CN102229647B - Method for synovium-derived mesenchymal stem cell affinity peptide modifying stent implanted in human body - Google Patents

Method for synovium-derived mesenchymal stem cell affinity peptide modifying stent implanted in human body Download PDF

Info

Publication number
CN102229647B
CN102229647B CN 201110153632 CN201110153632A CN102229647B CN 102229647 B CN102229647 B CN 102229647B CN 201110153632 CN201110153632 CN 201110153632 CN 201110153632 A CN201110153632 A CN 201110153632A CN 102229647 B CN102229647 B CN 102229647B
Authority
CN
China
Prior art keywords
mesenchymal stem
stem cells
polypeptide
affine
synovial mesenchymal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN 201110153632
Other languages
Chinese (zh)
Other versions
CN102229647A (en
Inventor
敖英芳
邵振兴
皮彦斌
张辛
周春燕
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Peking University Third Hospital
Original Assignee
Peking University Third Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Peking University Third Hospital filed Critical Peking University Third Hospital
Priority to CN 201110153632 priority Critical patent/CN102229647B/en
Publication of CN102229647A publication Critical patent/CN102229647A/en
Priority to PCT/CN2011/084337 priority patent/WO2012167587A1/en
Application granted granted Critical
Publication of CN102229647B publication Critical patent/CN102229647B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses an amino acid sequence of synovium-derived mesenchymal stem cell affinity peptide, a screening method and an application and belongs to the field of bio-pharmaceuticals. The screening method comprises the following steps: utilizing a phage display process to respectively perform negative screening of human fibroblasts and positive screening of a synovium-derived mesenchymal stem cell; after screening, extracting the synovium-derived mesenchymal stem cell, and then decomposing the cell; extracting a phage segment which has specific affinity to the synovium-derived mesenchymal stem cell; and sequencing the phage segment, thereby acquiring a peptide segment which has high affinity to the synovium-derived mesenchymal stem cell. The amino acid sequence of synovium-derived mesenchymal stem cell affinity peptide can be used for promoting the specific affinity of biological material to the synovium-derived mesenchymal stem cell and has profound significance and wide application prospect in the field of repairing tissue engineered cartilages.

