CN110025768A - A kind of construction method of eye disease animal model and its application - Google Patents
A kind of construction method of eye disease animal model and its application Download PDFInfo
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- CN110025768A CN110025768A CN201910476999.XA CN201910476999A CN110025768A CN 110025768 A CN110025768 A CN 110025768A CN 201910476999 A CN201910476999 A CN 201910476999A CN 110025768 A CN110025768 A CN 110025768A
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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
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1858—Platelet-derived growth factor [PDGF]
- A61K38/1866—Vascular endothelial growth factor [VEGF]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
Abstract
The present invention provides a kind of construction method of eye disease animal model and its applications, and specifically, the present invention provides a kind of modeling reagents, comprising: (a) VEGF sustained release preparation；(b) VEGFR or its expression vector.(a) VEGF sustained release preparation and (b) VEGFR or its expression vector, which is applied in combination, in the present invention can effectively construct eye disease animal model.
Description
Technical field
The present invention relates to field of biotechnology, in particular it relates to a kind of building side of eye disease animal model
Method and its application.
Background technique
One of the reason of formation of ocular angiogenesis is blinding on clinical ophthalmology, mainly with cause intraocular ischemia anoxic
Angiogenic diseases are related.Cause the disease of new vessels very much, clinically common includes diabetes, arteria retina resistance
Plug, ischemic retinal central retinal vein occlusion etc..New vessels may betide in many tissues of eyes, such as cornea rebirth blood
Pipe, iris neovascularization, choroidal neovascularization, ciliary body new vessels and retinal neovascularization etc..Therefore, research influences
Its key molecule generated, or find the newtype drug of its progress of interference, it will significant impact is generated to the progress of clinical ophthalmology.
Iris neovascularization (neovascularization of the iris, NVI) is also known as rubeosis of iris, mostly secondary to
The disease and systemic disease at other positions of eye.Studies show that, in diabetic population, the incidence of iris neovascularization is
1%-17%, and in proliferating diabetic retinopathy change, incidence is up to 65%.The western general, VEGF Trap of Compaq, shellfish
The drug for cutting down the treatment retinal neovascularization such as monoclonal antibody, Lucentis is widely used in clinic, and new bio peptide intervenes cornea
The research of generation and the development of new vessels has also just obtained effect, but more about the research of iris neovascularization mechanism and treatment
It is poor.Currently, one of the problem that NVI correlative study faces is a lack of ideal animal model.Currently, classical animal NVI mould
The foundation of type mostly uses photochemical method, and cardinal principle is after photochemical drugs Bengal rose red is injected into animals iv
Animal eyes are irradiated using laser, the oxygen in blood is excited to be changed into oxygen radical.The oxygen radical inspired destroys blood
Endothelial cell and platelet membrane, then start clotting system.At the same time, a series of blood coagulations of broken intra platelet free calcium because
Son accelerates the formation of thrombus.This model uses rabbit, cat, pig, monkey, rat etc. as experimental animal more.But it is medium-and-large-sized dynamic
Although object has biggish eyeball, due to higher cost, be not suitable for large-scale use, this becomes the very important disadvantage of above-mentioned model
End.
Therefore, there is an urgent need in the art to provide new animal experimental model.
Summary of the invention
The object of the present invention is to provide new animal experimental models.
First aspect present invention provides a kind of modeling reagent, comprising:
(a) VEGF sustained release preparation；
(b) VEGFR or its expression vector.
In another preferred example, the VEGF sustained release preparation is selected from the group: VEGF microsome, VEGF suspension, or combinations thereof.
In another preferred example, the VEGF sustained release preparation includes VEGF microsome.
In another preferred example, the expression vector includes viral vectors.
In another preferred example, the viral vectors is selected from the group: adenovirus vector, gland relevant viral vector, slow virus
Carrier, or combinations thereof.
In arranging another preference, the microsome includes nanoparticle.
In another preferred example, the diameter of the microsome is 30-300nm, preferably 50-200nm, more preferably 100~
150nm。
In another preferred example, total drugloading rate of the microsome is 2-50 μ g, preferably, 5-40 μ g, more preferably, 6-
20, more preferably, 8-15 μ g.
In another preferred example, in the modeling reagent, the drug concentration of the microsome is 20-90ng/ μ l, preferably
Ground, 30-70ng/ μ l, more preferably, 40-55ng/ μ l.
In another preferred example, the modeling reagent is liquid preparation.
In another preferred example, the modeling reagent is nanoparticle body suspension.
In another preferred example, the modeling reagent substantially (>=90%, 95%, 96%, 97%, 98%, 99%,
99.5%, 99.9%) it or is all made of (a) VEGF sustained release preparation and (b) VEGFR or its expression vector.
