CN107496348A - A kind of hydrogel and preparation method for tissue damage reparation - Google Patents
A kind of hydrogel and preparation method for tissue damage reparation Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
- A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
Abstract
The invention discloses a kind of hydrogel and preparation method for tissue damage reparation, its preparation method comprises the following steps：Hydrogel material is dissolved using sterile distilled water, swelling, obtains hydrogel；PGE2 powder is dissolved in phosphate buffer PGE2 solution is made, PGE2 solution is added drop-wise in hydrogel, under the conditions of 28 DEG C, stirred 35 hours；Preserve or freeze, the PGE2 is writing a Chinese character in simplified form for prostaglandin E2.The hydrogel of the present invention is capable of a certain degree of extension PGE2 release time, is directly used in the treatment of tissue damage, can promote the recovery of injured cutaneous tissue and function assessment.It can be polarized with induced damage site macrophage to M2 types and suppress inflammatory reaction, accelerate the healing of damage, and the angiogenesis of damage location in early promotion.In addition, the hydrogel no cytotoxicity of the present invention, can be applied directly to wound surface, and it is easy to use, it is cheap, and there is effective therapeutic action.
Description
Technical field
The present invention relates to a kind of hydrogel and preparation method for tissue damage reparation.
Background technology
Skin injury (Skin Wound) has become a kind of serious health problem.Wound, burn, chronic ulcer etc. are made
Into large skin defect, the surface of a wound for being difficult to heal is commonly formed, is not only caused suffering to patient, and reduces quality of life.
Currently it is proved to effective treatment method (including stem-cell therapy and dermatoplasty) and the defects of medical treatment cost is high is present, it is used
It is restricted.Prostaglandin E2 (Prostaglandin E2, PGE2) is a kind of important cell growth and regulatory factor, and it is made
With to expand blood vessel, increase organic blood flow volume, while there is immunosupress and antiinflammatory action.And it has been reported that mesenchyma is dry thin
Born of the same parents (Mesenchymal stem cells, MSCs) can play the work of promotion skin healing by regulating and controlling local inflammation reaction
With.Mechanism of action therein is probably that the PGE2 of MSCs secretions polarizes induction of macrophage to M2 types, promotes IL-10's
Secretion, and then play anti-fibrosis and reduce the effect of collagen deposition.Therefore, this makes us want to determine in exogenous PGE2
Stimulation under whether can make macrophage occur M2 types polarization.However, simple PGE2 half-life shorts, during contact with cell
Between it is shorter, this characteristic greatly reduce PGE2 exclusive use value.
Chitosan, collagen, fibrin and hyaluronic acid have preferably tissue as a kind of natural macromolecular compound
Compatibility, biodegradability and abundant biological activity.Polytype based on these natural macromolecular compounds
Timbering material shows important application value in regenerative medicine field, therefore, has using this kind of biomaterial netted
The feature of structure, the upper specific bioactive molecule of hydrogel coating formed, so as to which bioactivity hydrogel be made, this
Kind hydrogel can slowly discharge particular growth factor, and growth factor and the histocyte interaction of release can improve part
Microenvironment, strengthens the biological action of cell, and accelerates the reparation and regeneration of tissue.At present, there has been no will contain PGE2 water-settings
The report of glue.
The content of the invention
The purpose of the present invention is overcome the deficiencies in the prior art, there is provided a kind of hydrogel for tissue damage reparation.
Second object of the present invention is to provide a kind of preparation method of the hydrogel for tissue damage reparation.
Third object of the present invention be to provide a kind of hydrogel for tissue damage reparation prepare skin injury
Application in preparation for repairing.
