Background technology
The agglutination of the surface of a wound is a complicated process, and it is related to the re-epithelialization at defect of skin position, granulation groupThe processes such as the remodeling of hyperplasia, inflammatory reaction, blood vessel hyperplasia and the surface of a wound knitted, one process of any of which goes wrong, and can all leadCause wound healing obstacle.At present, the method for wound repairing is mainly operation, will open wound and is changed into by operation stitching and be closedWound is closed, so that accelerate wound healing, but postoperative scar, the wound overtension left occurs function barrier occur after splitting or heal againThe complication such as hinder.For some complex surface of a wound, such as the radioactivity surface of a wound, diabetes, the surface of a wound being used for a long time after hormoneUsually occur point of local blood circulation obstacle, the reduction of fibroblastic bioactivity, mescenchymal stem cell Deng, these surface of a woundChange and paracrine action be suppressed waits negative effect so that surface of a wound promoting epidermization is slow, inflammatory reaction reduction, the anti-infective energy of the surface of a woundPower declines, even if the surface of a wound is smaller, closes the surface of a wound using skin-grafting or the method directly sutured, is also often difficult healing.In recent years,There is the cell factor such as recombinant bovine or human fibroblastic growth factor (b-FGF), recombined human EGF, rhPDGF-BB through priorityThe launch such as gel, spray or dressing are simultaneously applied to clinic, are shown by the clinical test results of multicenter large sample,To surface of a wound such as the burn wound of light degree Ⅱ~III degree, varicose ulcer of lower extremity, diabetes, radiation ulcer and bedsoresThe therapeutic intervention of cell factor is used alone or in combination, can accelerate wound healing, and can significantly improve the healing quality of the surface of a wound.ButIt is that there are still many deficiencies：The effect of these growth factors is single；Act on after the surface of a wound, quickly by the protein kinase institute in the surface of a woundDegraded, it is impossible to effectively continuingly act on specificity target cell, so as to cause the reduction of its bioavilability, it is impossible to obtain satisfactionClinical effectiveness.
In recent years research report shows mescenchymal stem cell to the repair function of histoorgan except relying on its self-replacationOutside the ability of directed differentiation, more is to rely on a large amount of trophic factors of its secretion to adjust microenvironment in body, and activation is suffered fromThe stem cell of dormancy in person's body, so that reaching improves the effect of body pathology situation.U.S. Stemnion, Inc. etc. pass through animalThe wound healing correlation factor being rich in experiment reference's amnion multipotential stem cell culture supernatant can be effectively facilitated woundHealing, to burn, wound and diabetic ulcer etc. have preferable curative effect.There is scholar in umbilical cord Derived Stem Cells supernatantComposition carried out careful research, its supernatant, user's growth factor antibodies chip are collected to the umbilical cord stem cells of cultureAnalyzed with ELISA experiments, as a result show that supernatant contains different cell factor and chemokines, such as substantial amounts of leucocyteInterleukin 6, interleukin 8, MCP-1, TIMP-1, GM-CSF, TGF-1 etc., these factors are in general woundIt is extremely important.
HEGF (human EGF, hEGF) is that one kind is present in human multiple tissue, with extensive biologyThe polypeptide of activity, it is by the single chain polypeptide of 53 Amino acid profiles, also known as oligopeptides -1.EGF has been obtained extensively at presentCorneal transplantation, skin burn and wound, skin ulcerous, mouth after general application, such as ophthalmic cornea damage, cataract extractionChamber ulcer, gastrointestinal ulceration and glioma, squamous cell carcinoma etc.；The cosmeceutical containing EGF can also be made, promotesHuman epidermal cell metabolism.Currently with prokaryotic system express Urogastrone (rhEGF) major drawbacks be expression quantity compared withLow, bioactivity is not high, and the eukaryotic expression cycle is long, cumbersome, and the shortcomings of cost is high is difficult large-scale production.
The content of the invention
The technical problem of solution：It is an object of the invention to provide a kind of stem cell culture supernatant gel combination and its preparationMethods and applications, the stem cell culture supernatant gel combination optimizes stem cell culture supernatant and human epidermal growth factor geneThe protein binding obtained afterwards, for skin wound reparation, effect significantly, and does not stay scar.
