CN107320788A - A kind of SEMA4D and CXCL12 bimoleculars collaboration promotees the preparation method and applications of endothelialization coating in situ - Google Patents
A kind of SEMA4D and CXCL12 bimoleculars collaboration promotees the preparation method and applications of endothelialization coating in situ Download PDFInfo
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/08—Materials for coatings
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/02—Inorganic materials
- A61L31/022—Metals or alloys
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/16—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/0005—Use of materials characterised by their function or physical properties
- A61L33/0011—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/06—Use of macromolecular materials
- A61L33/08—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/23—Carbohydrates
- A61L2300/236—Glycosaminoglycans, e.g. heparin, hyaluronic acid, chondroitin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/42—Anti-thrombotic agents, anticoagulants, anti-platelet agents
Abstract
The invention discloses the preparation method and applications that a kind of collaboration of SEMA4D and CXCL12 bimoleculars promotees endothelialization coating in situ, comprise the following steps：（1）It will be activated after base material polished and cleaned with NaOH, single steaming water immerses after being cleaned by ultrasonic and 12h is reacted at distilled water, 80 DEG C, it is dry after single steaming water is cleaned by ultrasonic；（2）Base material is handled with PL200；（3）SEMA4D solution is mixed in equal volume with heparin solution, reaction obtains SEMA4D/Heparin compounds；（4）Base material immerses step（3）Obtain the base material of SEMA4D/Heparin compounds modification；（5）Base material is again dipped into CXCL12 solution, both obtains target product after reaction after PBS；The present invention can preferably simulate body sequential response blood vessel endothelialization process in situ, and the biotic factor contained can cooperate with promotion endothelialization process in situ；A variety of factor collaborations play a role, and preparation method is simple, strong applicability.
Description
Technical field
The present invention relates to cardiovascular implant, the surface biological method of modifying of cardiovascular servicing unit, and in particular to a kind of
The collaboration of SEMA4D and CXCL12 bimoleculars promotees the preparation method and applications of endothelialization coating in situ.
Background technology
Endothelialization in situ can be realized by two ways, when implant surface be not inoculated with endothelial cell (ECs) situation
Under, can be by promoting the ECs of peripheral vascular tissue to migrate, adhere to and Reproduction methods are in implant surface one layer of anti-freezing of formation
Endodermis；Another is to arrive implant surface by the endothelial progenitor cells (EPCs) mobilized and capture in peripheral blood；Adhesion, propagation
And ECs is finally divided into, so as to promote the formation of endodermis；These EPCs also assist in the injury repair and blood of vascular tissue simultaneously
Pipe is rebuild；The cell factor and chemotactic factor (CF) for promote EPCs to mobilize, going back to the nest and breaking up have a lot, such as CSF3, Simvastatin
(simvastatin), CCL2, EPO, TYMP, EGF, CXCL12 (C-X-C chemokine ligands 12, i.e. SDF-1 α)；Wherein
CXCL12 is the chemotactic factor (CF) that a kind of widely used promotion EPC is mobilized, gone back to the nest and be divided into EC；Soluble Semaphorin
4D (SEMA4D) is a kind of cell factor of promotion EC migrations, can promote the generation of vascularization.
Heparin (Heparin) is the glycosaminoglycan being widely present in extracellular matrix (ECM), passes through pentasaccharide structure
Combined with antithrombase, suppress thrombin activity, and then suppress fibrin and relevant coagulation Factors to be formed, clinically often by with
Make anti-coagulants；It is mainly used in treatment DVT, thrombophlebitis and thromboembolism；Contain substantial amounts of sulfate radical in heparin structure
Group, is the elecrtonegativity having now been found that most strong biomolecule, and this strong elecrtonegativity causes heparin to combine multiple protein, blood
Platelet, ECs and other circulating cells, play and promote vascularization or the effect of anti-angiogenetic therapy；But the rush blood vessel shape of heparin
Into or anti-angiogenetic therapy effect be strongly depend on the protein classes of combination, when with angiogenesis factor, Fibroblast Growth because
The albumen knots such as son (FGFs), placenta growth factor (PIGF), tissue factor (TF) and VEGF-A (VEGFA)
It will be played during conjunction and promote vascularization effect；But work as and angiostatin, endostatin, the interferon-γ-(IP- of inducible protein -10
10) effect of anti-angiogenetic therapy will be produced when, thrombospondin I and II (TSP- I and TSP- II) is combined；Work as vascular injury
When, the blood platelet of activation can discharge 1000 various active molecules, wherein including SEMA4D and CXCL12；It is used as extracellular matrix
(ECM) constituent component, heparin has an opportunity to combine and produce corresponding with SEMA4D, CXCL12 simultaneously under normal physiological condition
Physiologic function；Heparin can promote into the identification and combination between angiogenesis factor and corresponding acceptor, activate corresponding acceptor
Signal path, so as to promote the endothelialization on body vessel transplanting surface；There is presently no find by heparin simultaneously with SEMA4D and
CXCL12 combines the technology for painstaking effort tube material.
