CN109813911A - A Quantitative and Traceable Method for Determination of Mussel Mucin Degradation - Google Patents
A Quantitative and Traceable Method for Determination of Mussel Mucin Degradation Download PDFInfo
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- CN109813911A CN109813911A CN201810925327.8A CN201810925327A CN109813911A CN 109813911 A CN109813911 A CN 109813911A CN 201810925327 A CN201810925327 A CN 201810925327A CN 109813911 A CN109813911 A CN 109813911A
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- mussel mucin
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- degradation
- mucin
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to field of biotechnology, being related to one kind can quantify and traceable sea-mussel mucin Study on degradation method, comprising steps of (1) by sea-mussel mucin in conjunction with radioactive element or fluorescent reagent after, condensate is formed by intermolecular cross-linking, or is coated on biomedical material and is formed by curing sea-mussel mucin microballoon.(2) in vitro and in vivo model is established.(3) using internal or/and external model, qualitative and quantitative determination increase of the marker in supernatant and the reduction amount on sea-mussel mucin condensate/coating microballoon, so that it is determined that the degradation characteristic and degradation rate of sea-mussel mucin in different environments.The present invention compensates for the missing of biomedical macromolecules degradation research methodology, provides a kind of dynamic, quantitative research method.
Description
Technical field
The present invention relates to medical biotechnology macromolecules degradation fields, more particularly, to for measuring sea-mussel mucin degradation
Condensate and the method degraded using condensate measurement sea-mussel mucin.
Background technique
Sea-mussel mucin (Mussel adhesive protein, MAP), also referred to as mussel byssus protein (Mytilus
Edulis foot protein, Mefp), it is seashells Mytilus galloprovincialis (Mytilus edulis Linnaeus), Trachyostracous mussel
The special protein of one kind of the secretions such as (Mytilus coruscus), Perna viridis (Perna viridis).Mussel usually at
Group ground is attached on longshore reef or the bottom of steamer, is had and is resistant to the ability of wave stroke in coastal waters.Actually mussel
It almost can extremely be securely attached in the substrate of any material, such as metal, timber, glass.Mussel has above-mentioned characteristic
The main reason for be to be produced in its byssus gland and store this special mucoprotein, mussel passes through byssus release mucoprotein to rock
On the surface of solids of stone one kind, the combination of water resistant is formed, so that oneself be fixed.
Identification obtains 11 kinds of mucoprotein subclass from mussel at present, including mefp1, mefp-2, mefp-3, mefp-4,
Mefp-5, mefp-6, pre-COL-P, pre-COL-D, pre-COL-NG, byssus stromatin PTMP and DTMP (Zhu's sunlight sunlight etc.,
Marine Sciences progress, 2014,32 (4): 560-568).Sea-mussel mucin has 2 design features: (1) containing lysine, make egg
Leukorrhea has high carrying capacity positive charge；(2) (DOPA, DOPA) containing 3,4 dihydroxyphenylalanines.The cell and tissue of human body have negative electricity
Lotus.Sea-mussel mucin passes through itself positive charge and human body between cell and tissue negative electrical charge electrostatic interaction and cell and
Tissue tight combines, and plays the effect of protection and treatment.In addition, dopa oxidase generate ortho position diquinone, can with it is not oxidized
DOPA is cross-linked with each other to form film or network, promotes that protein is even closer, is firmly attached to human body surface, plays guarantor
Shield effect.
Sea-mussel mucin has from a wealth of sources, low immunogenicity, biofacies as a kind of novel animal derived biomaterial
The advantages that capacitive is good, easy and negatively charged cell interacts, has obtained in clinic as new bio Wound dressing
Application, future also act as novel tissue engineering rack or coating material for medical instruments etc..But research both at home and abroad at present is still not
The degradation characteristic and immunogenicity for understanding sea-mussel mucin are especially the absence of effective Study on degradation method and lack degradation production
Report of the object to body effect, making this kind of product, there are potential risks in clinical use.
As large biological molecule especially protein molecule is more and more applied in the medical field, it is necessary to establish one
The method that kind can quantitative determine and characterize its degradation rate and degradation characteristic under human body use environment.