Description

The method of Synovial Mesenchymal Stem Cells is affine peptide modified implant into body support
Technical field
The invention belongs to biomedical sector, particularly aminoacid sequence, screening method and the application of the affine polypeptide of a kind of Synovial Mesenchymal Stem Cells.
Background technology
Through Development In recent 20 years, organizational project (Tissue Engineering, TE) art has been widely used in clinical every field, comprises regeneration and the reparation of osseous tissue, cartilage, nerve, blood vessel, skin and gi tract and urogenital system.In the evolution of organizational project, for the support of various tissues update and renewal is crucial, comprise renewal, the improvement of making method and the constantly bringing forth new ideas of Construction Idea of material.And the transformation on recent another apparent in view theory of organizational engineering, be exactly " abbreviation ", namely with its composition that goes as much as possible to simulate healthy tissues in external means by various complexity and technology, and then implant, not as the support of a self-regeneration is provided for human body, allow this natural " bio-reactor " of human body depend on this support and carry out self-regeneration.Because the human internal environment is very complicated, thus just can cause a lot of experiment imaginations to differ greatly with the result, and support implantation step complicated also can be limited its promotion and application clinically.But such support will specifically be modified according to needs in rooting, thereby plays the effect of inducing specific cells to adhere to, breed and break up.
In the healthy tissues, cell peripheral matrix has great importance to the biological function of cell, functional domain by the albumen in the matrix or peptide molecule is combined with cell surface receptor, the signal path of complexity in the activating cells, the biological functions such as genetic expression, adhesion, migration, propagation and differentiation to cell are regulated and control, and perhaps these functional domains only only have the length of several amino acid fragments.This control methods for we make up the active polypeptide sequence, are carried out finishing to tissue engineering bracket, provide theoretical foundation with matrix around the analog cell to the adjusting function of cell itself.Adopt dissimilar polypeptide that support is modified, also give support different biological functions.There are some researches show, adopt the cell peripheral stromatin that man-made support is carried out finishing, can improve cell to attaching rate and the proliferation rate of support, further research is found, in these cell peripheral stromatins, fibronectin (fibronectin) is to promote that cell attaches, the main component of migration.What in fact, really affect the cell biological function is the functional domain that several amino acid fragments consist of in the fibronectin.For example, adopt rgd peptide that support is modified, can improve the myocyte to the attaching rate of support, also improved the proliferate efficiency of cell, illustrate that the structural domain that only needs three amino acid tablet segment length just can cellular function make a significant impact.4 people such as Duan also have similar discovery, after utilizing corneal cell pathoklisis peptide sequence that support is modified, can improve corneal epithelial cell to the attaching rate of support, and induce corneal cell to arrange at the support higher slice, the weave construction under the physiological conditions occur being similar to.Simultaneously, utilize polypeptide that support is modified, also can regulate and control the gathering of protein molecular on tissue engineering bracket, the small molecular protein that the people such as Ryadnov utilize the multiple polypeptides fragment to make up is modified support, the affinity of utilizing polypeptide that the specific protein molecule is shown, give the function that support is caught these protein moleculars, make protein molecular in a frame peripheral enrichment, and then the biological function of regulation and control support inner cell.The people such as Shah utilize the highly affine peptide sequence of TGF-β that the facultative molecule nano support of polypeptide (PA) is modified, greatly improved the repairing effect of support to cartilage defect, this support strengthens greatly to the inducibility of stem cell to the cartilage directed differentiation, and the expression degree of mucopolysaccharide also significantly improves.Support after peptide modified is described; can load, protect; and slowly-releasing somatomedin; this slow releasing function; closely related to the degree of modification of support with degradation time and the affine polypeptide of support; when degree of modification was 5%-10%, after the 4th week that implanted, support still can be brought into play good growth factor slow-release function.And most critical is, the support that loads exogenous TGF-β and do not reprint exogenous TGF-β does not have marked difference to the final repairing effect of cartilage defect, this illustrates the affine support after peptide modified of this TGF-β, in the situation that do not load the exogenous growth factor, the a large amount of endogenic TGF-β of enrichment somatomedin, improve the microenvironment in the support, realize that induced dry-cell is to the function of cartilage differentiation.
Synovial Mesenchymal Stem Cells (SMSC) is as the newcomer in mescenchymal stem cell (MSC) family, from synovial tissue, successfully extracted first in calendar year 2001 by De Bariet al. the earliest, passed through the research in nearly ten years, Synovial Mesenchymal Stem Cells has with the similar multi-lineage potential of mescenchymal stem cell and is confirmed by numerous experiments, and Synovial Mesenchymal Stem Cells is compared other tissue-derived mescenchymal stem cells and is had stronger proliferation activity, its multi-lineage potential is subjected to donor age, the impact of passage number and preservation preserving type is less also, the position of drawing materials is also comparatively wide in range, the demonstration of what is more important the experimental results, Synovial Mesenchymal Stem Cells has the stronger potential to Chondrocyte Differentiation compared to other tissue-derived mescenchymal stem cells.
In realizing process of the present invention, the contriver finds that there is following problem at least in prior art: the screening method that does not also have the affine polypeptide of a kind of Synovial Mesenchymal Stem Cells in the prior art, the method can improve biomaterial for Synovial Mesenchymal Stem Cells specificity affinity, and profound significance and application prospect are widely arranged in tissue engineering bone/cartilage reparation field.
Summary of the invention
The purpose of the embodiment of the invention is the defective for above-mentioned prior art, provides a kind of biomaterial that can improve for the screening method of the affine polypeptide of Synovial Mesenchymal Stem Cells of Synovial Mesenchymal Stem Cells specificity affinity.
Another purpose of the present invention provides the aminoacid sequence of the affine polypeptide of Synovial Mesenchymal Stem Cells.
Another object of the present invention provides the application of the affine polypeptide of Synovial Mesenchymal Stem Cells.