The content of the invention
The present invention provide the preparation method that a kind of collaboration of SEMA4D and CXCL12 bimoleculars promotees endothelialization coating in situ and its
Using.
The technical solution adopted by the present invention is：A kind of SEMA4D and CXCL12 bimoleculars collaboration promotees endothelialization coating in situ
Preparation method, comprises the following steps：
(1) 8-16h will be activated with 2-3mol/L NaOH after base material polished and cleaned, single steam immerses double steam after water is cleaned by ultrasonic
Water, reacts 12h at 80 DEG C, single to steam after water is cleaned by ultrasonic, and dries；
(2) by the PL200 of treated base material 2-5mg/ml in step (1), the Immersion treatment under the conditions of 4 DEG C
It is stand-by after 12h, PBS；
(3) Heparin heparin solutions that the SEMA4D solution for being 100-400ng/ml by concentration is 10mg/ml with concentration etc.
Volume mixture, reaction 1-3h obtains SEMA4D/Heparin compounds under the conditions of 37 DEG C；
(4) base material of processing in step (2) is immersed in the SEMA4D/Heparin compounds that step (3) is obtained, at 4 DEG C
Under the conditions of reaction 12-24h after base material is cleaned；
(5) base material in step (4) is immersed in 200-400ng/ml CXCL12 solution, 12- is reacted under the conditions of 4 DEG C
Both target product is obtained after 24h, PBS.
Further, the base material includes titanium or titanium alloy.
Further, the application of the coating, for cardiovascular implantation instrument surface.
Further, the application of the coating, for anti-freezing.
Further, the application of the coating, the endothelialization in situ for promoting blood vessel.
The beneficial effects of the invention are as follows：
(1) present invention can preferably simulate body sequential response blood vessel endothelialization process in situ, and heparin is used as anti-coagulants
Internal thrombosis can be suppressed, suppress the activation and adhesion of blood platelet and fibrin in material surface；SEMA4D、CXCL12
Molecule is the biotic factor of the promotion endothelial regeneration discharged after platelet activation, promotes the effect of endothelialization with collaboration,
Quick in situ endothelialization can be promoted；
(2) heparin and a variety of factors are combined and are fixed on material surface by the present invention, and heparin can delay the half of biotic factor
Decline the phase, protection can be played under physiological environment to biotic factor, it is to avoid biotic factor is easily degraded by proteases, at the same promote it is biological because
Identification and combination between son and acceptor, it is ensured that the performance of biotic factor function；
(3) preparation technology of the present invention it is simple, easily operated, without equipment costly, it is adaptable to the various complicated hearts
Vascular implantation apparatus such as intravascular stent, thrombus filter etc. have the surface of anti-freezing/promotion endothelial migration requirement.
Brief description of the drawings
Fig. 1 is coating preparation flow schematic diagram of the present invention.
Coating and the XPS collection of illustrative plates of comparative example 1 that Fig. 2 is prepared for embodiment 1 in the present invention.
The high-resolution collection of illustrative plates of coating and C1s in the XPS of comparative example 1 that Fig. 3 is prepared for embodiment 1 in the present invention.
Fig. 4 is that coating and the fluorescence of comparative example 1 and the platelet adhesion reaction situation of comparative example 2 prepared by the embodiment of the present invention 1 shows
Micro mirror figure.