Currently, in terms of the research degraded in protein and peptide body is concentrated mainly on pharmacokinetic.The medicine studied
Product largely belong to Formulations for systemic administration, and cracking be rapidly absorbed into blood reaches peak plasma concentrations, then into different
In tissue.Research method has biologic assay, Isotope tracer labelling method, immunoassay, liquid chromatogram (LC), capillary electricity
Swimming (CE), gas-chromatography (GC), mass spectrum (MS), nuclear magnetic resonance (NMR), liquid chromatography-mass spectrography (LC-MS) and Capillary Electrophoresis-
Mass spectrum (CE-MS) joint technology etc..These methods largely require cumbersome sample acquisition and processing step, and cannot obtain
Obtain real-time data.
And medical instrument is different from drug, most of is all local use, and longer using the time.Therefore, degradation is ground
Study carefully and be different from whole body system medication, degradation process is one and continues to slow process, catabolite or is only limitedly applied to portion
It is gradually absorbed around position, or blood circulation and its hetero-organization is continued into extremely low concentration.Therefore, the above method or
Measurement accuracy is inadequate, cannot obtain the degradation situation under the true use state of instrument；Or be unable to real-time in-situ measurement and can not
Obtain true degradation data.It therefore, is always the difficult point studied for the degradation process of protide medical instrument.
Summary of the invention
To overcome defect of the existing technology, what the present invention provided a kind of novel measurement protein degradation has efficacious prescriptions
Method, and in particular to a method of the degradation of detection sea-mussel mucin more particularly to a kind of mussel based on marker identification glue egg
The detection method of white degradation.This method can it is simple, quickly, carry out sea-mussel mucin degradation dynamic mistake under physiological environment in real time
The measurement of journey.
Method of the invention utilizes the markers such as radioactive isotope or fluorescent chemicals in conjunction with sea-mussel mucin, and leads to
The condensate that is cross-linked to form between protein molecule is crossed, or solidification forms microballoon in biomedical material surface, by condensate or packet
It is placed under use environment by good microballoon, timing detects marker in free marker concentrations or condensate or microballoon out
Concentration is quantified hence for the sea-mussel mucin that marker is coupled.
Specifically, the method for measurement sea-mussel mucin degradation according to the present invention comprising step: (1) mussel is glued into egg
It is white in conjunction with marker after, condensate is formed by intermolecular cross-linking, or is coated on to be formed by curing on biomedical material and make a gift of
Shellfish mucoprotein microballoon.(2) external and/or In vivo model is established.(3) qualitative and/or quantitative using internal or/and external model
Increase of the marker in supernatant and the reduction amount on sea-mussel mucin condensate/coating microballoon are measured, so that it is determined that
The degradation characteristic and degradation rate of sea-mussel mucin in different environments.
In one embodiment, the method for measurement sea-mussel mucin degradation of the invention comprising step: (1) will make a gift of
After shellfish mucoprotein is in conjunction with marker, condensate is formed by intermolecular cross-linking.(2) external and/or In vivo model is established.(3)
In the external and/or In vivo model of foundation, it is evenly mixed in sea-mussel mucin condensate in system with free form, or solely
It is vertical to be present in another space and realize the mass exchange with system with some form.(3) continuous timing sampling, it is qualitative and/or
The marker increase in supernatant and/or the marker reduction amount on sea-mussel mucin condensate are quantitative determined, thus really
Determine the degradation characteristic and degradation rate of sea-mussel mucin in different environments.
In another embodiment, the method for measurement sea-mussel mucin degradation of the invention comprising step: (1) will
After sea-mussel mucin is in conjunction with marker, it is solidificated in formation sea-mussel mucin microballoon on biomedical material.(2) it establishes external
And/or In vivo model.(3) in the external and/or In vivo model of foundation, mix sea-mussel mucin microballoon uniformly with free form
It closes in system, or is independently present in another space and realizes the mass exchange with system with some form.(3) continuous timing
Sampling, marker that is qualitative and/or quantitative determining in supernatant increase and/or are coated with the label on microballoon in sea-mussel mucin
Object reduction amount, so that it is determined that the degradation characteristic and degradation rate of sea-mussel mucin in different environments.