The technical scheme taked of the present invention is to achieve these goals:
A kind ofly utilize the screening of improved display technique of bacteriophage can improve biomaterial for the method for the affine polypeptide of Synovial Mesenchymal Stem Cells of Synovial Mesenchymal Stem Cells specificity affinity, may further comprise the steps:
(1) the former culture of people's Synovial Mesenchymal Stem Cells and human fibroblasts: the former culture by people's Synovial Mesenchymal Stem Cells also passes a generation, obtains the positive-selecting cell; Former culture by the human fibroblasts also passes more than ten generations, obtains negative screening cell;
(2) negative screening: in feminine gender screening cell, add phage library, remove the polypeptide fragment of being combined for the ACL inoblast with P10 in the phage library;
(3) positive-selecting: in the positive-selecting cell, add the phage-displayed library bacterium liquid that obtains after the negative screening in the step (2), the Synovial Mesenchymal Stem Cells of acquisition behind positive-selecting, this Cell binding in the phage library with the affine polypeptide fragment of its specific binding;
(4) phage amplification: extraction step 3 gained Synovial Mesenchymal Stem Cells, the preparation cell pyrolysis liquid increases to the phage titre in it;
(5) titre of the phage extracting solution after the measurement amplification, 4 ℃ of preservations;
Above (1)～(5) steps repeats 4 and takes turns, and the 1st takes turns screening adds phage library stoste, later every take turns add last
Phage extracting solution after the amplification of wheel cells lysate;
(6) extract that each takes turns gained with the affine phage DNA fragment of Synovial Mesenchymal Stem Cells specificity, check order, filter out the peptide sequence that Synovial Mesenchymal Stem Cells is had high affinity: i.e. SEQ ID NO.1:LTHPRWP in the sequence table, its dna sequence dna is shown in the SEQ ID NO.2 in the sequence table: CTTACGCATCCTCGTTGGCCT.
Another technical scheme of the present invention is application and the modifying method thereof of the affine peptide modified implant into body support of Synovial Mesenchymal Stem Cells, may further comprise the steps: with 3 ' C-terminal of the affine polypeptide of Synovial Mesenchymal Stem Cells selected, connect a halfcystine (C), utilize this halfcystine (C) residue and the surface-NH of polycaprolactone (PCL) electrospinning cortina after ammonification 2Covalency is coupled, so that PCL nanofiber membrane support has the performance of specific enrichment Synovial Mesenchymal Stem Cells.
Specifically may further comprise the steps:
1) the PCL nano fibrous membrane is placed Virahol configuration 10%w/v 1, in the 6-hexanediamine, 37 ℃, place 1h;
2) by linking agent (4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC)) 4-[N-maleimide ylmethyl] hexanaphthene-1-carboxyl sulfosuccinimide is connected the amino on PCL nano fibrous membrane surface with affine polypeptide.
Step is more specifically:
1) the PCL nano fibrous membrane is trimmed to the circle that equates with the hole area of 24 orifice plates, then puts it in the hole of 24 orifice plates, every hole add 500 μ l 10%w/v 1,6-hexanediamine/aqueous isopropanol, is placed 1h by 37 ℃;
2) rinsed with deionized water is 3～5 times, distilled water rinsing 3～5 times, PBS(0.01M, pH7.4) wash 3 times;
3) every hole adds linking agent 4-(the N-maleimide ylmethyl) hexanaphthene of 400 μ l-1-carboxylic acid-3-sulfosuccinimide ester (1mg/ml SMCC, Thermo company), places 1h under the room temperature;
4) with damping fluid (PBS+18.612gEDTA of 500ml 0.01M/pH 7.4, pH 7.2～7.5) flushing that contains EDTA 3 times
5) polypeptide solution of adding 0.1mg/ml (with the above-mentioned damping fluid configuration that contains EDTA), every hole 400 μ l, 4 ℃ are spent the night;
6) rinsed with deionized water is 3 times ,-20 ℃ of pre-freezes, 4 ℃ of preservations behind the vacuum-freeze-dry.
The beneficial effect that the technical scheme that the embodiment of the invention provides is brought is:
1, obtains the specific affine polypeptide fragment of Synovial Mesenchymal Stem Cells by display technique of bacteriophage, enable targeting ground and Synovial Mesenchymal Stem Cells height affinity.
2, by being carried out covalency, affine polypeptide fragment and PCL electrospinning cortina be coupled, thereby PCL nano fibrous membrane surface is modified, enable specifically enrichment Synovial Mesenchymal Stem Cells, so that this material carries out regenerating bone or cartilage when repairing in vivo as support, can routinely adhere to Synovial Mesenchymal Stem Cells, and produce normal cell epimatrix (ECM), thereby reach better repairing effect.
3, find that in the process of this polypeptide being carried out the species specificity evaluation this polypeptide there is no obvious species specificity, so this affine peptide sequence also can be widely used in various cytologic experiments and the experimentation on animals, as a kind of affiliation carrier that screens Synovial Mesenchymal Stem Cells, can screen more efficiently, the purifying Synovial Mesenchymal Stem Cells.
Description of drawings
Fig. 1 is as a result figure of the former culture of people's Synovial Mesenchymal Stem Cells that provides in the embodiment of the invention 1;
Fig. 2 a, Fig. 2 b and Fig. 2 c are the phage locus coeruleus experimental results that provides during the phage titre of the embodiment of the invention 1 is measured; Phage 10 μ l behind the doubling dilution are added in the 200 μ l Escherichia coli bacteria liquids hatched 1-5 minute, add the rapid mixing of top layer substratum, on the LB-tet culture plate of tiling tetracyclin resistance, 37 ℃, 5%CO 2Spend the night, calculate the phage locus coeruleus number on the flat board.Then multiply by plaque forming unit (pfu) titre that dilution factor namely obtains per 10 μ l phages with this number.And the single locus coeruleus of picking increases the order-checking of extraction phage DNA in LB/ intestinal bacteria nutrient solution.Fig. 2 a: locus coeruleus quantity 10 0Fig. 2 b: locus coeruleus quantity 10 1Fig. 2 c: locus coeruleus quantity 10 2
Fig. 3 is the rate of recovery of the phage of the four-wheel screening that provides in the screening of the affine polypeptide of the phage of the embodiment of the invention 1;
Fig. 4 a is the fluidic cell detected result figure of the people's that provides of the embodiment of the invention 2 the affine polypeptide of MSC;
Fig. 4 b be for Fig. 4 a the quantitative analysis results figure of fluorescence intensity;
Referring to Fig. 4 a and Fig. 4 b, the affine polypeptide of the fluorescently-labeled Synovial Mesenchymal Stem Cells of the FITC of 1nM (SMSC-affinity peptide sequence) and mispairing polypeptide (Scramble peptide) carry out flow cytometry after hatching 1 hour with Synovial Mesenchymal Stem Cells respectively; By Fig. 4 b as seen, mispairing polypeptide and the Synovial Mesenchymal Stem Cells of FITC mark are hatched jointly, and average fluorescent strength is 5; Affine polypeptide and the Synovial Mesenchymal Stem Cells of FITC mark are hatched jointly, and average fluorescent strength is 174, and the fluorescence intensity of affine polypeptide group (174) is far above the fluorescence intensity (5) of mispairing polypeptide group;
