CN107551314A - A kind of E7 collagem membranes for promoting mesenchymal stem cells MSCs adhesion and preparation method thereof - Google Patents
A kind of E7 collagem membranes for promoting mesenchymal stem cells MSCs adhesion and preparation method thereof Download PDFInfo
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- CN107551314A CN107551314A CN201710860289.8A CN201710860289A CN107551314A CN 107551314 A CN107551314 A CN 107551314A CN 201710860289 A CN201710860289 A CN 201710860289A CN 107551314 A CN107551314 A CN 107551314A
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Abstract
The invention discloses a kind of E7 collagem membranes for promoting mesenchymal stem cells MSCs (BMSCs) adhesion and preparation method thereof.The present invention is grafted E7 polypeptides using natural biologic material collagen as raw material, by bifunctionality crosslinking agent sulfo SMCC in collagen surface.The collagem membrane prepared using this method has the advantages of can remarkably promoting BMSCs adhesions, and has good biocompatibility.Stem cell has versatility, is studied for repairing the structure and function of Various Tissues, and this material promotes the method for modifying of stem cell adhesion, can be widely applied to Various Tissues repair materials, have a good application prospect.
Description
Technical field
It is more particularly to a kind of to promote mesenchymal stem cells MSCs adhesion the present invention relates to E7- collagem membranes and preparation method thereof
E7- collagem membranes and preparation method thereof.
Background technology
In recent years, there is stem cell such as embryonic stem cell, epidermal stem cells, fat stem cell and the bone of more differentiation potentials
Bone marrow-drived mesenchymal stem (BMSCs) etc. is widely used in the Regeneration and Repair of skin.Wherein, MSCs is because of its abundance, separation
With cultural method maturation and with promotion skin healing and reduce the advantages such as immunity of organism repulsion, it is considered to be realize skin again
One of raw important stem cell.Graftskin of the structure based on MSCs generally requires a number of MSCs.At present, in vitro training
Foster method is widely used in MSCs purifying and amplification.However, time-consuming for ex vivo expansion process；What is more important, leave
After internal microenvironment, MSCs easily loses its stem cell properties.The MSCs death rates that are implanted into outside numerous studies display body are high by (75%
MSCs is dead in two weeks), only a small amount of MSCs directly participates in skin regeneration with rebuilding.In recent years, with the hair of regenerative medicine
Exhibition, the thought of tissue in situ regeneration induction are accepted and paid attention to, and in the Regeneration and Repair of the tissues such as bone, corium and nerve
It is proven and applies.The regeneration induction in situ of tissue or organ, it is using body normal healing and regenerative system, to regenerate doctor
Material is regeneration template, is passed through material property (such as pore structure, chemical composition, specific proteins site and activation signal)
The knot of defective tissue or organ is rebuild in behavior, the regeneration such as adhesion, growth and the differentiation of original position induction surface of a wound area body cell/stem cell
Structure and function.E7- collagem membranes can remarkably promote BMSCs adhesion, and good adhesion has established base for follow-up cell behavior
Plinth, and its good biocompatibility, it is expected to the reparation applied to Various Tissues.
The content of the invention
It is an object of the invention to provide a kind of E7- collagem membranes for promoting mesenchymal stem cells MSCs adhesion and its preparation side
Method, the E7 polypeptides of material surface grafting, mesenchymal stem cells MSCs adhesion can be remarkably promoted.
A kind of E7- collagem membranes of promotion mesenchymal stem cells MSCs adhesion of the present invention, are in collagen film surface grafting
The E7 polypeptides that mesenchymal stem cells MSCs can be promoted to adhere to；Wherein collagem membrane can use the I type glue picked up by oneself from fresh beef tendon
Original, it is dissolved in after acid flux material is configured to collagen solution, is spun on surface of glass slide and is made；E7 polypeptides are crosslinking with sulfo-SMCC
Agent, graft on collagem membrane surface.