Technical scheme is summarized as follows：
A kind of preparation method of hydrogel for tissue damage reparation, comprises the following steps：
(1) using sterile distilled water dissolving hydrogel material, 2-8 DEG C of swelling 12-24 hour, hydrogel is obtained；
(2) PGE2 powder is dissolved in the PGE2 solution for being made that concentration is 0.1mg/ml-5mg/ml in phosphate buffer, pressed
Mass ratio is 1:1-4 ratio, the PGE2 solution is added drop-wise in the hydrogel of step (1) acquisition, under the conditions of 2-8 DEG C,
Stir 3-5 hours；2-8 DEG C preserves or is frozen at -18--22 DEG C, and the PGE2 is writing a Chinese character in simplified form for prostaglandin E2.
The hydrogel material is preferably at least one of chitosan, collagen, hyaluronic acid and fibrin.
The mass content of hydrogel material is 0.01%-5% in the hydrogel.
Hydrogel for tissue damage reparation prepared by the above method.
Application of the above-mentioned hydrogel for tissue damage reparation in skin injury preparation for repairing is prepared.
Advantages of the present invention：
The hydrogel for tissue damage reparation of the present invention is capable of a certain degree of extension PGE2 release time.Directly
For the treatment of tissue damage, the recovery of injured cutaneous tissue and function assessment can be promoted.Can be with induced damage site macrophage
Cell polarizes to M2 types suppresses inflammatory reaction, accelerate the healing of damage, and the angiogenesis of damage location in early promotion.
In addition, the hydrogel no cytotoxicity of the present invention, can be applied directly to wound surface, and it is easy to use, it is cheap, and have
The therapeutic action of effect.
The hydrogel of the present invention, PGE2 slow release can be realized by being applied to skin lesion sites, extend PGE2 and damage
The action time of traumatic part bit organization cell, improve the microenvironment of damage location, so as to be provided newly for the repairing and treating of skin injury
Visual angle.
The present invention has with chitosan, collagen, fibrin and the coated PGE2 hydrogels of hyaluronic acid, this kind of hydrogel
Slowly release PGE2 and promote the characteristic that is polarized to M2 types of macrophage, further relate to this kind of PGE2 hydrogels using skin injury as
In the partial smearing treatment of the disease of feature, damage location inflammation can be inhibited, strengthens damage location angiogenic factor table
Reach, improve damage skin heart new life, promote the effect of skin injury reparation.
Brief description of the drawings
Fig. 1 represents the temperature stability of the hydrogel for tissue damage reparation.
Fig. 2 handles cytoscopy different time points nutrient solution PGE2 levels with PGE2, PGE2 hydrogel.Wherein A PGE2
It is horizontal to handle cytoscopy different time points nutrient solution PGE2, B handles cytoscopy different time points culture with PGE2 hydrogels
Liquid PGE2 is horizontal
Fig. 3 assesses the suitable hydrogel concentration for tissue damage reparation.
Fig. 4 represent hydrogel for skin injury reparation in vitro can inducing macrophage polarized to M2 types；(A) will
After macrophage co-cultures 48 hours with PBS, aquagel (CS hydrogels), PGE2, PGE2 hydrogel, LPS and IL-4,
Cellular immunofluorescence detects CD206 and CD68 expression.(B) to the quantitative analytical data of A figures.PGE2 hydrogels can significantly improve
CD206 expression.IL-4 is as the positive control for improving CD206 expression.
Fig. 5 is the influence that PGE2 hydrogels are expressed macrophage inflammatory factor；(A) with PBS, CS hydrogel, PGE2 and
PGE2 hydrogels (being CS+PGE2) handled macrophage after 48 hours, M2 type macrophage related gene IL-10 and M1 type macrophages
Cell relating gene-1 IL-6 Western detections.(B) quantitative data of IL-10 protein expressions.PGE2 hydrogels are to macrophage
Inflammatory factor IL-10 expression has obvious promote.(C) quantitative data of IL-6 protein expressions.
Fig. 6 is therapeutic action of the PGE2 hydrogels to skin injury, (A) with PGE2 hydrogels, PGE2, CS hydrogel and
PBS treatments excision property skin injury animal model.The damage location area of every 3 days measurement animal models.(B) quantitative analysis each group
The speed of wound healing, display PGE2 hydrogels can be obviously promoted wound healing；(C) the 7th day and the 14th day skin after treating are taken
Tissue carries out HE dyeing.