Fig. 5 is that coating and the SEM of comparative example 1 and the platelet adhesion reaction situation of comparative example 2 prepared by the embodiment of the present invention 1 is schemed.
Fig. 6 is coating and comparative example 1 and the fluorescence of the surface endothelial cell migration of comparative example 2 prepared by the embodiment of the present invention 1
Figure.
Fig. 7 is the fluorescence that coating prepared by the embodiment of the present invention 1 is captured with comparative example 1 and the endothelial progenitor cell of comparative example 2
Micrograph.
Fig. 8 is the SEM that coating prepared by the embodiment of the present invention 1 is captured with comparative example 1 and the endothelial progenitor cell of comparative example 2
Figure.
Fig. 9 is the hematoxylin-eosin that coating prepared by the embodiment of the present invention 1 implants with comparative example 1 and comparative example 2
(HE) colored graph.
Figure 10 is the immunofluorescence after coating prepared by the embodiment of the present invention 1 implants with comparative example 1 and comparative example 2
Figure.
Embodiment
The present invention will be further described with specific embodiment below in conjunction with the accompanying drawings.
As shown in figure 1, a kind of SEMA4D and CXCL12 bimoleculars collaboration promotees the preparation method of endothelialization coating in situ, including
Following steps：
(1) 8-16h will be activated with 2-3mol/L NaOH after base material polished and cleaned, single steam immerses double steam after water is cleaned by ultrasonic
Water, reacts 12h at 80 DEG C, single to steam after water is cleaned by ultrasonic, and dries；
(2) by the 2-5mg/ml of treated base material in step (1) PL200 (PLL, MW 150-300KDa),
It is stand-by after Immersion treatment 12h, PBS under the conditions of 4 DEG C；
(3) Heparin heparin solutions that the SEMA4D solution for being 100-400ng/ml by concentration is 10mg/ml with concentration etc.
Volume mixture, reaction 1-3h obtains SEMA4D/Heparin compounds under the conditions of 37 DEG C；
(4) base material of processing in step (2) is immersed in the SEMA4D/Heparin compounds that step (3) is obtained, at 4 DEG C
Under the conditions of reaction 12-24h after base material is cleaned；
(5) base material in step (4) is immersed to 200-400ng/ml CXCL12 solution (solvent is pH=7.4 PBS)
In, reacted under the conditions of 4 DEG C and target product is both obtained after 12-24h, PBS.
Further, the base material includes titanium or titanium alloy.
Further, for cardiovascular implantation instrument surface.
Further, for anti-freezing.
Further, for promoting the endothelialization process of blood vessel.
Embodiment 1
A kind of SEMA4D and CXCL12 bimoleculars collaboration promotees the preparation method of endothelialization coating in situ, comprises the following steps：
(1) 8-16h will be activated with 2-3mol/L NaOH after pure titanium-based material polished and cleaned, single steam after water is cleaned by ultrasonic is immersed
Distilled water, reacts 12h at 80 DEG C, single to steam after water is cleaned by ultrasonic, and dries；
(2) by the 2mg/ml of treated base material in step (1) PL200 (PLL, MW 150-300KDa), 4
It is stand-by after Immersion treatment 12h under the conditions of DEG C, PBS；
(3) it is concentration the SEMA4D solution for being 200ng/ml and Heparin heparin solutions that concentration is 10mg/ml is isometric
Mix, reaction 1-3h obtains SEMA4D/Heparin compounds under the conditions of 37 DEG C；
(4) base material of processing in step (2) is immersed in the SEMA4D/Heparin compounds that step (3) is obtained, at 4 DEG C
Under the conditions of reaction 12-24h after base material is cleaned；
(5) base material in step (4) is immersed in 200ng/ml CXCL12 solution (solvent is pH=7.4 PBS),
Reacted under the conditions of 4 DEG C and target product is both obtained after 12-24h, PBS.
Sample manufactured in the present embodiment is represented with C200.