In the present invention, by sea-mussel mucin binding label in the step (1), marker therein can be radiation
Property element, including the radioactive isotopes such as 14C, 3H hydrogen (tritium), 125I, 131I, 35S, 32P；Marker therein is also possible to
Fluorescent dye, such as fluorescein isothiocynate (FITC), hydroxyl fluorescein (FAM), tetrachlorofluorescein (TET), R101, tetraethyl sieve
Red bright (RB200) and carboxyl tetramethylrhodamine (TAMRA), thiazole orange (thiazole orange, TO), oxazole orange
(oxazole orange, YO), talan, naphthalimide, Coumarins, acridine, pyrene class etc.；Marker therein can also
To be green fluorescent protein, phycoerythrin, other phycocyanin, perdinin phyllochlorin etc..
In the present invention, by sea-mussel mucin binding label in the step (1), wherein combining is with covalent bond
Form is crosslinked or is combined by intermolecular physical action.Wherein the weight ratio of sea-mussel mucin and marker is 10:1 to 10:
5。
In the present invention, sea-mussel mucin by the condensate that intermolecular cross-linking is formed can be colloidal, sediment or
Manufactured powder, particle, film etc. after drying.Polymerization methods can be to be crosslinked by autoxidation, can also be crosslinked by addition
Agent mode forms polymerization.
In the present invention, sea-mussel mucin and/or labeled sea-mussel mucin are cured to the side of biomedical material
Formula can be spraying, immersion etc..The dosage for being solidificated in the sea-mussel mucin on biomedical material should be able to uniform fold or infiltration
The biomedical material.It can be arbitrary for cured temperature, preferably 10-40 DEG C.It can be 1- for the cured time
24 hours.
The biomedical material being related in the present invention is formed according to material and property may include titanium, tantalum, niobium, zirconium, no
Become rusty the metal materials such as steel, cobalt-base alloys and titanium-base alloy, the inorganic material such as ceramics, glass, carbon, polyethylene, polypropylene, poly- third
Olefin(e) acid ester, polysiloxanes, polyformaldehyde, collagen, linear aliphatic adoption ester, chitin, cellulose, polyaminoacid, gathers aromatic polyester
Vinyl alcohol, the degradable or non-degradable high molecular material such as gather own propyl ester, biomedical composite material and bio-derived material etc.,
Wherein biomedical composite material is the material being combined by two or more above-mentioned material, and bio-derived material is
The biomedical material formed by the natural biological tissue Jing Guo specially treated.
In the present invention, establishing external model may include the culture system in vitro for establishing humans and animals tissue and cell,
It especially include fibroblast, cardiac muscle, smooth muscle, osteoblast, intravascular including primary cell, passage cell and cell strain
Chrotoplast, skin epidermis and its derivative (sweat gland, sebaceous glands) alimentary canal epithelium, liver, pancreas, alveolar epithelium, further include migration type
Cell and pleomorphism cell etc..Establishing external model can also include the body fluid and artificial body fluid system for establishing humans and animals, especially
It includes blood plasma, serum, tissue homogenate, various enzyme complex liquids etc..Establish In vivo model may include establish mouse, rat,
The experimental animal models such as cavy, rabbit, dog, monkey.
The invention further relates to a kind of sides for introducing the condensate for being coated with sea-mussel mucin/microballoon and recycling in vivo and all
Method.Including but not limited to by condensate/microballoon direct injection in it is subcutaneous, by condensate/microballoon in netted microcapsules, utilize
Bag filter is wrapped in hydrogel by condensate/microballoon package, by condensate/microballoon or locally embeds condensate/microballoon
The methods of in the tissues such as skin, muscle or organ.
In the present invention, the detection to marker may include radiological measuring, including but not limited to use crystal scintillator
Number method and liquid scintillation counting.The detection of marker is also possible to fluorescence detection, including but not limited to uses spectrophotometry.
In the present invention, the sea-mussel mucin can be the method acquisition of natural origin or biosynthesis, including the use of
Known method is extracted, preparation, separates and purify and obtain, such as with mixing and absorption chromatography (see China Patent No.