Fig. 7 represents that the hydrogel for skin injury reparation enhances skin injury site anti-inflammatory power, and molecular image shows
Influence of the track PGE2 hydrogels to skin lesion sites active oxygen.(A) BLI detect PGE2 hydrogels, PGE2, CS hydrogel and
The ROS of different time points injury site after PBS treatments.(B) height of the different group ROS activity of the quantitative analysis of BLI signals.
Fig. 8 represents that the aquagel evoked macrophage in vivo for skin injury reparation polarizes to M2 types, and quantitative analysis is each
The quantity of the 1st, 4,7 day injury site CD206 positive cell after group treatment.
The influence horizontal to skin lesion sites vegf expression of Fig. 9 molecular images tracer PGE2 hydrogels, (A) controls in each group
The the 0th, 4,7,10 and 14 day after treatment, with bioluminescent detection Vegfr2 expression.(B) quantitative analysis of bioluminescence signal.
The newborn evaluation of skin heart after Figure 10 PGE2 Hydrogel In Treatings.
Embodiment
The present invention is described in further details with reference to specific embodiment..
Embodiment 1
A kind of preparation method of hydrogel for tissue damage reparation, comprises the following steps：
(1) using sterile distilled water dissolving chitosan, 6 DEG C are swelled 18 hours, obtain the water that chitosan mass content is 1%
Gel；
(2) PGE2 powder is dissolved in the PGE2 solution for being made that concentration is 1mg/ml in neutral phosphate buffer liquid, by quality
Than for 1:2 ratio, the PGE2 solution is added drop-wise in the hydrogel of step (1) acquisition, under the conditions of 6 DEG C, stirring 4 is small
When；6 DEG C of preservations, the PGE2 are writing a Chinese character in simplified form for prostaglandin E2.
2 DEG C can also be used to preserve for the present embodiment or 8 DEG C preserve.
Embodiment 2
A kind of preparation method of hydrogel for tissue damage reparation, comprises the following steps：
(1) using sterile distilled water dissolving hydrogel material (mass ratio 1：1 collagen and hyaluronic acid), 2 DEG C of swellings
24 hours, obtain the hydrogel that hydrogel material mass content is 0.01%；
(2) PGE2 powder is dissolved in the PGE2 solution for being made that concentration is 0.1mg/ml in neutral phosphate buffer liquid, by matter
Amount is than being 1:1 ratio, the PGE2 solution is added drop-wise in the hydrogel of step (1) acquisition, under the conditions of 2 DEG C, stirring 5 is small
When；- 18 DEG C freeze, and the PGE2 is writing a Chinese character in simplified form for prostaglandin E2.
Embodiment 3
A kind of preparation method of hydrogel for tissue damage reparation, comprises the following steps：
(1) sterile distilled water solution fibrin is used, 8 DEG C are swelled 12 hours, and it is 5% to obtain fibrin quality content
Hydrogel；
(2) PGE2 powder is dissolved in the PGE2 solution for being made that concentration is 5mg/ml in neutral phosphate buffer liquid, by quality
Than for 1:4 ratio, the PGE2 solution is added drop-wise in the hydrogel of step (1) acquisition, under the conditions of 8 DEG C, stirring 3 is small
When；- 22 DEG C freeze, and the PGE2 is writing a Chinese character in simplified form for prostaglandin E2.
In herein below, the hydrogel abbreviation PGE2 hydrogels for tissue damage reparation.
Experiment 1：
The characteristic of PGE2 hydrogels (prepared by experimental example 1)
The temperature stability of PGE2 hydrogels
For influence of the evaluation temperature to PGE2 hydrogel stability, by different temperatures (- 80 DEG C, -20 DEG C, 0 DEG C, 4 DEG C and
37 DEG C) under the conditions of the PGE2 hydrogels (prepared by embodiment 1) deposited add 96 orifice plate culture macrophages, carry out CCK-8 after 24h
Dyeing.Cell is according to 3 × 104The concentration paving disk (4 holes/group) (Fig. 1) in/hole.