Technical scheme：Stem cell culture supernatant gel combination, by stem cell culture supernatant, hEGF's albumenWith methylcellulose composition, solvent is water；The stem cell culture supernatant is that mescenchymal stem cell is used into doing without serumPurifying is collected after cell culture medium culture to obtain；HEGF's albumen is by human epidermal growth factor gene sequenceInduced expression is obtained after optimization.
Further, the sequence after the human epidermal growth factor gene sequence optimisation is as shown in SEQIDNO.1.
The preparation method of the stem cell culture supernatant gel combination, comprises the following steps：
Step 1, mescenchymal stem cell is used into the stem cell media culture without serum, collects supernatant, centrifuged,Ammonium sulfate is added in gained supernatant, stirring, standing, centrifugation take precipitation, are dissolved in water, filters, obtain albumen filtrate；
Step 2, step 1 gained albumen filtrate is purified through gel column, then freezed, obtain lyophilized supernatant；
Step 3, the gene optimization of e. coli codon preferences is carried out to the code area of human epidermal growth factor gene,Nde I and the restriction enzyme sites of Xho I are added respectively in gained optimization N-terminal and C-terminal, and full genome conjunction is carried out to obtained composition sequenceInto, and it is connected into the prokaryotic expression plasmid pET-30a-hEGF that prokaryotic expression carrier pET-30a is recombinated；
Step 4, step 3 gained recombinant plasmid transformed is obtained with inclusion bodies into Escherichia coli after induced expressionHEGF's albumen of expression, obtains hEGF's albumen after isolating and purifying；
Step 5, step 2 obtained freeze-drying supernatant sterilized water is dissolved, obtained aqueous solution and hEGF's albumenThe aqueous solution, methylated cellulose aqueous solution mixing, obtain stem cell culture supernatant gel combination.
Further, ammonium sulfate step is added in step 1 to carry out in 0 DEG C of ice bath.
Further, static conditions are 4 DEG C, 12h-16h in step 1.
Further, the gel column used in step 2 is 0.1M-0.15M for glucan G-25 gel columns, eluant, eluentThe NaCl aqueous solution.
Further, the concentration that the supernatant aqueous solution is freezed in step 5 is 19mg/mL, and hEGF's albumen is water-solubleThe concentration of liquid is 160-640ng/mL, and the mass concentration of methylated cellulose aqueous solution is 3%.
Application of the above-mentioned stem cell culture supernatant gel combination in skin wound reparation.
Beneficial effect：The albumen knot that the present invention is obtained after free serum culture supernatant human epidermal growth factor gene is optimizedClose, worth gel combination is used for skin wound reparation, effect significantly, and does not stay scar.
Embodiment 1
1. the preparation of lyophilized supernatant
The collection of 1.1HU-MSCs supernatants
The preparation of mescenchymal stem cell：Fresh and healthy neonatal umbilical cord is taken, is rinsed well umbilical cord with PBS, blood vessel is removed,The Fahrenheit glue tissue of the inside is stripped out, gained tissue is fully shredded to 1mm3Size, add serum free medium, in 37 DEG C, 5%CO2Cultivate, after cell is covered with, passed on 0.25% Trypsin Induced in incubator.When the umbilical cord Derived Stem Cells in the 3rd generationWhen covering with 70% in blake bottle, it is further cultured for 48 hours, collects supernatant standby.
Serum free medium main formula：Human serum albumins：4g/L-10g/L, glycine：30.00mg/L, it is nonessentialAmino acid：1mg/L-50mg/L, rh-insulin：1mg/L-10mg/L, human transferrin：5mg/L-20mg/L.
1.2HU-MSCs supernatants are slightly purified
1.2.1 cells and supernatant salt precipitation
500mL step 1.1 gained supernatants are taken, 8000rpm, 4 DEG C of centrifugation 15min remove tissue block therein and other are miscellaneousMatter, is placed in 1L beaker, is slowly added to the 304g ammonium sulfate solids weighed in advance (in terms of 85% saturation degree, with reference to ammonium sulfatePrecipitating reagent table), side edged magnetic stirrer, at least 1h is added, and above operation is completed in 0 DEG C of frozen water mixing bath.4Continue to stir to being completely dissolved in DEG C chromatography cabinet, 12h is stood in chromatography cabinet, 9000rpm, 4 DEG C of centrifugation 10min collect albumen and sunkForm sediment, be dissolved in water and be settled to 400mL, rushed with filter (plus 2 0.45 μm of filter membranes) filtrate protein solution, then with sterile deionized waterWash several times that filter membrane is to reduce the loss of albumen, final albumen filtrate volume is about 540mL.