Fig. 5 a is the fluidic cell detected result figure of the affine polypeptide of MSC of the rat that provides of the embodiment of the invention 2;
Fig. 5 b be for Fig. 5 a the quantitative analysis results figure of fluorescence intensity;
Fig. 5 c is the fluidic cell detected result figure of the affine polypeptide of MSC of the rabbit that provides of the embodiment of the invention 2;
Fig. 5 d be for Fig. 5 c the quantitative analysis results figure of fluorescence intensity;
Referring to Fig. 5 a and Fig. 5 b, rat (Rat): mispairing polypeptide and the lymphocyte of adjurant arthritis rat mescenchymal stem cell of FITC mark are hatched jointly, and average fluorescent strength is 4; Affine polypeptide and the lymphocyte of adjurant arthritis rat mescenchymal stem cell of FITC mark are hatched jointly, and average fluorescent strength is 99, and the fluorescence intensity of affine polypeptide group (99) is far above the fluorescence intensity (4) of mispairing polypeptide group;
Referring to Fig. 5 c and 5d, rabbit (Rabbit): mispairing polypeptide and the rabbit Synovial Mesenchymal Stem Cells of FITC mark are hatched jointly, and average fluorescent strength is 5; Affine polypeptide and the rabbit Synovial Mesenchymal Stem Cells of FITC mark are hatched jointly, and average fluorescent strength is 92, and the fluorescence intensity of affine polypeptide group (92) is far above the fluorescence intensity (5) of mispairing polypeptide group;
Wherein, among Fig. 4 a, Fig. 5 a and Fig. 5 c, what the peak on the left side represented is the mispairing polypeptide, and what the peak on the right represented is affine polypeptide.
Fig. 6 is the confocal laser scanning microscope result that the embodiment of the invention 2 provides; The affine polypeptide of flag F ITC and mispairing polypeptide are hatched 1h jointly with Synovial Mesenchymal Stem Cells respectively, and Phalloidine is redyed cytoskeleton, and hochest redyes karyon, and the confocal laser scanning microscope cell is combined situation with polypeptide; The result shows that affine peptide sequence (LTHPRWP) is significantly higher than mispairing polypeptide (PRHLPTW) for the avidity of Synovial Mesenchymal Stem Cells;
Fig. 7 be the embodiment of the invention 3 provide utilize the covalent attachment mode, affine polypeptide fragment is connected to the schematic diagram on polycaprolactone (PCL) nanometer electricity spinning fibre film surface;
Fig. 8 is that the polypeptide that PCL nanometer electricity spinning fibre film that the Synovial Mesenchymal Stem Cells specific polypeptide that provides of the embodiment of the invention 3 is modified is observed under laser confocal microscope connects;
Show among the figure: connected polycaprolactone (PCL) nanofiber of the affine polypeptide fragment (LTHPRWP) of FITC mark, the visible green fluorescence intensity is higher, points out higher joint efficiency;
Fig. 9 a and Fig. 9 b are the effects that the Synovial Mesenchymal Stem Cells specific polypeptide that provides of the embodiment of the invention 3 is modified PCL nanometer Electrospun support; Fig. 9 a is for having connected the affine polypeptide fragment of Synovial Mesenchymal Stem Cells; Fig. 9 b is for having connected the mispairing polypeptide fragment;
Polycaprolactone (PCL) nano fibrous membrane that will connect respectively the affine polypeptide fragment of Synovial Mesenchymal Stem Cells (LTHPRWP) and mispairing polypeptide fragment (PRHLPTW) places the Synovial Mesenchymal Stem Cells suspension, co-cultivation, the result shows: the PCL nano fibrous membrane that has connected affine polypeptide is compared adhesion and the growth that the PCL nano fibrous membrane that connects the mispairing polypeptide more is conducive to Synovial Mesenchymal Stem Cells.(blue 1:hochest-karyon; Green 2: the PCL nano fibrous membrane that connects polypeptide; Red 3: Phalloidine-cytoskeleton).
Embodiment
For making the purpose, technical solutions and advantages of the present invention clearer, embodiment of the present invention is described further in detail below in conjunction with accompanying drawing.
Embodiment 1
The affine peptide sequence of display technique of bacteriophage screening Synovial Mesenchymal Stem Cells:
(1) the former culture of people's Synovial Mesenchymal Stem Cells and human fibroblasts:
A. the former culture of people's Synovial Mesenchymal Stem Cells and going down to posterity
From the corrective surgery process of accepting total knee replacement (TKA), obtain the synovial tissue in the knee joint suprapatellar bursa, put into PBS(0.01M, pH7.4) wash 2 times, synovial tissue is shredded with eye scissors, add pancreatin, 37 ℃ of digestion 30min remove film sex organization; Centrifugal, PBS(0.01M, pH7.4) washing, abandon supernatant liquor, add 37 ℃ of digestion of 0.2% NTx enzyme 2 hours; Centrifugal, PBS(0.01M, pH7.4) wash 2 times, add the DMEM(LG that contains 10%FBS) perfect medium, changed nutrient solution one time in 2～3 days, and pass a generation, as positive-selecting cell (referring to Fig. 1).Wherein DMEM is a kind of substratum that contains each seed amino acid and glucose.
B. the former culture of human fibroblasts and going down to posterity
From the corrective surgery process of accepting total knee replacement (TKA), obtain ACL (people's anterior cruciate ligament) tissue, PBS(0.01M, pH7.4) flushing, remove film sex organization and the blood clot on surface, with eye scissors it is shredded, 37 ℃ lower to trysinization 30min removal impurity, adds perfect medium and end digestion, PBS(0.01M, pH7.4) washing is 2 times, add 0.2% NTx enzyme (preparing with DMEM), 37 ℃ digested 2-3 hour again, per hour put 37 ℃ of constant temperature oscillation casees vibration 5min, until the tissue block major part is macroscopic floss, observation of cell is most of under the inverted microscope separate after, blow and beat gently with elbow straw, add isopyknic perfect medium with in and collagenase, postdigestive cell suspension is through centrifugal, after abandoning supernatant liquor, resuspended with perfect medium, bed board, and pass more than ten generations.As feminine gender screening cell.
(2) negative screening (removing the polypeptide fragment of being combined for the ACL inoblast with P10):
A. get a ware P10 for the ACL inoblast, with trysinization, PBS(0.01M, pH7.4) wash 2～3 times; Add blocking buffer(sealing damping fluid, 0.1M NaCO 3(pH8.6), 5mg/ml BSA, 0.02%NaN 3) be resuspended in the 1mlEP pipe, 4 ℃ of sealing 1h reduce non-specific binding;
B. centrifugal (1500rpm, 5min) abandons supernatant liquor, adds the serum-free DMEM(HG of 0.5ml), cell counting (2 * 10 6);
C. the phage library (PH.D.-7TM PHage Display Library, NEB) (1 * 10 that adds 1 μ l 11PFU/10 μ l phage stoste, 100 μ l), room temperature is placed 30min;
D. centrifugal (10000rpm, 5min) draws supernatant liquor, obtains the phage-displayed library bacterium liquid after feminine gender is screened, and moves in the new EP pipe for subsequent use;
(3) positive-selecting:
A. get a ware P1 for people's SMSC(Synovial Mesenchymal Stem Cells of originating), use trysinization, PBS(0.01M, pH 7.4) washing 2～3 times; Add blocking buffer and be resuspended in the 1ml EP pipe, 4 ℃ of sealing 1h reduce non-specific binding;
B. centrifugal, abandon supernatant liquor, add the serum-free DMEM(HG of 0.5ml), cell counting (2 * 10 6);
C. add the phage-displayed library bacterium liquid after step (2) gained feminine gender is screened, room temperature is placed 30min;