Specific colouring method is as follows：
(1) CCK-8 dyeing liquors are prepared (according to CCK-8 stostes:Complete medium=1:9 are diluted)；
(2) culture medium is suctioned out；
(3) 100 μ l dyeing liquors are added per hole, orifice plate is put into (37 DEG C) incubation 4h of incubator；
(4) suction out supernatant (100 μ l) and be transferred in new orifice plate；
(5) using ELIASA detection (absorbance) OD450 values；
EUSA detection PGE2 releases are (see Fig. 2)
Time is set to 0.5h to 48h.It is thin that each group is collected with PGE2, PGE2 hydrogel and after cell culture certain time respectively
Born of the same parents' culture supernatant.Operated according to PGE2 enzyme linked immunological kit specifications, ELIASA detects the suction at wavelength 412nm
Shading value.PGE2 quantitative result is represented with mass concentration.As a result show：(Fig. 2A) PGE2 is under the concentration straight line of nutrient solution
Drop.(Fig. 2 B) handles cell with PGE2 hydrogels, and the PGE2 concentration in nutrient solution reached peak value at the 10th hour, then slowly under
Drop, the maintenance of valid density, which is substantially better than, directly uses PGE2.
Hydrogel for tissue damage reparation extends PGE2 release time.
The selection of PGE2 hydrogels (prepared by experimental example 1) concentration
(1) the PGE2 hydrogels of various concentrations are added into cell culture fluid to be incubated jointly with macrophage；
(2) selection concentration is respectively 0,0.35,0.70,1.00,2.00 and 5.00 μm of ol/L PGE2 hydrogels；
As a result show, by peritoneal macrophage 24 hours in the PGE2 of various concentrations.VEGF gene expressions are with dense
Degree dependence mode increases, and reaches peak value at 1 μM.Most suitable PGE2 hydrogel concentrations are 1 μm of ol/L (Fig. 3).
Experiment 2：
PGE2 hydrogels (prepared by experimental example 1) external evoked macrophage polarizes to M2 types
The immunofluorescence dyeing of cell climbing sheet
(1) macrophage for extracting mouse ascites counts after screening, with 1 × 104It is inoculated in 48 holes of prior sheet glass
Plate.
(2) after cell attachment, with different condition (LPS, IL-4, PBS, independent hydrogel, independent PGE2 solution and
PGE2 hydrogels) pretreatment macrophage.
(3) cell is placed in incubator and cultivated, abandoned supernatant after 48 hours, cell will have been climbed in culture plate
Sheet glass is embathed 3 times with PBS.
(4) creep plate 15min, PBS are fixed with 4% paraformaldehyde and embathes sheet glass 3 times.
(5) (antigen expressed on cell membrane omits 0.5%Triton X-100 (PBS preparations) penetrating 15-20min of room temperature
This step).
(6) PBS embathes sheet glass 3 times, and each 3min, blotting paper blots PBS, and normal goats blood is added dropwise on the glass sheet
Clearly, room temperature closing 30min.
(7) blotting paper sops up confining liquid, does not wash, and the primary antibody diluted of sufficient amount is added dropwise on every sheet glass and is put into wet
Box, 4 DEG C of overnight incubations.
(8) fluorescence secondary antibody is added：PBS embathes creep plate 3 times, each 3-5min, and blotting paper blots to drip on creep plate after surplus liquid
Add the fluorescence secondary antibody diluted, 20-37 DEG C of lucifuge is incubated 2h in wet box, and PBS embathes creep plate 3 times, each 3min.
(9) core is redyed：DAPI lucifuges are added dropwise and are incubated 5min, dye core are carried out to sample, PBS washes 3 times and washes away unnecessary DAPI.