Described acid solution is the acetic acid of mass concentration 3%, and collagen solution concentration is 0.5% (W/V)；37 DEG C of stirrings are equal
It is even, obtain collagen solution.Collagen solution is spun on surface of glass slide, is placed in 37 degree of baking ovens and fully dries, obtain collagem membrane；
Described E7 polypeptides graft on collagem membrane surface with sulfo-SMCC and can be prepared as follows：
The first step：Sulfo-SMCC concentrates are configured with a small amount of pure water, and are diluted with pH 7.2 PBS, are obtained
Sulfo-SMCC reaction solutions；
Second step：Collagem membrane is soaked in above-mentioned sulfo-SMCC reaction solutions, soaking at room temperature at least 1 hour；
3rd step：Collagem membrane is cleaned with pH7.0 PBS and removes unnecessary sulfo-SMCC, and E7 polypeptides are dissolved in pH
E7 reaction solutions are configured in 7.0 PBS, above-mentioned collagem membrane is soaked in E7 reaction solutions into 37 DEG C reacts at least 2 hours；
The sequence of described E7 polypeptides is EPLQLKM；
4th step：Collagem membrane, nitrogen drying are cleaned with pure water.
Sulfo-SMCC is 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinyls in the first step
Imines ester sodium salt, its concentration are controlled in 0.25-1mg/mL；Most preferably, its concentration is 1mg/mL；
The PBS of the first step pH 7.2 be 0.1mol/L PBS add 0.15mol/L NaCl, regulation pH to
7.2。
In 3rd step pH 7.0 PBS be 0.1mol/L PBS add 0.15mol/L NaCl and
0.1mol/L EDTA, adjust pH to 7.0.
The E7- collagem membranes for promoting mesenchymal stem cells MSCs adhesion of the present invention, E7 polypeptides are crosslinked by sulfo-SMCC
Agent is grafted to collagem membrane surface, by E7 it is peptide modified after, adhesion rates, adhesion, adhered area of the BMSCs on collagem membrane surface
Dramatically increase, and three is lifted as the lifting of E7 grafting amounts is stable.
The raw material promoted used in the E7- collagem membranes of mesenchymal stem cells MSCs adhesion of the present invention has good biological shape
Capacitive, obtained aerogel dressing have certain mechanical property, and the antibacterial characteristics with ultraviolet light response, can controlled
Antibiotic is discharged, so as to avoid antibiotic largely using the side effect brought, suitable for the protection and treatment of the surface of a wound.
Brief description of the drawings
Fig. 1 prepares schematic diagram for the E7- collagem membranes of promotion mesenchymal stem cells MSCs adhesion；
Fig. 2 shows that collagem membrane is notable to promotion BMSCs adhesiving effect after E7 polypeptides are grafted, and (a) is on pure collagem membrane
BMSCs effect diagram is adhered to, (b) is the effect diagram that BMSCs is adhered on E7- collagem membranes；
Fig. 3 is the surface chemical composition (a) of the E7- collagem membranes that the promotion mesenchymal stem cells MSCs adheres in example 1-3
And E7 grafting amounts (b) characterize；
Fig. 4 is that BMSCs is incubated on the E7- collagem membranes in example 1-3, characterizes its capability analysis for promoting BMSCs adhesions,
It is followed successively by cell adherence rate (a), cell adhesion forces (b), 3h cell adherence areas (c) and 24h cell adherence areas (d)；
Fig. 5 is that BMSCs is placed in flow model the E7- collagem membranes surface flowed through in example 1-3, characterizes E7- collagem membranes and catches
Obtain BMSCs ability.
Embodiment
The present invention is elaborated with reference to example.
Example 1
A) 0.1g type i collagens (being picked up by oneself from fresh beef tendon) are taken, are cut into small pieces, the acetic acid solutions of 20mL 3% are dissolved in, in 37 DEG C
Swelling dissolving overnight, obtains collagen solution.20 μ L collagen solutions are coated on 1 × 1cm surface of glass slide, sprawled with spin coating instrument
It is even, it is positioned over 37 DEG C of baking ovens and fully dries, obtains collagem membrane；
B) 8g NaCl, 0.2g KCl, Na are weighed2HPO4·H2O 1.56g,KH2PO40.2g, it is added in 1000ml distilled water and matches somebody with somebody
0.1mol/L PBS are set to, then, add 8.77g NaCl, solution are divided into two parts fully after dissolving, first part of regulation
PH to 7.2, second part of addition 14.61g EDTA, adjusts pH to 7.0；
C) collagem membrane is placed in 24 orifice plates, be soaked in the above-mentioned pH 7.2 of 1mL cushioning liquid, soaking at room temperature 1 hour；This
Afterwards, clean collagem membrane 3 times with pH 7.0 PBS, collagem membrane is soaked in 37 DEG C of reactions in pH 7.0 PBS
2 hours；Finally, collagem membrane is cleaned 3 times with distilled water, nitrogen drying.