D. centrifugal, abandon supernatant liquor, PBS washing 2～3 times obtains the cell behind the positive-selecting;
(4) phage amplification (referring to PH.D.-7TM PHage Display Peptide Library Kit operational manual):
A. the cell behind step (3) the gained positive-selecting is prepared cell pyrolysis liquid, the phage titre in it is increased: the lysis extracting solution is added in the 20ml ER2738 nutrient solution (being in early-log), rock at 37 ℃, hatched 4.5 hours;
B. the nutrient solution in the step 1) is added in the centrifuge tube, under 4 ℃, centrifugal 10 minutes of 10,000rpm moves into supernatant in the new pipe, recentrifuge (10,000rpm, 10min);
C. extract 80% supernatant, move on in the new centrifuge tube, add the PEG/NaCl of 1/6 volume, allow phage spend the night 4 ℃ of lower precipitations;
D. next day, 10,000rpm, phage bacterium liquid is centrifugal under 4 ℃, 15 minutes, abandon supernatant, recentrifuge (10,000rpm, 10min) is abandoned supernatant liquor;
E is with 1ml TBS(50mM Tris-HCl (pH7.5), 150mM NaCl) Eddy diffusion phage precipitation, under 4 ℃, centrifugal 5 minutes.
F. supernatant is moved on in the new centrifuge tube, again precipitate with the PEG/NaCl of 1/6 volume.Hatched 60 minutes on ice.4 ℃, centrifugal 10 minutes of 10,000rpm abandons supernatant liquor, and is again of short duration centrifugal, again abandons supernatant.
G. use the TBS(pH7.5 of 200 μ l, contain 0.02%NaN 3) the Eddy diffusion precipitation, centrifugal 1 minute, supernatant is moved on in the new centrifuge tube.This is the phage extracting solution after the amplification.
(5) phage titre is measured (referring to PH.D.-7TM PHage Display Peptide Library Kit operational manual):
A. inoculate the single bacterium colony of ER2738 in 5-10ml LB substratum, 37 ℃, the 250rpm shaking table is hatched to mid-log phase (OD 600: ~ 0.5).
B. microwave-oven-heating melts top-layer agar, is divided into 3ml/ part and divides and install in the sterilizing test tubes each dilution phage one pipe.Be stored in 45 ℃ for subsequent use.
C.37 ℃ pre-temperature LB/IPTG/Xgal is dull and stereotyped, and it is for subsequent use that each phage dilution gradient is got a flat board.
D. with the LB nutrient solution phage is carried out the dilution of 10 multiple proportions row.Suggestion dilution range: the phage culture supernatant of amplification: 10 8-10 11The elutriation eluate that does not increase: 10 1-10 4Each extent of dilution changes a fresh suction nozzle, and suggestion uses band filter core suction nozzle to avoid crossed contamination.
E is divided into 200 μ l/ equal portions in Eppendorf tube when Escherichia coli bacteria liquid reaches mid-log phase, and each phage extent of dilution is managed with one.
F. add respectively the phage of the different extension rates of 10 μ l in every pipe Escherichia coli bacteria liquid, shake fast mixing, room temperature incubation 1-5min.
G. the Escherichia coli bacteria liquid with phage-infect adds in the top-agar culture tube of 45 ℃ of pre-temperature, each pipe, and mixing is poured on the LB/IPTG/Xgal flat board of 37 ℃ of pre-temperature immediately fast.Suitably tilt flat plate evenly spreads out top-layer agar.
H. behind dull and stereotyped cooling 5min, be inverted in 37 ℃ of incubators, overnight incubation.
I checks dull and stereotyped, counting has ~ and 10 2Spot number on the flat board of individual plaque (referring to Fig. 2 a, Fig. 2 b and Fig. 2 c).Then, multiply by plaque forming unit (pfu) titre that dilution factor namely obtains per 10 μ l phages with this number.
Above (1)～(5) step repeats 4 and takes turns, and then each phage extracting solution of taking turns is checked order.
(6) phage polypeptide sequencing fragment (referring to PH.D.-7TM PHage Display Peptide Library Kit operational manual):
A. the amplification of plaque: intestinal bacteria are expanded enrichment liquid (OD:0.5) press 1:100 LB nutrient solution dilution, each plaque to be checked order clone packing one pipe, 1ml/ pipe;
B. with sterilizing toothpick or suction nozzle, the blue bacterial plaque of picking mono-clonal is put into above-mentioned 1ml culture tube in the plate of bacterial plaque number between 10-100.Be total to 20 mono-clonals of picking.
C.37 ℃, the 250rpm shaking table is hatched 4.5-5h;
D. the phage bacterium liquid after hatching changes in the centrifuge tube, centrifugal 30 seconds.Supernatant moves into new pipe, and is centrifugal again.Draw 80% supernatant liquor and change new centrifuge tube over to, this is amplification phage storage liquid, can 4 ℃ stores several weeks and little on the titre impact.After long storage is used sterile glycerol 1:1 dilution ,-20 ℃ of storages;
E. from the mono-clonal phage bacterium liquid of above-mentioned amplification, draw 500 μ l in new centrifuge tube;
F.Add 200 μ l PEG/NaCl, put upside down mixing, room temperature is placed 10min;
G. centrifugal 10min abandons supernatant liquor, and of short duration centrifugal (10000rpm, 30sec) sucks remaining supernatant liquor again;
H. the phage precipitation thoroughly is resuspended in the 100 μ l iodide damping fluids, adds 250 μ l ethanol.Incubated at room 10min
I. centrifugal 10min abandons supernatant liquor.Ethanol with 70% is washed precipitation, of short duration vacuum-drying;
J. precipitation is resuspended in the 20 μ l distilled waters, is phage masterplate dna solution;
K. order-checking through the four-wheel screening, obtains one group of peptide sequence that Synovial Mesenchymal Stem Cells is had high affinity.
Embodiment of the invention test-results:
A, the screening of phage is affine polypeptide: utilize phage display library (PH.D.-7TM PHage Display Peptide Library Kit; New England Biolabs), carried out altogether 4 and taken turns screening, after each screening, mensuration is obtained phage titre, multiply by bacterium liquid cumulative volume, is the phage total amount that reclaims after the screening, simultaneously, the phage after the screening is increased, adopt the phage after increasing to carry out the next round screening.First round screening: the phage stoste (titre: 1 * 10 that adds 1 μ l 13PFU/10 μ l), behind the lysing cell, extracting phage titre is 1 * 10 3PFU/10 μ l, after the phage amplification of extracting, phage titre is 1 * 10 11PFU/10 μ l; Second takes turns screening: add the phage 10 μ l after increasing, after the screening, reclaiming phage titre is 5.3 * 10 4PFU/10 μ l, titre is 1 * 10 after the amplification 11PFU/10 μ l; The third round screening: after the screening, reclaiming phage titre is 1.2 * 10 6PFU/10 μ l, titre is 6.0 * 10 after the amplification 10PFU/10 μ l; The fourth round screening: after the screening, reclaiming phage titre is 9.0 * 10 5PFU/10 μ l.
Table 1: the phage rate of recovery:
Figure GDA00002093511600091
Referring to Fig. 3, after four-wheel screening, obtain and comprise the affine phage of Synovial Mesenchymal Stem Cells (LTHPRWP), calculate the phage rate of recovery, and compare with phage stoste.Contain the affine polypeptide phage rate of recovery: 1.50E-05; The rate of recovery of phage stoste: 1.00E-09.The Synovial Mesenchymal Stem Cells of obtaining is affine phage body and prophage liquid phase have relatively improved 15000 times (1.50E-06/1.00E-09) to the affinity of Synovial Mesenchymal Stem Cells
B, peptide sequence the selection result