(10) liquid on creep plate is blotted with blotting paper, with the mounting fluid-tight piece containing anti-fluorescence quenching, then in fluorescence
Micro- Microscopic observation gathers image.
For the LPS and IL-4 of addition as positive control, the PGE2 hydrogel concentrations of application are 1 μm of ol/L.Result of study shows
Show, PGE2 hydrogels substantially can polarize (Fig. 4) by inducing macrophage to M2 types：(A) by macrophage and PBS, CS hydrogel,
After PGE2, PGE2 hydrogel, LPS and IL-4 are co-cultured 48 hours, cellular immunofluorescence detects CD206 and CD68 expression.(B)
To the quantitative analytical data of A figures.PGE2 hydrogels can significantly improve CD206 expression.IL-4 is as the sun for improving CD206 expression
Property control.
In addition, being pre-processed using real-time quantitative RT-PCR detection different condition, (LPS, IL-4, PBS, independent hydrogel are single
Only PGE2 solution and PGE2 hydrogels) macrophage M1/M2 related genes expression, as a result show：After LPS is stimulated,
The expression rise of M1 phenotypic markers gene (TNF-α, IL-6, iNOs, IL-1 β) is the most obvious in Macrophage Cell；IL-4 is external
After stimulation, the expression highest of M2 phenotypic markers gene (CD206, IL-10, IL-1ra, Arg-1)；The macrophage that PBS is stimulated
The macrophage that expression M1/M2 mark of correlations gene level is handled close to LPS, through the post-stimulatory macrophage phases of independent PGE2
The macrophage of correlation gene expression convergence IL-4 processing, and the macrophage related gene expression handled through PGE2 hydrogels
Closer to M2 type macrophages.And for IL-10 (the high expression of M2 types macrophage) and IL-6, (M1 types are huge with Western Blot
The high expression of phagocyte) two kinds of albumen are detected, as a result the expression variation tendency of two kinds of albumen and immunofluorescence dyeing and
RT-PCR results are consistent (Fig. 5)：(A) macrophage is handled after 48 hours with PBS, CS hydrogel, PGE2 and PGE2 hydrogels, M2
Type macrophage related gene IL-10 and M1 type macrophage related gene IL-6 Western detections.(B) IL-10 albumen table
The quantitative data reached.PGE2 hydrogels have obvious promote to macrophage inflammatory factor IL-10 expression.(C) IL-6 albumen
The quantitative data of expression.PGE2 hydrogels have obvious suppress to macrophage inflammatory factor IL-6 expression.
Experiment 3：
PGE2 hydrogels (prepared by experimental example 1) can speed up the healing of skin injury
Skin excision damage is carried out to 8-10 week old FVB male mices.It is grouped according to animals received treatment type (every
Group 3)：PBS groups (are subjected to skin injury+injury site and smear 20 microlitres of PBS)；Independent hydrogel group (is subjected to skin injury+damage
Hinder site and smear 20 microlitres of hydrogels)；Independent PGE2 groups (be subjected to skin injury+injury site and smear 20 microlitres of PGE2 solution)；
PGE2 hydrogels group (is subjected to skin injury+injury site and smears 20 microlitres of PGE2 hydrogels).
Excision property skin injury model
(1) autoclave sterilization operating theater instruments (scissors, sharp tweezer, microvessel clamp, needle holder), uses medicinal alcohol sterile surgical
Taiwan area domain；
(2) chloral (4%, 350mg/kg) anesthetized mice is closed through abdominal cavity injection water, mouse is fixed on operation with prone position
On heating cushion, back hair, iodophor disinfection art area skin are removed using shaver；
(3) cut with Sterile ophthalmic and a diameter about 1cm skins holostrome damage surface of a wound, depth and manadesma are formed at its back, it is sterile
Gauze stops blooding；
(4) the cyclic organic film of 3 mm of thickness is sewn on wound with nylon suture and put, to prevent wound from receiving
Contracting；
(5) mouse is placed in rewarming on heating cushion, rearging cage is put back to after reviving.