Embodiment 2
A kind of SEMA4D and CXCL12 bimoleculars collaboration promotees the preparation method of endothelialization coating in situ, comprises the following steps
(1) 8-16h will be activated with 2-3mol/L NaOH after pure titanium-based material polished and cleaned, single steam after water is cleaned by ultrasonic is immersed
Distilled water, reacts 12h at 80 DEG C, single to steam after water is cleaned by ultrasonic, and dries；
(2) by the PL200 of treated base material 5mg/ml in step (1), the Immersion treatment under the conditions of 4 DEG C
It is stand-by after 12h, PBS；
(3) it is concentration the SEMA4D solution for being 400ng/ml and Heparin heparin solutions that concentration is 10mg/ml is isometric
Mix, reaction 1-3h obtains SEMA4D/Heparin compounds under the conditions of 37 DEG C；
(4) base material of processing in step (2) is immersed in the SEMA4D/Heparin compounds that step (3) is obtained, at 4 DEG C
Under the conditions of reaction 12-24h after base material is cleaned；
(5) base material in step (4) is immersed in 400ng/ml CXCL12 solution, 12-24h is reacted under the conditions of 4 DEG C,
Both target product is obtained after PBS.
Embodiment 3
A kind of SEMA4D and CXCL12 bimoleculars collaboration promotees the preparation method of endothelialization coating in situ, comprises the following steps：
(1) 8-16h will be activated with 2-3mol/L NaOH after pure titanium-based material polished and cleaned, single steam after water is cleaned by ultrasonic is immersed
Distilled water, reacts 12h at 80 DEG C, single to steam after water is cleaned by ultrasonic, and dries；
(2) by the 2.5mg/ml of treated base material in step (1) PL200 (PLL, MW 150-300KDa),
It is stand-by after Immersion treatment 12h, PBS under the conditions of 4 DEG C；
(3) it is concentration the SEMA4D solution for being 200ng/ml and Heparin heparin solutions that concentration is 10mg/ml is isometric
Mix, reaction 1-3h obtains SEMA4D/Heparin compounds under the conditions of 37 DEG C；
(4) base material of processing in step (2) is immersed in the SEMA4D/Heparin compounds that step (3) is obtained, at 4 DEG C
Under the conditions of reaction 12-24h after base material is cleaned；
(5) base material in step (4) is immersed in 200ng/ml CXCL12 solution (solvent is pH=7.4 PBS),
Reacted under the conditions of 4 DEG C and target product is both obtained after 12-24h, PBS.
Comparative example 1
(1) 8-16h will be activated with 2-3mol/L NaOH after pure titanium-based material polished and cleaned, single steam after water is cleaned by ultrasonic is immersed
Distilled water, reacts 12h at 80 DEG C, single to steam after water is cleaned by ultrasonic, and dries；
(2) by the 2mg/ml of treated base material in step (1) PL200 (PLL, MW 150-300KDa), 4
It is stand-by after Immersion treatment 12h under the conditions of DEG C, PBS；
(3) it is concentration the SEMA4D solution for being 200ng/ml and Heparin heparin solutions that concentration is 10mg/ml is isometric
Mix, reaction 1-3h obtains SEMA4D/Heparin compounds under the conditions of 37 DEG C；
(4) base material of processing in step (2) is immersed in the SEMA4D/Heparin compounds that step (3) is obtained, at 4 DEG C
Under the conditions of reaction 12-24h after base material is cleaned；
(5) base material in step (4) is immersed in 0ng/ml CXCL12 solution (solvent is pH=7.4 PBS), 4
Reacted under the conditions of DEG C and target product is both obtained after 12-24h, PBS.
Sample manufactured in the present embodiment is represented with C0.
Comparative example 2
Comparative example 2 be pure titanium-based material without any processing, represented with Ti.
Obtained sample in embodiment 1, comparative example 1 is subjected to XPS tests, as a result as shown in Figures 2 and 3, can from figure
Modification to find out CXCL12 can reduce the amount of surface heparin, increase surface protein content, show SEMA4D-CXCL12 coatings
Modify successfully.
Freshman source blood is centrifuged into 15min in 1500r/m and obtains the blood plasma of platelet rich, by blood plasma and embodiment 1,
Obtained sample is incubated jointly in comparative example 1 and comparative example 2；Blood platelet is observed 1 hour at 37 DEG C, after cleaning not same
The adhesion activation situation of product material surface；Fig. 4 is its fluorescence microscopy figure, and Fig. 5 is its scanning electron microscope diagram SEM；From figure
As can be seen that because CXCL12 modification can consume the heparin on surface, therefore blood platelet can be caused by introducing C200 samples after CXCL12
The increase of adhesion；Totally say that the platelet adhesion reaction quantity of C0 and C200 modified samples is less than pure titanium surface, show modified sample
Blood compatibility is improved.