In another preferred example, in the modeling reagent, the concentration of component (a) is 20ng/ μ l -100ng/ μ l, preferably
Ground, 30ng/ μ l-60ng/ μ l, more preferably, 50ng/ μ l-55ng/ μ l.
In another preferred example, in the modeling reagent, the concentration of component (b) is 1 × 107A/l -6 × 10 μ7A/μ l,
Preferably, 2 × 107A/l-5 × 10 μ7A/μ l, more preferably, 3 × 107A/l-3.5 × 10 μ7A/μ l.
In another preferred example, the VEGFR albumen includes full-length proteins or protein fragments.
In another preference, the VEGFR albumen source is more preferably (such as small from rodent in mammal
Mouse, rat), Primate and people.
In another preferred example, the VEGFR albumen further includes the derivative of VEGFR albumen.
In another preferred example, the derivative of the VEGFR albumen includes modified VEGFR albumen, amino acid sequence
With natural VE GFR albumen homology and with the protein molecular of VEGFR protein active, the dimer of VEGFR albumen or polymer, contain
There is the fusion protein of VEGFR protein amino acid sequence.
It is in another preferred example, described that " amino acid sequence is with natural VE GFR albumen homology and with natural VE GFR albumen
Active protein molecular " refers to that its amino acid sequence has >=85% homology compared with VEGFR albumen, preferably >=90%
Homology, homology more preferably >=95%, most preferably >=98% homology；And with natural VE GFR protein active
Protein molecular.
In another preferred example, the modeling reagent is the reagent for preparing eye disease disease animal model.
In another preferred example, the animal model includes the animal model of non-human mammal.
In another preferred example, the eye disease disease includes iris neovascularization related disease.
In another preferred example, the iris neovascularization related disease is selected from the group: retinal vein obstruction is concurrently new
The concurrent neovascular glaucoma of angiogenic glaucoma, diabetic retinopathy, or combinations thereof.
Second aspect of the present invention provides a kind of purposes of modeling reagent described in first aspect present invention, is used to prepare eye
The drug or preparation of portion's disease animal model.
In another preferred example, the preparation is liquid formulation.
In another preferred example, the eye disease disease includes iris neovascularization related disease.
In another preferred example, the iris neovascularization related disease is selected from the group: retinal vein obstruction is concurrently new
The concurrent neovascular glaucoma of angiogenic glaucoma, diabetic retinopathy, or combinations thereof.
Third aspect present invention provides a kind of preparation method of eye disease disease animal model, comprising:
(a) mammal is provided；
(b) ocular tissue of Xiang Suoshu mammal injects the first component and the second component, after cultivating a period of time T1, from
And obtain the eye disease disease animal model.
In another preferred example, the ocular tissue is selected from the group: anterior chamber, iris, or combinations thereof.
In another preferred example, first component includes VEGF sustained release preparation.
In another preferred example, second component includes VEGFR or its expression vector.
In another preferred example, first component and the second component are simultaneously or successively injected.
In another preferred example, when first component and second group are successively injected, the first component and the second component
Interval injection time be 12h-6 days, preferably, -4 days 1 day, more preferably, -4 days 3 days.
In another preferred example, the T1 is 7-21 days, preferably, 10-18 days, more preferably, 14-16 days.
In another preferred example, the mammal includes people or non-human mammal.
In another preferred example, the non-human mammal includes that rodent (such as rat, mouse, rabbit), primate are dynamic
Object (such as monkey).
In another preferred example, the receiving volume of anterior chamber's aqueous humor is 1 μ l-10 μ l, preferably, 1 μ l-5 μ l, more preferably
Ground, 3 μ of μ l -5 l.
In another preferred example, the concentration of first component is 10ng/ μ l -100ng/ μ l, preferably, 30ng/ μ l-
60ng/ μ l, more preferably, 50ng/ μ l-55ng/ μ l.
In another preferred example, the concentration of second component is 1 × 107A/l -6 × 10 μ7A/μ l, preferably, 2 ×
107A/l-5 × 10 μ7A/μ l, more preferably, 3 × 107A/l-3.5 × 10 μ7A/μ l.
Fourth aspect present invention provides a kind of purposes of the animal model of third aspect present invention the method preparation, will
The model is used as the animal model of research eye disease.
Fifth aspect present invention provides a kind of purposes of the animal model of third aspect present invention the method preparation, uses
It can reduce or treat the substance (therapeutic agent) of eye disease in screening or identification.