1.2.2 gel filtration desalination, decolouring
Glucan G-25 gel columns (5*60cm) are connected into AKTA protein purification instrument, with the deionized water of the NaCl containing 0.15MAs eluant, eluent, flow velocity 10mL/min, each sample introduction 80mL are adjusted, until ultraviolet absorption value substantially rises, starts to connect sampleUntil there is no UV absorption, by the sample being collected into preservation at 4 DEG C after 0.22um membrane filtrations.
1.3 lyophilized concentrations
1) by the purifying supernatant being collected into, freeze dryer is lyophilized into powder, freezes program such as table one；
Table one freezes program
2) freeze-dried powder is dissolved into the albumen condensed liquid that concentration is 19mg/mL with sterilized water, it is standby.
The preparation of 2.hEGF albumen
GeneBank is logged in, the gene order of hEGF's hEGF active peptides is searched, its length nucleic acid is159bp, hEGF are eucaryote peptide sequence, it is contemplated that follow-up research and development are expressed in protokaryon, therefore above-mentioned hEGF is trueThe coding codon of core peptide sequence is compared analysis with Escherichia coli Preference codon.According to the degenerate of codon andDo not change hEGF active peptide sequence of amino acid it is constant in the case of, with reference to biosoftware Vector NTI Suitor 7.0HEGF nucleotide sequences are analyzed, genetic modification optimization are carried out to known hEGF, mainly by hEGF protogene sequencesThe rare codon of Escherichia coli makes the codon of preference into, to improve high level expression of the target gene in Escherichia coli.AndTransgenosis checking is carried out, the optimization of hEGF genes is finally given as shown in SEQIDNO.1.
People hEGF gene optimization sequence N-terminals and C-terminal are added into Nde I and the restriction enzyme sites of Xho I respectively.Serve Hai Shenggong companiesFull genome synthesis is carried out, and is connected into prokaryotic expression carrier pET-30a (Military Medical Science Institute's present) Nde I and the digestions of Xho IBetween site, the prokaryotic expression plasmid pET-30a-hEGF recombinated.By recombinant plasmid transformed into Escherichia coli.IPTG is luredThe hEGF small peptides for obtaining expressing with inclusion bodies after expression are led, hEGF albumen is obtained after isolating and purifying.
3. the preparation of stem cell culture supernatant gel combination
Lyophilized supernatant sterilized water dissolves, and it is 19mg/mL to freeze protein concentration in the supernatant aqueous solution, with 160ng/mLHEGF protein solutions are mixed in equal volume, are then that 3% methylated cellulose aqueous solution is mixed in equal volume with mass concentration, are produced solidifyingJelly (low dosage)；
Lyophilized supernatant sterilized water dissolves, and it is 19mg/mL to freeze protein concentration in the supernatant aqueous solution, with 640ng/mLHEGF protein solutions are mixed in equal volume, are then that 3% methylated cellulose aqueous solution is mixed in equal volume with mass concentration, are produced solidifyingJelly (high dose)；
Lyophilized supernatant sterilized water dissolves, and it is 19mg/mL to freeze protein concentration in the supernatant aqueous solution, is with mass concentration3% methylcellulose is mixed in equal volume, produces gel (no hEGF).
The skin injury of 4.ICR mouse species is healed
4.1 packet
Experimental animal is randomly divided into three big groups of blank control group (10) and experiment material group (30), wherein testing materialThe sample number that material group is divided into 3 groups, every group again is 10.
1. blank control group：10, skin of back damage, wound does not give any processing during experiment；
2. experiment material group：Totally 30, it is divided into I, II, III group
I groups：10, skin of back damage, wound gives gel (low dosage) processing during experiment.
II groups：10, skin of back damage, wound gives gel (high dose) processing during experiment.
III groups：10, skin of back damage, wound gives gel (no hEGF) processing during experiment.