Sample preparation prepared by embodiment 1, comparative example 1 and comparative example 2 sample in 90 °, while being pure titanium another side
The sample of coating is prepared for for surface；Allowing endothelial cell to grow to 80%-90% in pure titanium side first expires；Then by sample
The sample that 90 ° of upsets allow surface to be prepared for coating tiles；Cell can continue to migrate growth to modified side, be incubated 12h；Fig. 6 is
Its fluorescence microscopy figure；From fig. 6, it can be seen that compared to pure titanium material, the migration distance of the upper cells of C0 improves, still
Introduce CXCL12 C200 samples can further improve migration of the endothelial progenitor cells in material surface, illustrate SEMA4D with
CXCL12 has collaboration facilitation well, it is possible to increase endothelialization process in situ.
Sample prepared by embodiment 1, comparative example 1 and comparative example 2 is respectively placed in conduit and is close to catheter wall, rabbit fiber crops
It is liquor-saturated and separate arteria carotis, by arteria carotis blood drainage after sample back to jugular vein, this process continues 90min；Clean table
Sample collection is fixed after the remained blood of face；It is all in experiment to be both needed to aseptic process with contacting blood article and use heparin
(150U/ml) rinse (except sample)；Fig. 7 is its fluorescence microscopy figure, and wherein CD34 is progenitor endothelial cell surface specific antibody,
Capture ability for expert evidence endothelial progenitor cell；Fig. 8 schemes for the SEM of sample；As can be seen from the figure with Ti and C0 phases
Than the capture of endothelial progenitor cell can be improved by introducing CXCL12 C200, illustrate that there is SEMA4D and CXCL12 collaboration to promote
The ability of endothelialization in situ；Wherein DAPI refers to fluorescent dye in figure, for the dyeing of nucleus, based on endothelial progenitor cells
Number；Merge figures are the composite diagram after DAPI and CD34 staining cells.
Base material in embodiment 1, comparative example 1 and comparative example 2 is prepared into 2-3cm sample；By sample through above-mentioned processing
It is implanted into inside mouse abdominal aorta and is affixed in abdominal aorta inwall afterwards；Sample is intercepted simultaneously together with abdominal aorta after 3 weeks
It is fixed, by histotomy and dyed after sample is carefully taken out before FFPE；Fig. 9 is hematoxylin-eosin (HE) colored graph；From
It can be seen from the figure that Ti can cause abdominal aorta excessive tissue to breed and sprawl, and have a strong impact on effective blood flow area of blood vessel；C0
Smaller on the hyperblastosis influence of blood vessel with C200 samples, blood vessel entire area ratio is smaller shared by neovascular tissue；C200
The cambium of formation is minimum, and the influence to normal blood vessels is minimum；Figure 10 is immunofluorescence figure, in figure, α-SMA and CD31 difference
For marking smooth muscle cell and endothelial cell；Wherein DAPI refers to fluorescent dye in figure, the dyeing for nucleus；Merge schemes
For the composite diagram after DAPI, α-SMA and CD31 staining cells；Compared to Ti, SEMA4D-CXCL12 C200 coating material energy shapes
Into more complete endothelial layer and less smooth muscle cell；Illustrate that C200 coating materials can speed up endothelialization in situ simultaneously
Reduce hyperblastosis；Relative to C200, the endodermis of C0 formation is more imperfect, and smooth muscle cell is more in hyperplastic tissue；
Generally speaking endothelialization effect in situ is not so good as C200, illustrates that sample is introduced after CXCL12, can be cooperateed with SEMA4D in promotion original position
Pi Hua.