The every wheel after the screening, the phage bacterium liquid that adopts screening to obtain carries out titer determination, respectively increases the 1ml Escherichia coli bacteria liquid from locus coeruleus number random 20 bacterium colonies of picking on the xgal culture plate between 10～100, extracts phage DNA and checks order.Through the four-wheel screening, obtain the one group of peptide sequence that Synovial Mesenchymal Stem Cells is had high affinity: LTHPRWP (table 2).
Table 2 peptide sequence the selection result
Figure GDA00002093511600092
Figure GDA00002093511600101
By the sequencing result of upper table as can be known, wherein second take turns in 20 phage mono-clonals of mensuration, 2 clones' peptide sequence is: LTHPRWP; In 20 phage mono-clonals that third round is measured, 3 clones' peptide sequence is: LTHPRWP; In 20 phage mono-clonals that fourth round is measured, 11 clones' peptide sequence is: LTHPRWP.First round the selection result lacks specificity, so do not detect the entrained polypeptide fragment of phage in the elutriant.Therefore, after the four-wheel screening, obtain the peptide sequence that Synovial Mesenchymal Stem Cells is had high affinity: LTHPRWP.
Embodiment 2
The affine polypeptide of Synovial Mesenchymal Stem Cells is identified the affinity of people and rat and rabbit source Synovial Mesenchymal Stem Cells
Cell aspect affinity is identified:
(1) flow cytometry
Method: get a ware Synovial Mesenchymal Stem Cells, with 0.05% trypsinase-0.02%EDTA, digested 5 minutes, until cell comes off fully, collect suspension, 1000rpm, centrifugal 5 minutes, abandon supernatant liquor, again use PBS(0.01M, pH7.4) suspension cell, repeat 3 times, behind the 400 mesh filter screen filtration cells, recentrifuge (2000rpm, 5min), abandon supernatant liquor, 0.1mlPBS(0.01M, pH7.4) behind the Eddy diffusion cell, add the affine polypeptide of SMSC (FITC-LTHPRWPC) and the mispairing polypeptide (FITC-PRHLPTWC) of 1nM, incubated at room 1h, centrifugal (2000rpm, 5min), PBS(0.01M, pH7.4) washing is 3 times, 0.5ml PBS(0.01M, pH7.4) move in the fluidic cell pipe behind the Eddy diffusion cell, carry out flow cytometry.
Result: people's Synovial Mesenchymal Stem Cells former culture on the 60mm plate of originating, after cell confluency is greater than 90%, after hatching 1 hour with the affine polypeptide of Synovial Mesenchymal Stem Cells (FITC-LTHPRWPC) of 1nM FITC mark and mispairing polypeptide (FITC-PRHLPTWC) respectively, do flow cytometry.The mispairing polypeptide is as the blank group, and average fluorescent strength is 5; With former generation Synovial Mesenchymal Stem Cells that the affine polypeptide of Synovial Mesenchymal Stem Cells is hatched, average fluorescent strength is 174.The fluorescence intensity of polypeptide group that Synovial Mesenchymal Stem Cells is affine is 34.8 times of mispairing polypeptide, illustrates that the affine polypeptide of Synovial Mesenchymal Stem Cells has significant affinity (referring to Fig. 4 a and Fig. 4 b) to people's Synovial Mesenchymal Stem Cells cell of originating.
Equally, Synovial Mesenchymal Stem Cells with rat and rabbit source is carried out flow cytometry, by the experimental result in rat and rabbit, illustrate that the affine peptide sequence that obtains does not have species specificity, not only the Synovial Mesenchymal Stem Cells in people source had affinity, the Synovial Mesenchymal Stem Cells in rat and rabbit source is also shown very high affinity (referring to Fig. 5 a, Fig. 5 b, Fig. 5 c and Fig. 5 d).
(2) confocal laser scanning microscope
Method: the cell climbing sheet of making Synovial Mesenchymal Stem Cells, 4% Paraformaldehyde 96 is 10min fixedly, PBS(0.01M, pH7.4) washing is 3 times, and the affine polypeptide of flag F ITC and mispairing polypeptide are hatched 1h jointly with cell climbing sheet respectively, under 37 ℃, Phalloidine is redyed cytoskeleton 1h, hochest redyes karyon, and room temperature is placed 10min, is combined situation with polypeptide by the confocal laser scanning microscope cell.
The result: employment source Synovial Mesenchymal Stem Cells is made cell climbing sheet, 4% Paraformaldehyde 96 is 10min fixedly, PBS washing 3 times, the affine polypeptide of flag F ITC and mispairing polypeptide are hatched 1h jointly with cell climbing sheet respectively, under 37 ℃, Phalloidine is redyed cytoskeleton 1h, under the room temperature, hochest redyes karyon 10min, be combined situation with polypeptide by the confocal laser scanning microscope cell, observe the distribution situation of FITC green fluorescence in the Synovial Mesenchymal Stem Cells cell, as seen the affine polypeptide of Synovial Mesenchymal Stem Cells can be gathered in around the Synovial Mesenchymal Stem Cells and inside in a large number, and the rondom polypeptide of mispairing then only has very on a small quantity by the random endocytosis of Synovial Mesenchymal Stem Cells cell.Illustrate that the affine polypeptide of Synovial Mesenchymal Stem Cells has very high affinity (referring to Fig. 6) to the Synovial Mesenchymal Stem Cells in people source.
Embodiment 3
The Synovial Mesenchymal Stem Cells specific polypeptide is modified PCL nanometer electricity spinning fibre film
(1) polypeptide is synthetic: the respectively affine polypeptide LTHPRWPC of Synovial Mesenchymal Stem Cells of synthetic screening acquisition and mispairing polypeptide PRHLPTWC(contrast) (Scilight-peptide Inc, China), adopt the FITC fluorescence dye that polypeptide is carried out mark, for material surface is carried out covalent modification, link a cysteine residues (C) at 3 ' of polypeptide and be beneficial to each material imino-(NH simultaneously 2) covalent attachment;
(2) PCL nanometer electricity spinning fibre film is trimmed to the circle that equates with the hole area of 24 orifice plates, then puts it in the hole of 24 orifice plates, every hole add 500 μ l 10%w/v 1, in 6-hexanediamine/aqueous isopropanol, 37 ℃, place 1h;
(3) rinsed with deionized water is 3～5 times, distilled water rinsing 3～5 times, PBS(0.01M, PH7.4) wash 3 times;
(4) every hole adds linking agent 4-(the N-maleimide ylmethyl) hexanaphthene of 400 μ l-1-carboxylic acid-3-sulfosuccinimide ester (1mg/ml SMCC, Thermo company), places 1h under the room temperature;
(5) with damping fluid (PBS+18.612gEDTA of 500ml 0.01M/pH 7.4, the pH7.2～7.5) flushing that contains EDTA 3 times;
(6) polypeptide solution of adding 0.1mg/ml (with the above-mentioned damping fluid configuration that contains EDTA), every hole 400 μ l, 4 ℃ are spent the night;
(7) rinsed with deionized water is 3 times ,-20 ℃ of pre-freezes, 4 ℃ of preservations behind the vacuum-freeze-dry;
(8) observe polypeptide connection (referring to Fig. 8) under the laser confocal microscope.
The Synovial Mesenchymal Stem Cells specific polypeptide is modified PCL nanometer Electrospun stented test result:
Utilize the covalent attachment mode, affine polypeptide fragment LTHPRWP and mispairing polypeptide fragment PRHLPTW are connected to polycaprolactone (PCL) nanometer Electrospun rack surface (referring to Fig. 7), place the Synovial Mesenchymal Stem Cells suspension, co-cultivation.The result shows: the PCL nano fibrous membrane that has connected affine polypeptide more is conducive to adhesion and the growth of Synovial Mesenchymal Stem Cells compared to the PCL nano fibrous membrane that connects the mispairing polypeptide.(referring to Fig. 9 a and Fig. 9 b)
The above only is preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Figure IDA00002093511900011

