CN108837140A - A kind of preparation method and application of pheretima protein microsphere nanometer wound compound - Google Patents
A kind of preparation method and application of pheretima protein microsphere nanometer wound compound Download PDFInfo
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- 241000237636 Pheretima Species 0.000 title claims abstract description 51
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 44
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 44
- 239000004005 microsphere Substances 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 150000001875 compounds Chemical class 0.000 title claims abstract description 27
- 239000002184 metal Substances 0.000 claims abstract description 18
- 229910052751 metal Inorganic materials 0.000 claims abstract description 18
- 230000007704 transition Effects 0.000 claims abstract description 18
- 229910021392 nanocarbon Inorganic materials 0.000 claims abstract description 11
- 206010052428 Wound Diseases 0.000 claims description 46
- 208000027418 Wounds and injury Diseases 0.000 claims description 46
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 239000000203 mixture Substances 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 12
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 9
- 229930006000 Sucrose Natural products 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 9
- 239000005720 sucrose Substances 0.000 claims description 9
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000002002 slurry Substances 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 230000001376 precipitating effect Effects 0.000 claims description 5
- 230000005779 cell damage Effects 0.000 claims description 4
- 208000037887 cell injury Diseases 0.000 claims description 4
- 238000011010 flushing procedure Methods 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 239000002504 physiological saline solution Substances 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- 206010013786 Dry skin Diseases 0.000 claims description 3
- 241001465754 Metazoa Species 0.000 claims description 3
- 238000013019 agitation Methods 0.000 claims description 3
- 235000019270 ammonium chloride Nutrition 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 230000008030 elimination Effects 0.000 claims description 3
- 238000003379 elimination reaction Methods 0.000 claims description 3
- 238000002270 exclusion chromatography Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 238000010907 mechanical stirring Methods 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 239000013049 sediment Substances 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 210000004876 tela submucosa Anatomy 0.000 claims description 3
- 150000002500 ions Chemical class 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 240000007594 Oryza sativa Species 0.000 claims 1
- 235000007164 Oryza sativa Nutrition 0.000 claims 1
- 238000005119 centrifugation Methods 0.000 claims 1
- 239000003610 charcoal Substances 0.000 claims 1
- 239000006185 dispersion Substances 0.000 claims 1
- 238000002156 mixing Methods 0.000 claims 1
- 235000009566 rice Nutrition 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 5
- 230000006698 induction Effects 0.000 abstract description 2
- 238000013268 sustained release Methods 0.000 abstract description 2
- 239000012730 sustained-release form Substances 0.000 abstract description 2
- 230000037314 wound repair Effects 0.000 abstract description 2
- 239000003814 drug Substances 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 229940079593 drug Drugs 0.000 description 9
- 239000013641 positive control Substances 0.000 description 9
- 230000001613 neoplastic effect Effects 0.000 description 8
- 239000011805 ball Substances 0.000 description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 3
- WTFXARWRTYJXII-UHFFFAOYSA-N iron(2+);iron(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Fe+2].[Fe+3].[Fe+3] WTFXARWRTYJXII-UHFFFAOYSA-N 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000002953 phosphate buffered saline Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 206010002091 Anaesthesia Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000035876 healing Effects 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000029663 wound healing Effects 0.000 description 2
- 241000243684 Lumbricus Species 0.000 description 1
- 241000361919 Metaphire sieboldi Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010039203 Road traffic accident Diseases 0.000 description 1
- 206010053615 Thermal burn Diseases 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 238000001949 anaesthesia Methods 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000035617 depilation Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000008556 epithelial cell proliferation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 239000011806 microball Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000036515 potency Effects 0.000 description 1
- 238000011137 process chromatography Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000037393 skin firmness Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
-
- 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
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/501—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/62—Leeches; Worms, e.g. cestodes, tapeworms, nematodes, roundworms, earth worms, ascarids, filarias, hookworms, trichinella or taenia
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Inorganic Chemistry (AREA)
- Dermatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of preparation method and application of pheretima protein microsphere nanometer wound compound, the preparation of preparation, nano carbon microsphere including ground dragon protein, the preparation of transition series metal hollow nano microballoon, the preparations of pheretima protein microsphere nanometer wound compound.Compared with prior art, albumen is physically embedded in transition series metal hollow nano microballoon by the present invention, so that preparing has sustained release, the medical preparation of induction and multidigit point effect, to achieve the purpose that promote wound repair.