Example 2
A) 0.1g type i collagens (being picked up by oneself from fresh beef tendon) are taken, are cut into small pieces, the acetic acid solutions of 20mL 3% are dissolved in, in 37 DEG C
Swelling dissolving overnight, obtains collagen solution.20 μ L collagen solutions are coated on 1 × 1cm surface of glass slide, sprawled with spin coating instrument
It is even, it is positioned over 37 DEG C of baking ovens and fully dries, obtains collagem membrane；
B) 8g NaCl, 0.2g KCl, Na are weighed2HPO4·H2O 1.56g,KH2PO40.2g, it is added in 1000ml distilled water and matches somebody with somebody
0.1mol/L PBS are set to, then, add 8.77g NaCl, solution are divided into two parts fully after dissolving, first part of regulation
PH to 7.2, second part of addition 14.61g EDTA, adjusts pH to 7.0；
C) collagem membrane is placed in 24 orifice plates, be soaked in the above-mentioned pH 7.2 of 1mL cushioning liquid, soaking at room temperature 1 hour；
D) 0.2mg E7 polypeptides are dissolved in the E7 solution that 0.2mg/mL is configured in 1mL pH 7.0 PBS.Use pH
After 7.0 PBS cleaning collagem membrane 3 times, it is soaked in 0.2mg/mL E7 solution 37 DEG C and is reacted 2 hours；Finally,
Collagem membrane is cleaned with distilled water 3 times, nitrogen drying.
Example 3
A) 0.1g type i collagens (being picked up by oneself from fresh beef tendon) are taken, are cut into small pieces, the acetic acid solutions of 20mL 3% are dissolved in, in 37 DEG C
Swelling dissolving overnight, obtains collagen solution.20 μ L collagen solutions are coated on 1 × 1cm surface of glass slide, sprawled with spin coating instrument
It is even, it is positioned over 37 DEG C of baking ovens and fully dries, obtains collagem membrane；
B) 8g NaCl, 0.2g KCl, Na are weighed2HPO4·H2O 1.56g,KH2PO40.2g, it is added in 1000ml distilled water and matches somebody with somebody
0.1mol/L PBS are set to, then, add 8.77g NaCl, solution are divided into two parts fully after dissolving, first part of regulation
PH to 7.2, second part of addition 14.61g EDTA, adjusts pH to 7.0；
C) by 0.25mg, 0.5mg, 1.0mg sulfo-SMCC are dissolved in 200 μ L distilled water respectively, then are separately added into 800 μ L pH
7.2 buffer solution, it is configured to 1mL 0.25mg/mL, 0.5mg/mL and 1.0mg/mL sulfo-SMCC solution.
D) collagem membrane is placed in 24 orifice plates, is soaked in 1mL above-mentioned 0.25mg/mL, 0.5mg/mL and 1.0mg/mL sulfo-
In SMCC solution (corresponding final sample is respectively labeled as CP1, CP2, CP3), soaking at room temperature 1 hour；
E) 0.2mg E7 polypeptides are dissolved in the E7 solution that 0.2mg/mL is configured in 1mL pH 7.0 PBS.Use pH
After 7.0 PBS cleaning collagem membrane 3 times, it is soaked in 0.2mg/mL E7 solution 37 DEG C and is reacted 2 hours；Finally,
Collagem membrane is cleaned with distilled water 3 times, nitrogen drying.
Pure collagem membrane (embodiment 1) and to be soaked in the reaction solution of E7 polypeptides pure collagem membrane (embodiment 2) right as two
According to group, Col, Col/E7 are respectively labeled as, performance comparison such as Fig. 3,4,5 institute with sample CP1, CP2, CP3 in embodiment 3
Show.