Wound healing rate determines
Postoperative 0,1,4,7,10,13d digital cameras record each group wound healing situation, the images of Image-Pro Plus 6.0
Analysis software measures surface of a wound area, calculates Wound healing rate.
Wound healing rate=[(original surface of a wound area-current point in time measurement area)/original surface of a wound area] × 100%
HE is dyed and Wound healing rate measurement result shows：In excision property skin injury model, PGE2 hydrogel energy
Enough accelerate the healing (Fig. 6) of the surface of a wound：(A) moved with PGE2 hydrogels, PGE2, CS hydrogel and PBS treatment excision property skin injury
Thing model.The damage location area of every 3 days measurement animal models.(B) speed of quantitative analysis each group wound healing, PGE2 is shown
Hydrogel can be obviously promoted wound healing.
HE is dyed
Respectively at postoperative 7 days and 10 mouse of every group of selection in 14 days, take surface of a wound neoplastic skin tissue and surrounding few after anesthesia
Perhaps normal skin, 1d, serial dehydration, FFPE, 5 μm of serial section, conventional hematoxylin-Yihong are fixed with 4% paraformaldehyde
Dyeing, neutral gum mounting, optical microphotograph Microscopic observation neoplastic skin pathological change (Fig. 6 C)：(C) take treatment after the 7th day and
Skin histology carries out HE dyeing within 14th day, observes the closure situation of wound.PGE2 hydrogels can preferably promote skin function
Recover.
Experiment 4：
PGE2 hydrogels (prepared by experimental example 1) enhance skin injury site anti-inflammatory power
Evaluate injury site ROS content
The anti-inflammatory power of injury site can be strengthened in order to evaluate PGE2 hydrogels, we are in mouse skin excision property damage
Different time points afterwards, injury site ROS content (Fig. 7) is evaluated using biodiversity resources technology：Molecular image tracer PGE2
Influence of the hydrogel to skin lesion sites active oxygen.(A) BLI detects PGE2 hydrogels, PGE2, CS hydrogel and PBS treatments
The ROS of different time points injury site afterwards.(B) height of the different group ROS activity of the quantitative analysis of BLI signals.PGE2 hydrogels
The reactive oxygen species of damage location can substantially be reduced.
Small animal living body biodiversity resources
(1) chloral (4%, 350 mg/kgs) anesthetized mice is closed through abdominal cavity injection water.
(2) to mouse through firefly luciferase substrate Lumino (100 mg/kg) is injected intraperitoneally.
(3) after substrate is injected 5 minutes, mouse is put into small animal living body imaging system camera bellows, is well placed position (prostrate
Position), its head is directed at venthole in camera bellows.
(4) time for exposure is adjusted according to fluorescence signal intensity, generally 0.5-2 minutes, continuous acquisition image is until signal
Intensity is begun to decline.
(5) data analysis：The fluorescence signal intensity of every mouse is determined using Living Image softwares, carries out statistics
Analysis.
Experiment 5：
PGE2 hydrogels (prepared by experimental example 1) polarize induction of injury site macrophage to M2 types
We to after treatment mouse skin frozen section carry out immunofluorescence dyeing, quantitative analysis each group treatment after the 1st,
4th, the quantity (Fig. 8) of 7 days injury site CD206 positive cells.It was found that change over time, huge in 1,4,7 day expression M2 type
Phagocyte surface molecular mark CD206 macrophage ratio rises, and PGE2 Hydrogel In Treatings group is apparently higher than other groups.
Skin histology frozen section immunostaining
(1) section is taken out from -20 DEG C of refrigerators, room temperature places 30min；
(2) section is put into (- 20 DEG C of precooling 30min) acetone stoste and fixes 10min；
(3) section is placed in air drying 30min；
(4) PBS washing slices (5min, 2 times) are used；
(5) rupture of membranes：0.1%Triton X-100, room temperature, 10min；PBS is washed, 5min, 2 times；
(6) serum is closed, 4 DEG C, 45min；
(7) add primary antibody, 4 DEG C, stay overnight；
(8) secondary morning rewarming, 1h；PBS is washed, 5min, 4 times；
(9) secondary antibody (lucifuge), room temperature, 2h are added；PBS is washed, 5min, 5 times；
(10) DAPI mountings, fluorescence microscopy Microscopic observation, take pictures.