Description
Technical field
The present invention relates to protein drug preparation technical field, especially a kind of pheretima protein microsphere nanometer wound compound
Preparation method and application.
Background technique
Modern society is because each caused by the reasons such as traffic accident, incident (scald, mechanical injuries etc.), medical operating
Kind trauma patient number is constantly being increased sharply.Studies have shown that the death rate as caused by wound is in various diseases in worldwide
It is middle to occupy the 4th.According to another statistics, China is every year 100000 people because of died of wounds number, and 1,000,000 people of disability causes direct economy to damage
Lose 10,000,000,000 RMB.With the development of society, this number is also constantly soaring.Therefore repair after wound is always medicine neck
The hot subject of domain research.Research in recent years discovery, traditional Chinese medicine pheretima (earthworm, Lumbricus) show in this regard
Unique curative effect.A large amount of result of study shows that Pheretima extract can reach rush by stimulating epithelial cell proliferation in recent years
Into the effect of wound healing.
However, albumen is very easy to degradation as a kind of polymer substance in nature, so ground dragon protein is using
It is big that there is also consumptions in the process, at high cost, the low problem of relative potencies.Nano-medicament carrier generally has porous, hollow, more
The architectural characteristics such as layer, are easy to the Controlled release of drug.By selecting the type and proportion of carrier material, the release of drug can control
Speed prepares the drug-carrying nanometer particle with slow release characteristic, greatly extends the half-life period of drug, reduces times for spraying and medication
Amount.In addition, the large specific surface area of nano-medicament carrier, the functional group of connection or carrier band or activated centre are more, it is easy to accomplish treat
The synchronization of effect tracking and treatment.Existing superparamagnetic nanoparticle SPIO (superparamagnetic iron oxide at present
Nanoparticles) commercial product (Endorem, Feridex, Resovist), these are four oxidations of polysaccharide covering
Three Fe nanometer particles, the partial size of entire particle is in 50nm, but these materials do not have the condition as Thermosensitive Material Used for Controlled Releasing of Medicine.
Summary of the invention
The invention aims to solve the deficiencies in the prior art, a kind of pheretima protein microsphere nanometer wound is provided
The preparation method and application of compound.
In order to achieve the above objectives, the present invention is implemented according to following technical scheme：
A kind of preparation method of pheretima protein microsphere nanometer wound compound, includes the following steps：
S1, dragon protein preparation：Fresh and alive pheretima is cleaned three times with physiological saline, removes body surface silt and dirt, cuts
Clasmatosis slurry is obtained with the mode of pressure breaking after broken, physiological buffer is added into clasmatosis slurry in 4 DEG C of progress low speed
Centrifugation 5 minutes obtains cell damage mixture after removing internal silt and digest；It is prepared in centrifuge tube slow containing Tris-HCl
10%~70% graded sucrose solutions of fliud flushing, gently set cell damage mixture in above-mentioned sucrose solution upper end, in 2500rpm from
The heart, the upper layer component in collection centrifuge tube, middle layer component, submucosa composition, then pass through conventional ion for middle layer component respectively
Exchange process and exclusion chromatography prepare pheretima protein mixture；
The preparation of S2, nano carbon microsphere：It weighs 40g sucrose to be dissolved in 100ml deionized water, ultrasonic agitation is stayed overnight, then
Being heated to 200 DEG C makes it all be carbonized, and spends example water and ethanol/acetone mixed liquor repeated flushing, except impurity in carbon elimination, then
Ultrasonic disperse 72 hours, Nano carbon ball is obtained, it is spare；
The preparation of S3, transition series metal hollow nano microballoon, specifically include：
S31,0.5mol transition series metal salt MXn is weighed, wherein M is transition series metal, and it is good to be added to ultrasonic disperse
Nano carbon ball in, continue ultrasonic disperse 72 hours in ethanol/water mixed liquor, then remove solution, add new ethanol/water
Mixed solution continues ultrasonic disperse 72 hours；
S32,1g ammonium chloride continuation ultrasonic disperse 1 hour is then added into S31, is then heated 12 hours at 150 DEG C,
Precipitating is obtained by filtration after natural cooling, obtains drying in 40 DEG C of low temperature dryings after being rinsed precipitating 3 times with ethanol/water mixed liquor and produces
Object；
S33, drying product made from S32 with the heating rate of 0.5 DEG C/min is risen to 700 DEG C, maintains 5 hours, then
Started with the rate of 1 DEG C/min cooling to get to transition series metal hollow nano microballoon in room temperature after being cooled to 50 DEG C；
The preparation of S4, pheretima protein microsphere nanometer wound compound：1g transition series metal hollow nano microballoon is weighed, is added
The pheretima mixed liquid of protein of 2mg/ml mixed by pheretima protein mixture and solvent PBS stirs 24 hours at 4 DEG C, from
The heart removes supernatant, and sediment fraction is the pheretima protein microsphere nanometer wound compound prepared, saves in 4 DEG C.