As seen from Figure 4, on the E7- collagem membranes obtained using the inventive method, with the increase of E7 grafting amounts, adhesion
BMSCs quantity and spreading area dramatically increase, and adhesion rate is by 20% lifting 4 times to 80% (see Fig. 4-a).Especially when adopting
During with 1.0mg/mL sulfo-SMCC solution, the E7 polypeptides being grafted on collagem membrane can remarkably promote BMSCs on film
Sprawl, make what it can reach 24h in 3h to sprawl state completely.In addition, we are surveyed using flow model under mobility status
Examination BMSCs sprawls state on E7- collagem membranes, and the test can mescenchymal stem cell is gone back to the nest with peripheral blood in analogue body mistake
Journey, as a result as shown in figure 5, with the lifting of E7 grafting amounts, the quantity of stem cell is lifted, and cell is gradually sprawled, the number of right figure
The stem cell population that amount statistics more clearly shows adhesion is lifted with the lifting of E7 grafting amounts, and its adhesiving effect is to use
The collagem membrane (such as Col, Col/E7) that pure collagem membrane or other method obtain is all unapproachable.The collagem membrane of the present invention can conduct
The tissue engineering material of a variety of soft tissue repairs.
Claims (9)
1. a kind of preparation method for the E7- collagem membranes for promoting mesenchymal stem cells MSCs to adhere to, it is characterised in that including following step
Suddenly：
(1) collagen solution that mass concentration is 0.5% is prepared in acid condition, and 37 DEG C stir, and obtain collagen solution.Will
Collagen solution is spun on surface of glass slide, is placed in 37 degree of baking ovens and fully dries, obtains collagem membrane；
(2) sulfo-SMCC concentrates are configured with pure water, and is diluted with pH 7.2 PBS, obtain crosslinking agent sulfo-
SMCC reaction solutions；
(3) collagem membrane is soaked in above-mentioned sulfo-SMCC reaction solutions, soaking at room temperature at least 1 hour；
(4) clean collagem membrane with pH7.0 PBS and remove unnecessary sulfo-SMCC, E7 polypeptides are dissolved in pH 7.0 PBS
E7 reaction solutions are configured in buffer solution, above-mentioned collagem membrane is soaked in E7 reaction solutions into 37 DEG C reacts at least 2 hours；Described E7
The sequence of polypeptide is EPLQLKM；
(5) collagem membrane, nitrogen drying are cleaned with pure water.
2. a kind of preparation method of the E7- collagem membranes of promotion mesenchymal stem cells MSCs adhesion according to claims 1,
Characterized in that, described acid condition is the acetum of mass concentration 3%.
3. a kind of preparation method of the E7- collagem membranes of promotion mesenchymal stem cells MSCs adhesion according to claims 1,
Characterized in that, sulfo-SMCC concentration is 0.25-1mg/mL in described crosslinking agent sulfo-SMCC reaction solutions.
4. a kind of preparation method of the E7- collagem membranes of promotion mesenchymal stem cells MSCs adhesion according to claims 1,
Characterized in that, sulfo-SMCC concentration is 1mg/mL in described crosslinking agent sulfo-SMCC reaction solutions.
5. a kind of preparation method of the E7- collagem membranes of promotion mesenchymal stem cells MSCs adhesion according to claims 1,
Characterized in that, E7 peptide concentrations are 0.2mg/mL in described E7 reaction solutions.
6. a kind of preparation method of the E7- collagem membranes of promotion mesenchymal stem cells MSCs adhesion according to claims 1,
Characterized in that, the PBS of the pH 7.2 described in step (2) adds 0.15mol/L NaCl for 0.1mol/L PBS,
Adjust pH to 7.2.
7. a kind of preparation method of the E7- collagem membranes of promotion mesenchymal stem cells MSCs adhesion according to claims 1,
It is characterized in that pH 7.0 PBS adds 0.15mol/L NaCl and 0.1mol/ for 0.1mol/L PBS in step (4)
L EDTA, adjust pH to 7.0.
8. a kind of preparation method of the E7- collagem membranes of promotion mesenchymal stem cells MSCs adhesion according to claims 1,
Characterized in that, described crosslinking agent sulfo-SMCC is 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group ambers
Amber imide ester sodium salt.
9. a kind of E7- collagem membranes for promoting mesenchymal stem cells MSCs adhesion, it is characterised in that appointed using such as claim 1-8
Method described in one is prepared.
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Application publication date: 20180109 |