Experiment 6：
PGE2 hydrogels (prepared by experimental example 1) promote the effect of damage location angiogenesis
VEGF-R2 transgenic mices evaluate angiogenesis
In order to evaluate PGE2 hydrogels damage location angiogenesis can be promoted to act on, we are small to VEGF-R2 transgenosis
Mouse carries out skin excision damage processing (method is as previously described).It is grouped (every group 3)：PBS groups (are subjected to skin injury+damage
Smear 20 microlitres of PBS in site)；Independent hydrogel group (be subjected to skin injury+injury site and smear 20 microlitres of hydrogels)；Individually
PGE2 groups (are subjected to skin injury+injury site and smear 20 microlitres of PGE2 solution)；PGE2 hydrogels group (is subjected to skin injury+damage
Hinder site and smear 20 microlitres of PGE2 hydrogels).Different time points after injury, evaluated using BLI technologies (method is as previously described)
The expression of VEGF-R2 genes.
Immunofluorescence dyeing method is shown in experiment 6
In order to illustrate the mechanism that PGE2 hydrogels promote injury site angiogenesis, to VEGF expression-R2-Fluc transgenosis
Mouse carries out skin excision damage, and is grouped according to therapeutic modality.Different time points after damage, supervised in real time using BLI technologies
Angiogenesis is surveyed, more each therapeutic modality promotes the situation (Fig. 9) of VEGF-R2 gene expressions.As a result show, receive PGE2 water-settings
The mouse of glue treatment, the animal (p ＜ 0.05) for receiving other treatment is significantly higher than in the expression of injury site VEGF-R2 genes.
Isosorbide-5-Nitrae after injury in treating, 7 days, using CD31 staining evaluation skin injuries site angiogenesis situation, find PGE2 Hydrogel In Treatings
Group angiogenesis quantity is significantly higher than other groups (p ＜ 0.05) (Figure 10).
It is demonstrated experimentally that what PGE2 hydrogel properties and effect prepared by embodiment 2 and embodiment 3 were prepared with embodiment 1
PGE2 hydrogels are similar.
Claims (5)
- A kind of 1. preparation method of hydrogel for tissue damage reparation, it is characterized in that comprising the following steps：(1) using sterile distilled water dissolving hydrogel material, 2-8 DEG C of swelling 12-24 hour, hydrogel is obtained；(2) PGE2 powder is dissolved in the PGE2 solution for being made that concentration is 0.1mg/ml-5mg/ml in phosphate buffer, by quality Than for 1:1-4 ratio, the PGE2 solution is added drop-wise in the hydrogel of step (1) acquisition, under the conditions of 2-8 DEG C, stirring 3-5 hours；2-8 DEG C preserves or is frozen at -18--22 DEG C, and the PGE2 is writing a Chinese character in simplified form for prostaglandin E2.
- 2. according to the method for claim 1, it is characterized in that the hydrogel material be chitosan, collagen, hyaluronic acid and At least one of fibrin.
- 3. method according to claim 1 or 2, it is characterized in that the mass content of hydrogel material is in the hydrogel 0.01%-5%.
- 4. the hydrogel for tissue damage reparation prepared by the method described in one of claim 1-3.
- 5. application of the hydrogel for tissue damage reparation of claim 4 in skin injury preparation for repairing is prepared.