The foundation of 4.2 skin injury animal models
Each group ICR mouse anesthesias, prostrate is fixed on operating table, is removed back hair with shaver, is beaten using 5mm skinsHole device prepares skin trauma model in mouse back.It is administered once at regular time and quantity daily (gel, 50 μ L/ times).
Take the tissue time：3rd, 6,8,15 days when select 5 mouse etherizations in each experiment, blank control group at random and put to deathAnd take pictures, leave and take skin histology of each group containing the surface of a wound and part normal structure.It is fixed in formalin, paraffin organization is made and cutsPiece, for follow-up HE dyeing and Masson dyeing.
Wound healing rate is calculated
Using ImageJ image analysis softwares, the size of the healing surface of a wound is calculated.
Wound healing rate=(initial surface of a wound size-size for the surface of a wound that do not heal)/initial surface of a wound size × 100%
The healing rate of the surface of a wound is as shown in the table in different time points, by three groups of Wound healing rates of different time points respectively withBlank control group carries out paired t-test, wound healing the 5th, 7 days, no hEGF groups and high dose group Wound healing rate are all higher thanControl group, the statistically significant (P of its difference<0.05)；And low dose group is the 5th, the 7 days difference between control group has poleNotable statistical significance (P<0.01).And the 3rd, 9 days, Wound healing rate each group is compared without significant difference.
The healing rate (%) of the surface of a wound of table two
Note：* represent compared with control group, the statistically significant (P of its difference<0.05)；* represented compared with control group, itsDifference has extremely notable statistical significance (P<0.01).
HE is dyed
3rd, 6,8,15 days leave and take each group incision content side 1cm scope ring layers skin histology and carry out paraffin section and makeHE dyeing is carried out, to assess the general condition of different group wound healings.
As shown in figure 1, dyeing visible, the 15th day, the visible obvious epidermis homogeneous of the blank control group surface of a wound, wound through conventional HEStill retain certain necrotic zone at mouthful, space and slight crack are still visible, skin texture is loose, and the hair follicle tissue in skin is not yetRestoration ecosystem, and the trend of collagenous fibres is irregular.Experimental group epidermis homogeneous is then relatively thin, hair follicle, sweat gland etc. has occurredSkin affiliated group.Wherein, no hEGF groups wound area is significantly less than blank group, and the newborn epithelium thickened begins with a small amount of skinThe differentiation of accessory structure；High dose and low dose group surface of a wound region complete epithelialization, and differentiate the attached knot of substantial amounts of skinCollagenous fibres in structure, but high dose group corium still have a little fracture；Comparatively low dose group surface of a wound recovery effects are best, itsIn order, skin texture is more close for arrangement of collagen fibers, and Skin appendages are the most clear, complete.
Masson is dyed
3rd, 6,8,15 days leave and take each group incision content side 1cm scope ring layers skin histology and carry out paraffin section and makeMasson dyeing is carried out, to assess the general condition of collagen formation during different group wound healings.
As shown in Fig. 2 visible through Masson dyeing, the 15th day, blank group neonatal dermal collagen slightly had accumulation, collagenous fibresArrangement is relatively unordered, and outer skin homogeneous is thicker, and incomplete epithelialization, skin level is less；It is (dark blue without hEGF group collagen accumulationsColor) clearly, skin texture is comparatively dense, it may be observed that a small amount of newborn Skin appendages；High dose and low dose groupThe surface of a wound complete epithelialization, it may be observed that substantial amounts of hair follicle, sweat gland.Particularly low dose group, repairing effect preferably, its skinStructure is rich in level, and the arrangement of collagenous fibres is clearly orderly, no fracture or mixed and disorderly phenomenon.
SEQUENCE LISTING
<110>Jiangsu Mai Jian biotechnologies Development stock Co., Ltd
<120>Stem cell culture supernatant gel combination and its preparation method and application
<130> 20170615
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 159
<212> DNA
<213>Artificial sequence
<400> 1
aacagcgact ctgaatgccc actgtcccac gatggttact gcctgcatga tggtgtgtgc 60
atgtatattg aagctctgga caagtatgca tgcaattgtg ttgtaggcta catcggcgag 120
cgttgtcaat accgtgacct gaaatggtgg gaactgcgc 159