |
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