Further, when the Nano carbon ball in the S31 continues ultrasonic disperse in ethanol/water mixed liquor, every 12 hours
80 degrees Celsius mechanical stirring 12 hours.
Further, the pH=7.5 of the pheretima mixed liquid of protein in the S4.
In addition, the present invention also provides a kind of application of pheretima protein microsphere nanometer wound compound, described ground dragon protein is micro-
Ball nanometer wound compound heals for wounds in animals.
Compared with prior art, albumen is physically embedded in transition series metal hollow nano microballoon by the present invention,
There is sustained release to prepare, the medical preparation of induction and multidigit point effect, to achieve the purpose that promote wound repair.
Detailed description of the invention
Fig. 1 is the hollow nano microballoon electron microscope of metallic iron obtained in the embodiment of the present invention 1.
Fig. 2 (A) is blank group (Blank) in the embodiment of the present invention 2, positive controls (JWH), pheretima nanometer group (EW)
18 days healing rate statistical charts；It (B) is blank group (Blank), positive controls (JWH), pheretima nanometer in the embodiment of the present invention 2
Mortality statistics figure when 3 days, 6 days after group (EW) wound；It (C) is the new life of blank group (Blank) wound in the embodiment of the present invention 2
The H&E colored graph of skin, (D) be the H&E colored graph of the neoplastic skin of positive controls (JWH) wound in the embodiment of the present invention 2,
It (E) is the H&E colored graph of the neoplastic skin of pheretima nanometer group (EW) wound in the embodiment of the present invention 2.
Specific embodiment
The invention will be further described combined with specific embodiments below, in the illustrative examples and explanation of the invention
For explaining the present invention, but it is not as a limitation of the invention.
Embodiment 1
A kind of preparation method of pheretima protein microsphere nanometer wound compound, includes the following steps：
S1, dragon protein preparation：Fresh and alive pheretima is cleaned three times with physiological saline, removes body surface silt and dirt, cuts
Pressure breaking is carried out with Constant pressure breaking machine after broken and obtains clasmatosis slurry, and physiological buffer is added into clasmatosis slurry
Liquid (Tris-HCl, 20mM, pH=7.5) 4 DEG C progress low-speed centrifugal 5 minutes, remove obtain after internal silt and digest it is thin
Born of the same parents' breakage mixture；10%~70% graded sucrose solutions that the buffer containing Tris-HCl is prepared in centrifuge tube, gently set cell
Damaged mixture is centrifuged in above-mentioned sucrose solution upper end in 2500rpm, collects upper layer component, middle layer group in centrifuge tube respectively
Divide, submucosa composition, then for middle layer component, by conventional ion exchange process, (with NaCl 1M, Tris-HCl 20mM is cationic
Elution) and exclusion chromatography (using 20mM Tris-HCl as eluent) preparation pheretima protein mixture；
The preparation of S2, nano carbon microsphere：It weighs 40g sucrose to be dissolved in 100ml deionized water, ultrasonic agitation is stayed overnight, then