ZL200710179491.0), carboxymethyl ion-exchange chromatography (see China Patent No. ZL200710179492.5), and/or saltout
With dialysis (China Patent No. ZL200910087567.6).It is also possible to the sea-mussel mucin of commercial source, including Jiangyin shellfish is auspicious
Gloomy biochemical technology Co., Ltd (while being also sea-mussel mucin medical instrument manufacturer), BD bioscience (U.S.),
Kollodis (South Korea), Biopolymer (Sweden).Include one or more of known 11 sea-mussel mucin subclass
Mixture；It is also possible to natural origin or artificial bio-membrane synthesizes the range of hydrolysed peptides that sea-mussel mucin obtains after hydrolysis.
Another aspect of the present invention provides a kind of microballoon degraded for measuring sea-mussel mucin, and the microballoon includes biology
The sea-mussel mucin that medical material and coating are solidificated on the biomedical material, wherein the sea-mussel mucin is through that can examine
The label substance markers of survey.
Sea-mussel mucin is creatively cured on biomedical material and forms microballoon by the present invention, avoids cumbersome electricity
The experimental implementations such as swimming, and can continuous under physiological status or simulation physiological status, quantitatively measure the knot of sea-mussel mucin
Conjunction/degradation, so as in real time, rapidly evaluate the use and safety of sea-mussel mucin and its medical product.
In order to make it easy to understand, below by the method by specific embodiment to measurement sea-mussel mucin degradation of the invention
It is described in detail.It should be pointed out that specific example is merely to explanation, only describes a wherein few portion in embodiment
Point, however should not be construed as limitation of the present invention.The present invention is further illustrated in following non-limiting examples
Product/method property and advantage.Of particular note is that obviously those skilled in the art can be according to saying herein
It is bright, various modifications and variations are made to the present invention within the scope of the invention, this hair is also included in these modifications and variations
In bright range.
Specific embodiment
Embodiment 1: the sea-mussel mucin polymer particles of radioiodination are for studying the mussel in cell culture fluid
The degradation of mucoprotein
The preparation of radioiodination sea-mussel mucin: being oxidant with Iodogen, carries out iodate mark to sea-mussel mucin
Note, 125I is directly introduced on the tyrosine residue in molecule.Before label, Iodogen is first dissolved in organic solvent, is applied to anti-
Tube bottom is answered, and is allowed to drying.1.0mg/ml sea-mussel mucin solution is placed in reaction tube to be placed in ice bath.When iodate, 125I
Weight ratio with protein molecule is 1~1.2.Mild continuously stirs 10min, and reaction mixing is transferred out of from reaction tube
Liquid stops reaction.
The preparation of sea-mussel mucin polymer particles: taking concentration is the sea-mussel mucin solution of the iodine labeling of 1.0mg/ml
10ml is persistently stirred 24 hours under the conditions of pH7.0, and sea-mussel mucin is made to be cross-linked to form condensate by autoxidation, and centrifugation is received
Collect sediment, forms sea-mussel mucin polymer particles after being dried to and be completely dried in 100 DEG C.
External model: taking 264.7 cell line of Raw frozen, and after 37 DEG C of quick-thawings, 1000rpm/min is centrifuged 5min,
It is cleaned 1-2 times with DMEM culture medium, is centrifuged again after being resuspended with DMEM basal medium.Complete medium is added to be cultivated, often
It changes a subculture.Cell growth condition is observed, is covered with rear spare.
Test method: the sea-mussel mucin polymer particles of 0.5mg iodine labeling are fitted into netted capsule, with fresh passage
Raw264.7 cell cultivate together, daily sample, put using gamma-rays scintillation counter to what is released in cell culture fluid
Penetrating property iodine is quantitative determined, to track the timeline of sea-mussel mucin degradation.Off-test after two weeks quantitative determines micro-
Radioactivity in capsule can obtain the quantitative result of sea-mussel mucin degradation comparing with intial value.