Claims (4)

1. application that the affine polypeptide of Synovial Mesenchymal Stem Cells is used for modifying the implant into body support, wherein, the aminoacid sequence of the affine polypeptide of Synovial Mesenchymal Stem Cells is the SEQ ID NO.1 in the sequence table.
2. method with the affine peptide modified implant into body support of Synovial Mesenchymal Stem Cells, it is characterized in that, may further comprise the steps: with 3 ' C-terminal of the affine polypeptide of Synovial Mesenchymal Stem Cells selected, connect a halfcystine (C), utilize this halfcystine (C) residue and the surface-NH of polycaprolactone (PCL) electrospinning cortina after ammonification 2Covalency is coupled, so that PCL nanofiber membrane support has the performance of specific enrichment Synovial Mesenchymal Stem Cells;
The aminoacid sequence of the affine polypeptide of described Synovial Mesenchymal Stem Cells is the SEQ ID NO.1 in the sequence table.
3. the method for the affine peptide modified implant into body support of Synovial Mesenchymal Stem Cells as claimed in claim 2 is characterized in that, may further comprise the steps:
1) the PCL nano fibrous membrane is placed Virahol configuration 10%w/v 1, in the 6-hexanediamine, 37 ℃, place 1h;
2) by linking agent 4-(N-maleimide ylmethyl) hexanaphthene-1-carboxylic acid-3-sulfosuccinimide ester the amino on PCL nano fibrous membrane surface is connected with affine polypeptide.
4. the method for the affine peptide modified implant into body support of Synovial Mesenchymal Stem Cells as claimed in claim 2 is characterized in that, may further comprise the steps:
1) the PCL nano fibrous membrane is trimmed to the circle that equates with the hole area of 24 orifice plates, then puts it in the hole of 24 orifice plates, every hole add 500 μ l 10%w/v 1,6-hexanediamine/aqueous isopropanol, is placed 1h by 37 ℃;
2) rinsed with deionized water is 3～5 times, distilled water rinsing 3～5 times, PBS flushing 3 times;
3) every hole adds linking agent 4-(the N-maleimide ylmethyl) hexanaphthene of 400 μ l-1-carboxylic acid-3-sulfosuccinimide ester, places 1h under the room temperature;
4) the PBS flushing is 3 times;
5) polypeptide solution of adding 0.1mg/ml, every hole 400 μ l, 4 ℃ are spent the night;
6) rinsed with deionized water is 3 times ,-20 ℃ of pre-freezes, 4 ℃ of preservations behind the vacuum-freeze-dry.
CN 201110153632 2011-06-09 2011-06-09 Method for synovium-derived mesenchymal stem cell affinity peptide modifying stent implanted in human body Active CN102229647B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN 201110153632 CN102229647B (en) 2011-06-09 2011-06-09 Method for synovium-derived mesenchymal stem cell affinity peptide modifying stent implanted in human body
PCT/CN2011/084337 WO2012167587A1 (en) 2011-06-09 2011-12-21 Synovial mesenchymal stem cell affinitive polypeptide, screening method and use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 201110153632 CN102229647B (en) 2011-06-09 2011-06-09 Method for synovium-derived mesenchymal stem cell affinity peptide modifying stent implanted in human body