Being heated to 200 DEG C makes it all be carbonized, and spends example water and ethanol/acetone mixed liquor repeated flushing, except impurity in carbon elimination, then
Ultrasonic disperse 72 hours, Nano carbon ball is obtained, it is spare；
The preparation of S3, transition series metal hollow nano microballoon, specifically include：
S31,0.5mol transition series metal salt MXn is weighed, wherein M is transition series metal, and it is good to be added to ultrasonic disperse
Nano carbon ball in, continue ultrasonic disperse 72 hours in ethanol/water mixed liquor, every 12 hours in 80 degrees Celsius of mechanical stirrings
12 hours, solution is then removed, new ethanol/water mixed solution is added and continues ultrasonic disperse 72 hours；
S32,1g ammonium chloride continuation ultrasonic disperse 1 hour is then added into S31, is then heated 12 hours at 150 DEG C,
Precipitating is obtained by filtration after natural cooling, obtains drying in 40 DEG C of low temperature dryings after being rinsed precipitating 3 times with ethanol/water mixed liquor and produces
Object；
S33, drying product made from S32 with the heating rate of 0.5 DEG C/min is risen to 700 DEG C, maintains 5 hours, then
Started with the rate of 1 DEG C/min cooling to get to transition series metal hollow nano microballoon, such as Fig. 1 in room temperature after being cooled to 50 DEG C
It is shown；
The preparation of S4, pheretima protein microsphere nanometer wound compound：1g transition series metal hollow nano microballoon is weighed, is added
2mg/ml's is mixed by pheretima protein mixture and the ground dragon protein of the solvent phosphate buffered saline solution PBS pH=7.5 mixed
Liquid is closed, is stirred 24 hours at 4 DEG C, supernatant is centrifuged off, sediment fraction is the pheretima protein microsphere nanometer wound prepared
Compound is saved in 4 DEG C.
Embodiment 2
Pheretima protein microsphere nanometer wound compound made from embodiment 1 can be used in wounds in animals healing.
Verify example
It is tested using 4 week old Balb/C mouse, after mouse adaptive feeding 1 week, is divided into 3 groups and is respectively designated as：It is empty
White group (Blank), positive controls (JWH), pheretima nanometer group (EW), are tested.Fiber crops are injected intraperitoneally in 10% chloraldurate
It is liquor-saturated, by experimental mouse prone position, it is placed in sterile list, skin of back iodophor disinfection.
Then machinery depilation is carried out to back of mice, is cut using Sterile ophthalmic and does a circular incision (diameter 2cm) at back,
Open wound model is caused, is put into and continues to raise in cage.
Coating is carried out to mouse wound according to wound area, dosage is 100mg/cm2(being calculated with albumen quality), it is empty
White group is only smeared physiological saline, and positive controls smear commercialization drug, and pheretima nanometer group smears pheretima egg made from embodiment 1
White microsphere nano wound compound.Single cage raising, every dressing in 3 days is primary, continuous treatment 27 days, every statistics wound healing in three days
Situation, carries out equal proportion to the wound of three groups of mouse and takes pictures, and analyzes wound area with ImageJ software；Then excessively anesthesia is put to death
After take wound tissue sample, carry out H&E dyeing, observe tissue recovery situation.