The experimental results showed that in whole experiment process, only in last two days detection radioiodine elements, micro- glue in supernatant
Capsule radioactivity determination result is not significantly different with initial value, illustrates sea-mussel mucin polymer particles in cell culture fluid
It has good stability, only micro sea-mussel mucin is degraded.
Embodiment 2: the sea-mussel mucin of radioiodination is coated with artificial osseous granules for studying in tissue homogenate
The degradation of sea-mussel mucin
The preparation of radioiodination sea-mussel mucin: being oxidant with Iodogen, carries out iodate mark to sea-mussel mucin
Note, 125I is directly introduced on the tyrosine residue in molecule.Before label, Iodogen is first dissolved in organic solvent, is applied to anti-
Tube bottom is answered, and is allowed to drying.1.0mg/ml sea-mussel mucin solution is placed in reaction tube to be placed in ice bath.When iodate, 125I
Weight ratio with protein molecule is 1~1.2.Mild continuously stirs 10min, and reaction mixing is transferred out of from reaction tube
Liquid stops reaction.
Sea-mussel mucin is coated with the preparation of artificial osseous granules: taking 5g diameter is the artificial skelecton particle (Shanghai of 0.25-1mm
Ha Yan Biotechnology Co., Ltd) and concentration be 1.0mg/ml iodine labeling sea-mussel mucin solution 5ml, in optimal pH item
Under part, artificial skelecton particle is mixed with sea-mussel mucin, sea-mussel mucin is made to adhere to bone particles surface.Mixing is equal
In 40 DEG C of solidification 12h after even, until osseous granules form the coated artificial osseous granules of sea-mussel mucin after being completely dried.
Tissue homogenate: SD rat is put to death, and takes out liver, is washed 2~3 times with physiology salt, is removed blood, is peeled surface off
Connective tissue fat.It shreds, is washed to no color and stopped repeatedly with physiological saline, then add physiological saline a little again, use group
It knits bruisher or homogenizer is made homogenate.Liver homogenate is packed into centrifuge tube (1/3 or so), interchangeably with 2~3 times of amount physiology salts
Water and acetone wash respectively three times repeatedly, until supernatant stops without color, are finished after first using 2000rpm/min centrifugation 15min every time,
Supernatant is removed again.- 20 degree refrigerators save.
Experimental method: taking the coated artificial osseous granules of the sea-mussel mucin of 0.5mg iodine labeling to be fitted into netted capsule, and new
The liver tissues of rats homogenate of fresh preparation is cultivated together, is sampled daily, using gamma-rays scintillation counter in cell culture fluid
The radioiodine element released is quantitative determined, to track the timeline of sea-mussel mucin degradation.Off-test after two weeks,
The radioactivity in microcapsules is quantitative determined, comparing with intial value, the quantitative result of sea-mussel mucin degradation can be obtained.
The experimental results showed that beginning with radioactivity detection in supernatant from the 6th day, subsequent radioactive intensity starts to increase.
Microcapsules radioactivity determination result comparing with intial value, has dropped about 12%, illustrates the coated artificial osseous granules of sea-mussel mucin
Have in liver tissue homogenate's liquid and is degraded on a small quantity.
Embodiment 3: the sea-mussel mucin of radioiodination is coated with artificial osseous granules for studying the mussel in Mice Body
The degradation of mucoprotein
The preparation of radioiodination sea-mussel mucin: being oxidant with Iodogen, carries out iodate mark to sea-mussel mucin
Note, 125I is directly introduced on the tyrosine residue in molecule.Before label, Iodogen is first dissolved in organic solvent, is applied to anti-
Tube bottom is answered, and is allowed to drying.1.0mg/ml sea-mussel mucin solution is placed in reaction tube to be placed in ice bath.When iodate, 125I
Weight ratio with protein molecule is 1~1.2.Mild continuously stirs 10min, and reaction mixing is transferred out of from reaction tube
Liquid stops reaction.
Sea-mussel mucin is coated with the preparation of artificial osseous granules: taking 5g diameter is the artificial skelecton particle (Shanghai of 0.25-1mm
Ha Yan Biotechnology Co., Ltd) and concentration be 1.0mg/ml iodine labeling sea-mussel mucin solution 5ml, in optimal pH condition
Under, artificial skelecton particle is mixed with sea-mussel mucin, sea-mussel mucin is made to adhere to bone particles surface.It is uniformly mixed
Afterwards in 40 DEG C of solidification 12h, until osseous granules form the coated artificial osseous granules of sea-mussel mucin after being completely dried.