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| CN201710752030.1A CN107496348A (en) | 2017-08-28 | 2017-08-28 | A kind of hydrogel and preparation method for tissue damage reparation |
| PCT/CN2018/080831 WO2019041799A1 (en) | 2017-08-28 | 2018-03-28 | Hydrogel for repairing tissue injury and preparation method therefor |
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| CN201710752030.1A CN107496348A (en) | 2017-08-28 | 2017-08-28 | A kind of hydrogel and preparation method for tissue damage reparation |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019041799A1 (en) * | 2017-08-28 | 2019-03-07 | 天津昂赛细胞基因工程有限公司 | Hydrogel for repairing tissue injury and preparation method therefor |
| CN110624112A (en) * | 2019-07-15 | 2019-12-31 | 天津昂赛细胞基因工程有限公司 | Hydrogel connected with prostaglandin E2, and preparation method and application thereof |
| CN110713727A (en) * | 2018-06-27 | 2020-01-21 | 中国科学院过程工程研究所 | Collagen hydrogel prepared at low temperature, and preparation method and application thereof |
| CN112155553A (en) * | 2020-09-27 | 2021-01-01 | 甘肃省人民医院 | Wound surface evaluation system and method based on structured light 3D measurement |
| CN113599574A (en) * | 2021-07-28 | 2021-11-05 | 苏州大学 | Regeneration material for muscle repair and preparation method thereof |
| CN114288464A (en) * | 2021-11-24 | 2022-04-08 | 中国科学院理化技术研究所 | Antibacterial healing-promoting hydrogel dressing and preparation method and application thereof |
| CN114948861A (en) * | 2022-05-26 | 2022-08-30 | 中国人民解放军西部战区总医院 | Multifunctional hydrogel for promoting healing of radioactive skin injury and preparation method and application thereof |
| CN115054569A (en) * | 2022-05-16 | 2022-09-16 | 四川大学华西医院 | DNA hydrogel for treating alveolar bone injury and preparation method and application thereof |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019041799A1 (en) * | 2017-08-28 | 2019-03-07 | 天津昂赛细胞基因工程有限公司 | Hydrogel for repairing tissue injury and preparation method therefor |
| CN110713727A (en) * | 2018-06-27 | 2020-01-21 | 中国科学院过程工程研究所 | Collagen hydrogel prepared at low temperature, and preparation method and application thereof |
| CN110624112A (en) * | 2019-07-15 | 2019-12-31 | 天津昂赛细胞基因工程有限公司 | Hydrogel connected with prostaglandin E2, and preparation method and application thereof |
| CN112155553A (en) * | 2020-09-27 | 2021-01-01 | 甘肃省人民医院 | Wound surface evaluation system and method based on structured light 3D measurement |
| CN112155553B (en) * | 2020-09-27 | 2023-05-23 | 甘肃省人民医院 | Wound surface evaluation system and method based on structured light 3D measurement |
| CN113599574A (en) * | 2021-07-28 | 2021-11-05 | 苏州大学 | Regeneration material for muscle repair and preparation method thereof |
| CN114288464A (en) * | 2021-11-24 | 2022-04-08 | 中国科学院理化技术研究所 | Antibacterial healing-promoting hydrogel dressing and preparation method and application thereof |
| CN114288464B (en) * | 2021-11-24 | 2023-07-07 | 中国科学院理化技术研究所 | Antibacterial healing-promoting hydrogel dressing and preparation method and application thereof |
| CN115054569A (en) * | 2022-05-16 | 2022-09-16 | 四川大学华西医院 | DNA hydrogel for treating alveolar bone injury and preparation method and application thereof |
| CN115054569B (en) * | 2022-05-16 | 2024-03-19 | 四川大学华西医院 | DNA hydrogel for treating alveolar bone injury and preparation method and application thereof |
| CN114948861A (en) * | 2022-05-26 | 2022-08-30 | 中国人民解放军西部战区总医院 | Multifunctional hydrogel for promoting healing of radioactive skin injury and preparation method and application thereof |
| CN114948861B (en) * | 2022-05-26 | 2023-08-25 | 中国人民解放军西部战区总医院 | Multifunctional hydrogel for promoting healing of radioactive skin injury as well as preparation method and application thereof |
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