Result of study discovery has smeared the pheretima nanometer group (EW) of compound in agglutination, after wound 18 days more
It closes speed and is faster than other two groups, as shown in Fig. 2 (A).The pheretima nanometer group of pheretima protein microsphere nanometer wound compound is smeared
(EW) compared with the blank group (Blank) for not smearing drug, 3 days death rates are declined slightly before mouse, and have smeared certain business
The positive controls (JWH) of chemical drug object, occur the higher death rate in early period, and statistics has smeared positive controls at 6 days
(JWH) mouse death rate is apparently higher than blank group (Blank) and has smeared the ground of pheretima protein microsphere nanometer wound compound
Imperial nanometer group (EW), as shown in Fig. 2 (B).In addition, by H&E colored graph such as Fig. 2 (C) of neoplastic skin of observation wound, (D),
(E), wherein H&E colored graph, (D) that (C) is the neoplastic skin of blank group (Blank) wound are positive controls (JWH) wound
The H&E colored graph of neoplastic skin, the neoplastic skin that (E) is pheretima nanometer group (EW) wound H&E colored graph, discovery smears
The pheretima nanometer group (EW) of pheretima protein microsphere nanometer wound compound grown chaeta earlier on neoplastic skin, and the 28th day
Cambium is taken to carry out the discovery of H&E Coloration experiment, the pheretima nanometer group through pheretima protein microsphere nanometer wound complex therapies
(EW) its order degree of mouse tissue is significantly stronger than the blank group of untreated and the positive controls (JWH) of JWH treatment, the
Mouse was all put to death using breathing anaesthesia in 30 days, the stretch-proof for having counted neoplastic skin for putting to death later half experience in quality is strong
Degree, skin firmness of the discovery by the experimental group of pheretima protein microsphere nanometer wound complex therapies is also above other two groups.
The limitation that technical solution of the present invention is not limited to the above specific embodiments, it is all to do according to the technique and scheme of the present invention
Technology deformation out, falls within the scope of protection of the present invention.
Claims (4)
1. a kind of preparation method of pheretima protein microsphere nanometer wound compound, which is characterized in that include the following steps：
S1, dragon protein preparation：Fresh and alive pheretima is cleaned three times with physiological saline, body surface silt and dirt are removed, after shredding
Clasmatosis slurry is obtained with the mode of pressure breaking, physiological buffer is added into clasmatosis slurry in 4 DEG C of progress low-speed centrifugals 5
Minute, cell damage mixture is obtained after removing internal silt and digest；Buffer containing Tris-HCl is prepared in centrifuge tube
10%~70% graded sucrose solutions, gently set cell damage mixture in above-mentioned sucrose solution upper end, be centrifuged in 2500rpm,
Upper layer component in collection centrifuge tube, middle layer component, submucosa composition respectively, then pass through conventional ion for middle layer component and exchange
Method and exclusion chromatography prepare pheretima protein mixture；
The preparation of S2, nano carbon microsphere：It weighs 40g sucrose to be dissolved in 100ml deionized water, ultrasonic agitation overnight, then heats
So that it is all carbonized to 200 DEG C, spends example water and ethanol/acetone mixed liquor repeated flushing, it is then ultrasonic except impurity in carbon elimination
Dispersion 72 hours, obtains Nano carbon ball, spare；
The preparation of S3, transition series metal hollow nano microballoon, specifically include：
S31,0.5mol transition series metal salt MXn is weighed, wherein M is transition series metal, is added to that ultrasonic disperse is good to be received
In rice charcoal ball, continue ultrasonic disperse 72 hours in ethanol/water mixed liquor, then remove solution, adds new ethanol/water mixing
Solution continues ultrasonic disperse 72 hours；
S32,1g ammonium chloride continuation ultrasonic disperse 1 hour is then added into S31, is then heated 12 hours at 150 DEG C, it is natural
Cooled and filtered is precipitated, and obtains drying product in 40 DEG C of low temperature dryings after being rinsed precipitating 3 times with ethanol/water mixed liquor；
S33, drying product made from S32 is risen to 700 DEG C with the heating rate of 0.5 DEG C/min, maintained 5 hours, then with 1
DEG C/rate of min start it is cooling to get to transition series metal hollow nano microballoon in room temperature after being cooled to 50 DEG C；
The preparation of S4, pheretima protein microsphere nanometer wound compound：1g transition series metal hollow nano microballoon is weighed, 2mg/ is added
The pheretima mixed liquid of protein of ml mixed by pheretima protein mixture and solvent PBS stirs 24 hours at 4 DEG C, and centrifugation removes
Supernatant is removed, sediment fraction is the pheretima protein microsphere nanometer wound compound prepared, is saved in 4 DEG C.
2. the preparation method of pheretima protein microsphere nanometer wound compound according to claim 1, it is characterised in that：It is described
When Nano carbon ball in S31 continues ultrasonic disperse in ethanol/water mixed liquor, every 12 hours in 80 degrees Celsius of mechanical stirrings 12
Hour.