Experimental method: the sea-mussel mucin coating osseous granules of radioiodination are subcutaneously injected into Mice Body, every
Mouse injects 6 sites, carefully marks injection point, in order to recycle all particles for detecting remaining radioactive intensity.
Sampling: each sample point puts to death 2 mouse, removes the tissue samples of each injection site, carefully to recycle
There are osseous granules, measures the radioactive intensity of each sample.
Sample time: (1) 1 hour, (2) 24 hours (1 day), (3) 48 hours (2 days), (4) 4 days, (5) 8 days, (6) 16 days
The experimental results showed that the radioactive intensity of the 4th day sample has dropped 3%, 5% was reached by the 8th day, is increased within the 16th day
It is added to 8%, illustrating sea-mussel mucin, there is also degradations in Mice Body, and degrade earlier than external model.
Embodiment 4: the sea-mussel mucin of fluorescent marker is coated with artificial osseous granules for studying the mussel in tissue homogenate
The degradation of mucoprotein
Sea-mussel mucin fluorescent marker: the sea-mussel mucin of 1.0mg/ml is fitted into bag filter.With same buffering
FITC is made into the solution of 0.1mg/ml by liquid, and by 10 times of 1% protein liquid volume, FITC dilution is contained in hydrostatic column
It is interior, and it is immersed in bag filter in FITC liquid.Container head covers tightly, and stirring rod is put in bottom, under 4 DEG C of electromagnetic agitations, dialysis mark
Note is for 24 hours.Marking fluid in bag filter is taken out, uses sephadex G-50 gel filtration at once, removes free fluorescein, packing, storage
In 4 DEG C
Sea-mussel mucin is coated with the preparation of artificial osseous granules: taking 5g diameter is the artificial skelecton particle (Shanghai of 0.25-1mm
Ha Yan Biotechnology Co., Ltd) and concentration be 1.0mg/ml fluorescent marker sea-mussel mucin solution 5ml, in optimal pH item
Under part, artificial skelecton particle is mixed with sea-mussel mucin, sea-mussel mucin is made to adhere to bone particles surface.Mixing is equal
In 40 DEG C of solidification 12h after even, until osseous granules form the coated artificial osseous granules of sea-mussel mucin after being completely dried.
Tissue homogenate: SD rat is put to death, and takes out liver, is washed 2~3 times with physiology salt, is removed blood, is peeled surface off
Connective tissue fat.It shreds, is washed to no color and stopped repeatedly with physiological saline, then add physiological saline a little again, use group
It knits bruisher or homogenizer is made homogenate.Liver homogenate is packed into centrifuge tube (1/3 or so), interchangeably with 2~3 times of amount physiology salts
Water and acetone wash respectively three times repeatedly, until supernatant stops without color, are finished after first using 2000rpm/min centrifugation 15min every time,
Supernatant is removed again.- 20 degree refrigerators save.
Experimental method: taking the coated artificial osseous granules of 0.5mg fluorescent marker sea-mussel mucin to be fitted into netted capsule, and new
The liver tissues of rats homogenate of fresh preparation is cultivated together, is sampled daily, and the fluorescence intensity in supernatant is measured, viscous to track mussel
The timeline of protein degradation can calculate the concentration of corresponding sea-mussel mucin compared with standard curve.It tests after two weeks
Terminate, quantitative determine the fluorescence intensity in microcapsules, comparing with intial value, and compare supernatant determination data, it is viscous to obtain mussel
The quantitative result of protein degradation.
The experimental results showed that fluorescence intensity gradually increased since the 8th day in supernatant.Microcapsules fluorescent strength determining knot
Fruit comparing with intial value, has dropped about 10%, illustrates that the coated artificial osseous granules of sea-mussel mucin have in liver tissue homogenate's liquid
It is degraded on a small quantity.
Claims (10)
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