Sixth aspect present invention provides the side of a kind of screening or the determining potential treatment agent treated or alleviate eye disease
Method, comprising steps of
(a) in test group, under the conditions of testing existing for compound, test compound is applied to claim 3 institute
The animal model of method preparation is stated, the severity Q1 of the eye disease of animal model described in test group is detected；And not
It applies in the test compound and the identical control group of other conditions, detects the eye disease of animal model described in control group
Severity Q2；With
(b) previous step severity Q1 detected and severity Q2 is compared, so that it is determined that the test
Whether compound is treatment or the potential treatment agent for alleviating eye disease；
Wherein, if severity Q1 is substantially less than severity Q2, then it represents that the test compound be treatment or
Alleviate the potential treatment agent of eye disease.
In another preferred example, the detection eye disease severity includes detecting one or more selected from the group below
The variation of index: intraocular pressure increases, whether iris tissue occurs whether visible new vessels, iris angiography under mirror fluorescence occur
Leakage.
In another preferred example, eye disease severity reduction shows themselves in that under intraocular pressure decline, iris tissue mirror
New vessels subside, iris angiography fluorescence leakage is reduced.
In another preferred example, described ratio≤1/2 for " substantially less than " referring to severity Q1/ severity Q2, preferably
Ground≤1/3, more preferably ,≤1/4.
In another preferred example, the method is non-diagnosing and treating.
In another preferred example, the method includes the steps (c), and the potential treatment agent that step (b) is screened or identified is applied
For the animal model of third aspect present invention the method preparation, so that it is tight to the eye disease of the animal model to measure it
The influence of weight degree.
Seventh aspect present invention provides a kind of non-human mammal model, described in the model third aspect present invention
Method preparation.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 shows that this research tissue samples VEGFR is overexpressed situation.
Fig. 2 shows this research each group rat intraocular pressure situation.
Fig. 3 shows anterior ocular segment situation after the mouse modeling of this research experiment.
Fig. 4 shows iris angiography result after the mouse modeling of this research experiment.
Fig. 5 shows that this research experiment mouse injection diameter is varieties of intraocular pressure situation after 10nm and 500nmVEGF microsome.
Fig. 6 shows varieties of intraocular pressure situation after this research experiment mouse injection 10ng/ μ l drug concentration VEGF microsome.
Specific embodiment
The present inventor after extensive and in-depth study, is found surprisingly that, (a) VEGF sustained release preparation and (b) VEGFR or
Its expression vector, which is applied in combination, can effectively construct eye disease animal model.Also, the present inventor is also found surprisingly that, (a)
VEGF sustained release preparation and (b) VEGFR or its expression vector are simultaneously or successively injected into can be effective in ocular tissue (such as anterior chamber)
Eye disease animal model is constructed, animal model of the invention can be used for studying eye disease, and can be used for certain drug
Screening and testing experiment.The present invention is completed on this basis.
Specifically, the present inventor, which constructs, is overexpressed VEGFR adenovirus (ADV) and VEGF microsome, injects in rat anterior chamber
It is overexpressed VEGFR adenovirus.The iris neovascularization and retinal vessel of laser enclosed experiment mouse after being clearly overexpressed, will
The injection of VEGF microsome has carried out in the experimental mouse anterior chamber of laser closing iris and retinal vessel.Test experience rathole pressure, it is preceding
Save the observation side reaction of camera iris angiography and new vessels.After VEGFR adenovirus injected into anterior chambers, in rat iris tissue
Middle be successfully overexpressed needs 48~72 hours, therefore we carry out VEGF microsome after rat injected into anterior chambers adenovirus 3 days
Injection.The biological half-life of VEGF is short, is only capable of lasting 4~6 hours, need to be injected repeatedly to maintain mould under anoxic conditions
Type constructs required effective concentration and is easy to appear ocular complications.Therefore, present invention application nanoparticle building VEGF sustained release
System, to solve the problems, such as above-mentioned to exist.In addition, adenovirus sustainable expression 1 week after function is dyed in rat iris tissue transfer, because
This, the present invention constructs the VEGF microsome of diameter about 150nm, about 10 days deenergized periods, to maintain the effective of model construction
Concentration, and delay the recession of NVI to the full extent.