3. the preparation method of pheretima protein microsphere nanometer wound compound according to claim 1, it is characterised in that：It is described
The pH=7.5 of pheretima mixed liquid of protein in S4.
4. a kind of application of pheretima protein microsphere nanometer wound compound as described in claim 1, it is characterised in that：Describedly
Dragon protein microsphere nano wound compound heals for wounds in animals.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109464709A (en) * | 2018-11-26 | 2019-03-15 | 陕西中医药大学 | A kind of preparation method and application of earthworm supernatant protein nanometre collagen repairing composite |
| CN109529012A (en) * | 2019-01-10 | 2019-03-29 | 陕西中医药大学 | A kind of metal oxide nano bletilla striata pheretima albumen composition preparation method and application |
| CN111743997A (en) * | 2020-07-08 | 2020-10-09 | 陕西中医药大学 | Paste traditional Chinese medicine composition based on earthworm protein paste and preparation method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1520829A (en) * | 2003-02-10 | 2004-08-18 | 杨宝峰 | Earthworm freeze dried powder injection and preparation method |
| CN101053812A (en) * | 2007-04-26 | 2007-10-17 | 华东理工大学 | Mesoporous iron oxide hollow microspheres with photoelectric catalytically active and preparation method thereof |
| KR100838644B1 (en) * | 2007-01-29 | 2008-06-16 | 충남대학교산학협력단 | Metal hallow sphere assemblies using carbon templates and process thereof |
| CN101607990A (en) * | 2009-07-03 | 2009-12-23 | 山东大学威海分校 | A kind of earthworm active protein and extracting method thereof and application |
| CN102464304A (en) * | 2010-11-12 | 2012-05-23 | 中国科学院过程工程研究所 | Multi-shell-layer metal oxide hollow ball and preparation method thereof |
| CN106629682A (en) * | 2016-12-27 | 2017-05-10 | 江汉大学 | Method for preparing hollow graphene nanospheres through confined catalysis |
-
2018
- 2018-06-26 CN CN201810673928.4A patent/CN108837140B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1520829A (en) * | 2003-02-10 | 2004-08-18 | 杨宝峰 | Earthworm freeze dried powder injection and preparation method |
| KR100838644B1 (en) * | 2007-01-29 | 2008-06-16 | 충남대학교산학협력단 | Metal hallow sphere assemblies using carbon templates and process thereof |
| CN101053812A (en) * | 2007-04-26 | 2007-10-17 | 华东理工大学 | Mesoporous iron oxide hollow microspheres with photoelectric catalytically active and preparation method thereof |
| CN101607990A (en) * | 2009-07-03 | 2009-12-23 | 山东大学威海分校 | A kind of earthworm active protein and extracting method thereof and application |
| CN102464304A (en) * | 2010-11-12 | 2012-05-23 | 中国科学院过程工程研究所 | Multi-shell-layer metal oxide hollow ball and preparation method thereof |
| CN106629682A (en) * | 2016-12-27 | 2017-05-10 | 江汉大学 | Method for preparing hollow graphene nanospheres through confined catalysis |
Non-Patent Citations (2)
| Title |
|---|
| 娄慧慧，等: "碳球为模板合成Fe3O4材料", 《河南科技学院学报》 * |
| 陈杰: "改性碳模板法制备氧化铁中空微球的研究", 《浙江工业大学硕士学位论文》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109464709A (en) * | 2018-11-26 | 2019-03-15 | 陕西中医药大学 | A kind of preparation method and application of earthworm supernatant protein nanometre collagen repairing composite |
| CN109464709B (en) * | 2018-11-26 | 2021-05-18 | 陕西中医药大学 | Preparation method and application of earthworm supernatant protein nano collagen repair compound |
| CN109529012A (en) * | 2019-01-10 | 2019-03-29 | 陕西中医药大学 | A kind of metal oxide nano bletilla striata pheretima albumen composition preparation method and application |
| CN111743997A (en) * | 2020-07-08 | 2020-10-09 | 陕西中医药大学 | Paste traditional Chinese medicine composition based on earthworm protein paste and preparation method thereof |
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