SEMA4D and heparin carry different electric charges under conditions of neutral ph in the present invention, and electrostatical binding forms specific albumen
Saccharide complex (i.e. SEMA4D/Heparin compounds), the PLL of rich amino can be fixed on material surface, PLL by-OH layers of richness
Amino can be combined with the heparin molecule in SEMA4D/Heparin composite surfaces by specific ionization so that will
SEMA4D/Heparin compounds are fixed on material surface；SEMA4D mainly acts on endothelial cell, can promote endothelial cell
Adhesion, migration and breed, and CXCL12 mainly has the mobilization of promotion endothelial progenitor cells (EPCs) and goes back to the nest and promotes endothelium ancestral
Cell (EPCs) is to the effect of endothelial cell differentiation, and two kinds of common presence of molecule can promote quick in situ endothelialization；Heparin is not
Molecule can be only fixed, moreover it is possible to which molecule is protected, and promote the identification between molecule and acceptor, the presence of heparin
The proteoglycans part in extracellular matrix is simulated, the strong elecrtonegativity of heparin can further attract and fixed CXCL12, and
CXCL12 is protected, while promoting the combination of CXCL12 and corresponding acceptor, the effect of fine analog cell epimatrix.
Heparin is combined and is fixed on material surface by the invention with a variety of factors, and heparin can delay biotic factor
Half-life period, protection can be played to biotic factor under physiological environment, it is to avoid biotic factor is easily degraded by proteases, while promoting life
Identification and combination between the thing factor and acceptor, it is ensured that the performance of biotic factor function；Build promote endothelialization coating in situ can be compared with
Good simulation body sequential response blood vessel endothelialization process in situ；First, heparin can suppress internal thrombus shape as anti-coagulants
Into suppressing the activation and adhesion of blood platelet and fibrin in material surface；Secondly, SEMA4D, CXCL12 molecule are that blood is small
The biotic factor of the promotion endothelial regeneration discharged after plate activation, promotes the effect of endothelialization in situ with collaboration；SEMA4D energy
Enough activation T cells start (T cell priming), promote B cell proliferation and antibody to produce, and produce positive immunoregulation effect,
Promote inner skin cell viscosity to echo propagation by acceptor PLEXINB1 simultaneously, promote injured blood vessel endothelialization；And CXCL12 can be moved
Member and capture EPCs, and promote EPCs Proliferation, Differentiations formation endothelium, so as to promote the endothelialization process of blood vessel.Promote endothelialization in situ
Apply layer building technique simply easily operated, without equipment costly, the method for submergence fixing biological molecules ensures sample table
Face biomolecule uniform fold, it is adaptable to which various complicated cardiovascular implantation instrument such as intravascular stents, thrombus filter etc. have anti-
The surface of solidifying/promotion endothelial migration requirement.
Claims (5)
1. a kind of SEMA4D and CXCL12 bimoleculars collaboration promotees the preparation method of endothelialization coating in situ, it is characterised in that including
Following steps：
(1) 8-16h will be activated with 2-3mol/L NaOH after base material polished and cleaned, single steam after water is cleaned by ultrasonic immerses distilled water,
12h is reacted at 80 DEG C, it is single to steam after water ultrasonic cleaning, dry；
(2) by the PL200 of treated base material 2-5mg/ml in step (1), the Immersion treatment 12h under the conditions of 4 DEG C,
It is stand-by after PBS；
(3) it is concentration the SEMA4D solution for being 100-400ng/ml and Heparin heparin solutions that concentration is 10mg/ml is isometric
Mix, reaction 1-3h obtains SEMA4D/Heparin compounds under the conditions of 37 DEG C；
(4) base material of processing in step (2) is immersed in the SEMA4D/Heparin compounds that step (3) is obtained, in 4 DEG C of conditions
Base material is cleaned after lower reaction 12-24h；
(5) base material in step (4) is immersed in 200-400ng/ml CXCL12 solution, 12-24h is reacted under the conditions of 4 DEG C,
Both target product is obtained after PBS.
2. a kind of SEMA4D and CXCL12 bimoleculars collaboration according to claim 1 promotees the preparation side of endothelialization coating in situ
Method, it is characterised in that the base material includes titanium or titanium alloy.
3. a kind of application of coating as claimed in claim 1, it is characterised in that for cardiovascular implantation instrument surface.
4. a kind of application of coating as claimed in claim 1, it is characterised in that for anti-freezing.
5. a kind of application of coating as claimed in claim 1, it is characterised in that the endothelialization in situ for promoting blood vessel.
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