Publications (2)

Publication Number Publication Date
CN102229647A CN102229647A (en) 2011-11-02
CN102229647B true CN102229647B (en) 2013-02-20

Family

ID=44842261

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 201110153632 Active CN102229647B (en) 2011-06-09 2011-06-09 Method for synovium-derived mesenchymal stem cell affinity peptide modifying stent implanted in human body

Country Status (2)

Country Link
CN (1) CN102229647B (en)
WO (1) WO2012167587A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102229647B (en) * 2011-06-09 2013-02-20 北京大学第三医院 Method for synovium-derived mesenchymal stem cell affinity peptide modifying stent implanted in human body

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009026218A2 (en) * 2007-08-17 2009-02-26 University Of Illinois Identification and use of peptide inhibitors of protein synthesis

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2439943T3 (en) * 2005-09-02 2014-01-27 ED. Geistlich Söhne AG für Chemische Industrie Meniscus tear repair method
CN102229647B (en) * 2011-06-09 2013-02-20 北京大学第三医院 Method for synovium-derived mesenchymal stem cell affinity peptide modifying stent implanted in human body

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009026218A2 (en) * 2007-08-17 2009-02-26 University Of Illinois Identification and use of peptide inhibitors of protein synthesis

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Fiber recruiting peptides: noncovalent decoration of an engineered protein scaffold;Ryadnov MG, Woolfson DN;《J Am Chem Soc.》;20040623;第126卷(第24期);全文 *
Ryadnov MG, Woolfson DN.Fiber recruiting peptides: noncovalent decoration of an engineered protein scaffold.《J Am Chem Soc.》.2004,第126卷(第24期),

Also Published As

Publication number Publication date
CN102229647A (en) 2011-11-02
WO2012167587A1 (en) 2012-12-13

Similar Documents

Publication Publication Date Title
CN102229646B (en) Amino acid sequence of bone marrow mesenchymal stem cell affinity peptide, screening method and application
Ding et al. Synthetic peptide hydrogels as 3D scaffolds for tissue engineering
Yan et al. Enhanced osteogenesis of bone marrow‐derived mesenchymal stem cells by a functionalized silk fibroin hydrogel for bone defect repair
Kretsinger et al. Cytocompatibility of self-assembled β-hairpin peptide hydrogel surfaces
CN108310467B (en) Assembled cell-derived extracellular matrix membrane composite bone repair material and preparation method and application thereof
Li et al. Functional regeneration of ligament-bone interface using a triphasic silk-based graft
Zhang et al. Human dental pulp progenitor cell behavior on aqueous and hexafluoroisopropanol based silk scaffolds
CN110237303B (en) Preparation method of acellular periosteum matrix gel material from natural tissue source
Pu et al. The use of flow perfusion culture and subcutaneous implantation with fibroblast-seeded PLLA-collagen 3D scaffolds for abdominal wall repair
Qiu et al. Cyclic tension promotes fibroblastic differentiation of human MSCs cultured on collagen‐fibre scaffolds
CN100571791C (en) The manufacture method of artificial joint
Wang et al. Self‐assembled peptide‐based hydrogels as scaffolds for proliferation and multi‐differentiation of mesenchymal stem cells
JPH04505717A (en) In vivo cartilage generation from cell cultures
Meng et al. Microfracture combined with functional pig peritoneum-derived acellular matrix for cartilage repair in rabbit models
WO2014190591A1 (en) Biphasic scaffold for tissue engineered joint, and preparation method and use thereof
CN109847098B (en) Composite biological scaffold material for repairing bone defect
Olvera et al. Spatial presentation of tissue-specific extracellular matrix components along electrospun scaffolds for tissue engineering the bone–ligament interface
CN110354311A (en) Extracellular matrix composite transparent matter acid gel and preparation method thereof, application and biomaterial
CN110478528A (en) A kind of preparation method and applications of novel rush tissue renovation material
Wu et al. Electrospun fibers immobilized with BMP-2 mediated by polydopamine combined with autogenous tendon to repair developmental dysplasia of the hip in a porcine model
Arredondo et al. Fibronectin-coating enhances attachment and proliferation of mesenchymal stem cells on a polyurethane meniscal scaffold
Gan et al. Three-dimensional, biomimetic electrospun scaffolds reinforced with carbon nanotubes for temporomandibular joint disc regeneration
Pan et al. Hydrogel modification of 3D printing hybrid tracheal scaffold to construct an orthotopic transplantation
WO2024037293A1 (en) Cellular matrix nerve graft for repairing peripheral nerve injury and preparation method therefor
CN102229647B (en) Method for synovium-derived mesenchymal stem cell affinity peptide modifying stent implanted in human body

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant