CN108295243A - Application of the soluble Jagged1 peptides in preparing the drug for promoting articular cartilage reparation - Google Patents
Application of the soluble Jagged1 peptides in preparing the drug for promoting articular cartilage reparation Download PDFInfo
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- CN108295243A CN108295243A CN201810162754.5A CN201810162754A CN108295243A CN 108295243 A CN108295243 A CN 108295243A CN 201810162754 A CN201810162754 A CN 201810162754A CN 108295243 A CN108295243 A CN 108295243A
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- peptides
- jagged1
- cartilage
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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
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
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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
Abstract
The present invention relates to soluble Jagged1 peptides to promote articular cartilage reparation preparing, or prepare treat various impacting injuries, degenerated, the not normal property disease of old cartilage metabolism drug in application.Soluble Jagged1 peptides handle placenta mesenchyma stem cell, promote it to after cartilage differentiation, the composition of soluble Jagged1 peptides and placenta mesenchyma stem cell is injected to knee joint cavity, inhibits articular chondrocyte apoptosis, promote Subchondral drilling, to repair the articular cartilage of damage.The present invention can be used for medicine and veterinary applications, can be administered alone to individual, or be applied together with other supplement activity ingredients, reagent, drug or hormone.
Description
Technical field
The present invention relates to a kind of methods of new promotion articular cartilage reparation.Particularly relate to soluble Jagged1 peptides
The placenta mesenchyma stem cell of processing carries out joint cavity injection, as various impacting injuries, degenerated, old cartilage metabolism
The application of not normal property disease treatment method.
Background technology
Chinese patent CN104162148A discloses FGF9 in 2014-11-26 and is preparing promotion cartilage of osteoarthritis reparation
Drug in application, the study found that FGF9 can play retardance articular cartilage in the articular chondrocytes in inflammatory environment
The effect of cell abnormal differentiation can alleviate the degradation of articular cartilage in mouse osteoarthritis, inhibit joint cartilage degradation
Key enzyme MMP-13, slow down the looseization process of articular cartilage, can be used for prepare promote cartilage of osteoarthritis reparation medicine
Object has potential good application prospect in osteoarthritis treatment field.
WO2016/112176 was disclosed to can be used for enhancing articular cartilage in mammals and be repaiied on 07 14th, 2016
Multiple, treatment joint injury or prevention, the inhibition or pharmaceutical composition for treating osteoarthritis.The composition may include a effective amount of
The protein of the separation of chemoattractant as Subchondral drilling progenitor cells and/or a effective amount of as the Subchondral drilling factor
The protein or encoding cartilage of separation form the nucleic acid sequence of the factor.
Chinese patent CN 104586876A disclose MicroRNA-29b in 2015-05-06 and are being closed as drug target
Save the application in repair of cartilage, the present invention passes through the study found that microRNA-29b is regulation and control MSCs at occurring after cartilage differentiation
The crucial miRNA that hypertrophyization changes, therefore, the present invention inhibit the sources MSCs cartilage by using microRNA-29b inhibitor
Cellular mast stablizes the chondrocyte phenotype in the sources MSCs, prevents tissue engineering bone/cartilage fibrosis and ectopic ossification, to
Tissue engineering bone/cartilage reparation is further realized, is provided for tissue engineering bone/cartilage structure, cartilage support material design and repair of cartilage
New approaches.
Articular cartilage damage belongs to clinical refractory disease, and the cell mainly with composition articular cartilage and ligament occurs
Permanent or semipermanent cell is tended to belong to, cell itself repair ability difference is related.
Stem cell is a kind of cell for keeping stablizing undifferentiated state and having multinomial differentiation potential under extraneous intervene,
It is had broad application prospects in terms of organ transplant, genetic modification.Placenta source property mescenchymal stem cell have abundance,
The characteristics of reliable in quality, shorter mention ethics problem, therefore can be as effective source of therapeutic stem cell.Hes1 albumen
By activating cartilage Notch accesses to promote cartilage apoptosis, and the composition of its inhibition ligand Jagged1 and PMSC then can be with
It induces PMSC to Chondrocyte Differentiation, promotes repair of cartilage.
Invention content
According to an embodiment of the invention, it is desirable to provide a kind of method that can effectively repair injured joint cartilage is realized simple
Effectively, cost-effective purpose, and its medical usage is provided.
According to embodiment, Notch signal paths inhibition ligand provided by the invention, soluble Jagged1 peptides can promote
Into placenta mesenchyma stem cell to Chondrocyte Differentiation, promote Subchondral drilling, the effective dose of Jagged1 is 10ug/ml.
According to embodiment, the present invention effectively repairs damage after proposing Jagged1 and placenta mesenchyma stem cell joint cavity injection
The articular cartilage of wound.Using the buffer solution of 8ul solubility containing 10ug/ml Jagged1 peptides and 200,000 placenta mesenchyma stem cells
Mouse intraarticular injection is carried out, is injected 1 time weekly, continuous injection 4 weeks.
Subsequent embodiment and test example is it can be proved that Jagged1 provided by the invention and placenta mesenchyma stem cell close
Chamber injection is saved, there is the purposes for the treatment of impacting injury, degenerative disorders, the not normal property disease of old cartilage metabolism.
By the per kilogram of body weight dosage conversions coefficient table of animal and human body.Mouse (20g) per injection 0.08ug is soluble
Jagged1 peptides, (60kg, w coefficient are 0.11) per injection 26.4ug solubilities Jagged1 peptides to people.By 10ug/ml solubilities
Jagged1 peptides configure, according to common people weight range 40-120kg, because the injection system of this person is scaled 1.96ml-5.28ml
The buffer solution of the Jagged1 peptides of solubility containing 10ug/ml and 200,000 placenta mesenchyma stem cells is into person joint's intracavitary administration, often
Week injection 1 time, continuous injection 4 weeks.
The dosage of other mammals, according to the per kilogram of body weight dosage conversions with human body.
Description of the drawings
Figure 1A, Figure 1B, Fig. 1 C, Fig. 1 D are after PMSCs expanded for 4 generations, and mechanocyte sample shape is presented in most of PMSCs
State, flow cytometry show PMSCs in overwhelming majority cell express human class leukocyte antigen CD29, CD73, CD90 and
CD166；Fig. 1 E are not express endothelial marker CD34；Fig. 1 F are the statistical chart of each cell sign object.
Fig. 2A -2D are that IgG control groups and JAG1 group PMSCs micelles HE, Alcian Blue/Orange G, Col II exempt from
Epidemic disease histochemical staining and Subchondral drilling marker gene expression.
Fig. 3 A-3C are control group, JAG1 injection group, JAG1/PMSC injections group, IgG/PMSC injection groups Alcian merely
Blue/Orange G dyeing, OARSI scorings and immunohistochemical staining.
Fig. 4 A-4D are control group, tri- groups of TUNEL dyeing of JAG1/PMSC, IgG/PMSC and statistics, inflammatory Cytokines Expression water
It is flat.
Specific implementation mode
In the following with reference to the drawings and specific embodiments, the present invention is further explained.These embodiments are interpreted as being only used for
It is bright the present invention rather than limit the scope of the invention.After having read the content of the invention recorded, art technology
Personnel can make various changes or modifications the present invention, these equivalence changes and modification equally fall into the claims in the present invention institute
The range of restriction.
In following embodiment of the present invention, the experimental method not indicated especially, i.e. flow cytometry, RT-PCR, Alcian
Blue/Orange G dyeing, immunohistochemical staining, Tunel dyeing are experimental method generally in the art.
Embodiment 1
The preparation of 1.1 placenta mesenchyma stem cells (PMSC)：
The removing of placental villi and base part under gnotobasis, remainder fully wash under PBS buffer solution, pancreas
Enzymic digestion remaining chorionic villi stands 90min at 37 DEG C in DMEM culture mediums, is stored in DMEM+10%FBS (calves later
Serum) in it is for use；PMSCs starts to grow after 3-5 days, and spreading 80% collection in culture dish falls behind, and cell secretes tryptophan.The IV phases
PMSCs with characteristic CD29-APC CD73-PE CD90-APC and CD166-APC express, and CD34-APC expression compared with
It is few.
1.2 solubility Jagged1 (JAG1) peptides handle PMSCs：
2.5x104PMSCs cells centrifuge after five minutes by 250g, form cell micelle, cell is divided into two groups, in no blood
Clearly at cartilage differentiation medium culture on the basis of, give 10ug/ml JAG1 peptides or 10ug/ml IgG processing respectively, handle
After 14 days, after cell micelle is fixed with 4% paraformaldehyde, it is dehydrated, paraffin embedding, passes through Alcian Blue/ after slice
Orange G and Col II (cartilaginous tissue specificity collagen) immunohistochemical staining is observed.And cell micelle is taken to extract
RNA, RT-PCR detect the expression of Subchondral drilling marker aggrecan, Col II and Sox9.
1.3 experimental result：
1.3.1PMSCS the characteristics of cell
For primary PMSCs after 4 generations were proliferated, mechanocyte sample form, fluidic cell point is presented in most of PMSCs
Analysis shows most cell express human class leukocyte antigen CD29, CD73, CD90 and CD166 (such as Figure 1A, figures in PMSCs
1B, Fig. 1 C, shown in Fig. 1 D), these cells still have very high.Without expressing endothelial marker CD34 (as referring to figure 1E).
Fig. 1 F are the statistical chart of each cell sign object.
1.3.2 soluble non-binding JAG1 peptides for PMSCs at the influence of cartilage differentiation.
We further observe the IgG or JAG1 populations of cells handled.Compared with IgG groups, soluble non-knot
The plastidogenetic cartilage cell's colonies of PMSCs for closing JAG1 inductions are more, and AlcianBlue dyeing is deeper, and II Collagen Type VIs are immune
Dyeing explanation cartilage matrix under JAG1 treatments generates apparent (as shown in Figure 2 A).RT-PCR data also illustrate Subchondral drilling mark
The expression of note object aggrecan, ColII and Sox9 obviously increase (as shown in Fig. 2 B-2D).
Embodiment 2
Right side of mice knee joint osseous arthritis (OA) model is established using MLI art formulas, i.e. 12 week old right side of mice knees close
After saving medial meniscus excision completely, a large amount of normal saline flushings remove tissue remnants, suture capsular ligament, are successfully manufactured after 4 weeks
Post-traumatic arthritis model.After right side of mice medial meniscus of knee joint removes operation 4 weeks, mouse is divided into 4 groups：control
Group, JAG1 groups, PMSC+IgG groups and PMSC+ JAG1 groups.Contain 10 μ g/ml JAG1 (or IgG) and 200,000 using 8 μ l for two groups afterwards
The PBS buffer solutions of PMSCs are injected into the successful right side of mice articular cavity of modeling, and control groups are injected with same volume PBS,
JAG1 only injects the PBS buffer solution that 8 μ l contain 10 μ g/ml JAG1.Arthroncus caused by order to reduce arthrocentesiS, in MLI
Week injection 1 time every time after performing the operation 4 weeks carries out Alcian Blue/Orange G dyeing to knee joint after injection 4 weeks (4 times), exempts from
Epidemic disease histochemical stain is analyzed.
Three examiners change degree (0- using osteoarthritis research common recognition (OARSI) scoring evaluation articulating joint surface cartilage
6 points)：0, normal cartilage；0.5, Alcian Blue dyeing is not normal, changes without cartilage structure；1, small range fibrosis is not
It is lost with cartilage；2, go deep into the crack of cartilage surface；The cracks 3-6 and cartilage surface calcification are simultaneously deposited, 25-50% (4 grades), 50-
75% (5 grades), ＞ 75% (6 grades).Classification is bigger, and lesion is more serious.Immunohistochemical method detects Col II and Col X tables
It reaches.
Experimental result：
Joint cavity injection JAG1+PMSCs promotes OA mouse articular cartilage reparations
The postoperative Alcian Blue/Orange G dyeing displayings (as shown in Figure 3A) of MLI, PBS control group occurs significant
Class OA lesions.Also there is the apparent of articular cartilage and strips off in simple JAG1 injections group, only small part Alcian indigo plants dyeing sun
Property.And it is more obvious in JAG1/PMSC injection group Alcian Blue stained positives.Compared with IgG/PMSCs groups, JAG1/
PMSC groups form deeper cartilage layers, while the dyeing of Alcian indigo plants becomes apparent.OARSI scorings are shown in JAG1 and PBS groups
Between OARSI score indifference, however the cartilage degeneration degree of JAG1/PMSC joint cavity injection groups is substantially less than IgG/PMSC
Group (as shown in Figure 3B).Immunohistochemical staining is shown, compared with IgG/PMSC groups, JAG1/PMSC group Col II obviously increase
Add (as shown in Figure 3 C), illustrates to be formed when JAG1 and PMSC combined treatments can be obviously promoted cartilage matrix.(cartilage increases Col X
Grow the marker of migration) be cartilage cell's hypertrophy regression marker, generally articular cartilage deep layer occur.Postoperative 8 weeks of MLI
Cartilage surface layer and middle level just disappear, and the visible Col X of control group only occur in the thin layer of articular cartilage, and the layer was answered originally
This is the position that initial joint cartilage occurs.IgG/PMSC group cartilage surface Col X obviously occur, and have injected JAG1/PMSC
Mouse group Col X expression reduces, and only (as shown in Figure 3 C) occurs in articular cartilage deep layer.
Embodiment 3
Right side of mice knee joint OA patterns are established using MLI art formulas, after 4 weeks, mouse is divided into 3 groups：Control groups, PMSC
+ IgG is organized and PMSC+JAG1 groups.Two groups of PBS bufferings using 8 μ l containing 10 μ g/ml JAG1 (or IgG) and 200,000 PMSCs afterwards
Liquid is injected into the successful right side of mice articular cavity of modeling, and control groups are injected with same volume PBS, after MLI performs the operation 4 weeks
Week injection 1 time every time, TUNEL dyeing observation articular chondrocyte apoptosis is carried out after injection 4 weeks (4 times) to knee joint, and extracting knee closes
Synovial tissue RNA is saved, detection inflammatory Cytokines Expression is horizontal.
Joint cavity injection JAG1+PMSCs inhibits OA mouse articular chondrocytes apoptosis and synovial membrane inflammation factor expression
The OA courses of disease include that the apoptosis of cartilage cell accelerates, and positive correlation is presented with OA severity in apoptosis.TUNEL
Dyeing display, compared with lgG/PMSCs groups, JAG1/PMSC joint injections can significantly reduce soft in MLI postoperative ankle cartilages
Apoptosis of bone cells (as illustrated in figures 4 a and 4b).Synovial tissue can generate pro-inflammatory cytokine TNF-α, IL-1 β, MMP1 (groups
Knit metalloproteinases 1) and MMP13 (tissue metal proteases 13), these inflammatory factors can promote local inflammation reaction,
Accelerate articular chondrocyte apoptosis in the OA courses of disease.PCR data shows two groups of MMP1 indifferences, PMSCs inject posterior joint intracavitary TNF-α,
IL-1 β and MMP13 are reduced, and JAG1/PMSC joint injections can more significantly inhibit the expression of the above-mentioned factor (as schemed
Shown in 4C).In addition to this gene Hes1 expression is substantially reduced under JAG1/PMSC groups synovial tissue Notch, discloses JAG1 tools
Having reduces the active effect (as shown in Figure 4 D) of Notch signal paths.
Claims (6)
1. solubility Jagged1 peptides are preparing promotion articular cartilage reparation, or treat various impacting injuries, regression preparing
Property, the old not normal property disease of cartilage metabolism drug in application.
2. the composition of solubility Jagged1 peptides and placenta mesenchyma stem cell composition, or solubility Jagged1 peptides stimulate tire
The composition that disk mescenchymal stem cell is formed promotes articular cartilage reparation preparing, or prepare treat various impacting injuries,
Degenerated, the old not normal property disease of cartilage metabolism drug in application.
3. purposes according to claim 1 or 2, characterized in that the dosage of soluble Jagged1 peptides is 10ug/ml.
4. purposes according to claim 2, characterized in that the composition includes pharmaceutically acceptable, suitable pharmaceutical
The drug of medicine and veterinary applications is made in the excipient and/or carrier on way.
5. purposes according to claim 2 or 4, characterized in that the composition is carried out joint cavity injection.
6. purposes according to claim 5, characterized in that by 1.96ml-5.28ml solubilities containing 10ug/ml Jagged1
The composition of peptide and placenta mesenchyma stem cell, or solubility Jagged1 peptides stimulate the group that placenta mesenchyma stem cell is formed
It closes object and carries out intraarticular injection, inject 1 time weekly, continuous injection 4 weeks.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110950927A (en) * | 2019-12-04 | 2020-04-03 | 广州领晟医疗科技有限公司 | Peptide GGS11 for promoting cartilage regeneration and application thereof |
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| CN101490245A (en) * | 2006-06-20 | 2009-07-22 | 建新公司 | Serum-free media and their uses for chondrocyte expansion |
| CN103550256A (en) * | 2013-10-21 | 2014-02-05 | 南京优而生物科技发展有限公司 | Application of autologous adipose-derived mesenchymal stem cells in preparation of medicines for treating joint diseases |
| CN106822876A (en) * | 2017-02-27 | 2017-06-13 | 山东景源生物科技有限公司 | A kind of medicine for blocking gonitis articular cartilage damage patient's inflammatory factor |
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2018
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101490245A (en) * | 2006-06-20 | 2009-07-22 | 建新公司 | Serum-free media and their uses for chondrocyte expansion |
| CN103550256A (en) * | 2013-10-21 | 2014-02-05 | 南京优而生物科技发展有限公司 | Application of autologous adipose-derived mesenchymal stem cells in preparation of medicines for treating joint diseases |
| CN106822876A (en) * | 2017-02-27 | 2017-06-13 | 山东景源生物科技有限公司 | A kind of medicine for blocking gonitis articular cartilage damage patient's inflammatory factor |
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| Title |
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| 孙俊魁: "调控VEGF与Notch信号通路对胎盘间充质干细胞修复骨关节炎软骨缺损的实验研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110950927A (en) * | 2019-12-04 | 2020-04-03 | 广州领晟医疗科技有限公司 | Peptide GGS11 for promoting cartilage regeneration and application thereof |
| CN110950927B (en) * | 2019-12-04 | 2022-05-20 | 广州领晟医疗科技有限公司 | Peptide GGS11 for promoting cartilage regeneration and application thereof |
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