Iris neovascularization (NVI)
As used herein, term " NVI " refers to iris neovascularization, also known as rubeosis of iris, refers under hypoxic-ischemic state,
The pathologic vessels that iris tissue surface is grown.NVI is not the primary disease of iris, but secondary to many eye disease and certain
Systemic disease.Since it can develop into or merge the formation of fibrovascular membranes, so that iridocorneal angle is closed and is occurred
Serious neovascular glaucoma, intraocular pressure are often difficult to control, and are eventually led to and are suffered from eye blindness, or even extract eye because of eyeball severe pain
Ball.Therefore, it finds and handles in time just particularly significant as early as possible.NVI early stage is the first phase, and new vessels come across iris first
Nearly pupil margin and some regions at room angle.The visible tiny bending in iris surface and irregular red line are in brown iris.Iridial angle
Film angle, which checks, sees that room angular breadth is still normal.This duration phase different, thrombosis of central vein of retina institute with pathogenic factor difference
Cause person quickly grows, this phase only maintains several weeks or several months.Second phase iris neovascularization continues growing, and merges into each other, until whole
A iris surface new vessels reticulate, and iridocorneal angle also has a more new vessels, but nothing or only a few regions iris week
Side anterior synechiae.NVI is developed to the third phase, and iris surface is generally covered by neovascular membranes；Since fibrovascular tissue is shunk, lead
Draw uvea and forms pupil margin ectropion of pigment layer forward；The extensive periphery anterior synechiae of iridocorneal angle, causes intraocular pressure sharply to increase
With the significant mixed congestion of neovascular glaucoma.Suffer from ophthalmagra, eyesight only deposits light sensation.Using the stage division of Miller to model
Mouse NVI is classified, and be divided into 0~5 grade, 0 grade: normal iris, a small amount of blood vessel is visible or invisible, ne-leakage；1 grade: visible
Increased vascularization, it is prominent, sinuous, discontinuous, but still ne-leakage；2 grades~3 grades: there is early stage, quickly new green blood in increased vascularization
Pipe leakage；4 grades: NVI is a large amount of, so that iris is invisible in preceding 35s, 5 grades: 4 grades of merging iridectropiums or glaucoma.
In the present invention, iris neovascularization related disease includes, but is not limited to: retinal vein obstruction is concurrently newborn
The concurrent neovascular glaucoma of neovascular glaucoma, diabetic retinopathy.
VEGF sustained release preparation
In the present invention, VEGF sustained release preparation refers to nanoparticle or suspension comprising VEGF, even if VEGF is in anoxic item
Under part, half-life period is also only capable of maintaining 4-6 hours, using VEGF sustained release preparation, can make the slow sustained release of VEGF, to maintain mould
Effective concentration needed for type constructs.
VEGFR albumen and polynucleotides
In the present invention, term " albumen of the present invention ", " VEGFR albumen ", " VEGFR polypeptide " are used interchangeably, and all refer to tool
There are the albumen or polypeptide of VEGFR amino acid sequence.They include the VEGFR albumen with or without initial methionine.In addition,
The term further includes the VEGFR and its segment of overall length.VEGFR albumen of the invention signified include its complete amino acid sequence,
Its secretory protein, its mutant and its functionally active segment.
VEGFR albumen is vascular endothelial growth factor receptor, can be combined with the multiple growth factors of VEGF family, thus
A series of angiogenesis are induced to react.
In the present invention, term " VEGFR gene ", " VEGFR polynucleotides " are used interchangeably, and are all referred to VEGFR core
The nucleic acid sequence of nucleotide sequence.
The full-length genome 5849bp of human VEGFR-3 gene (NCBI GenBank accession number is NC_000004.12).
The full-length genome 5892bp of rat VEGFR gene (NCBI GenBank accession number is NC_005113.4).
It is to be understood that the substitution of codon nucleotide is acceptable when encoding identical amino acid.In addition it needs
Understand, when being replaced by nucleotide and generating conservative amino acid substitution, the transformation of nucleotide is also can be received.
In the case where having obtained the amino acid fragment of VEGFR, its nucleic acid sequence of coding can be constructed according to it, and
Specific probe is designed according to nucleotide sequence.Nucleotide full length sequence or its segment can usually use PCR amplification method, recombination
Method or artificial synthesized method obtain.For PCR amplification method, can disclosed VEGFR nucleotide sequence according to the present invention, especially
It is that open reading frame sequence carrys out design primer, and with the commercially available library cDNA or presses conventional method institute well known by persons skilled in the art
The library cDNA of preparation expands as template and obtains related sequence.When sequence is longer, it is often necessary to carry out twice or repeatedly PCR
The segment that each time amplifies, is then stitched together by amplification by proper order again.
Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually will
It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.
In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In general, logical
After first synthesizing multiple small fragments, it is then attached the very long segment of available sequence again.
At present, it is already possible to obtain encoding albumen of the present invention (or its segment, derivative) completely by chemical synthesis
DNA sequence dna.Then the DNA sequence dna can be introduced into various existing DNA moleculars (such as carrier) as known in the art and cell.
By the recombinant dna technology of routine, it can be used to express or produce recombination using polynucleotide sequence of the invention
VEGFR polypeptide.In general there are following steps:
(1) polynucleotides (or variant) of encoding human VEGFR polypeptide of the invention, or with contain the polynucleotides
Recombinant expression carrier conversion or suitable host cell of transduceing；
(2) host cell that is cultivated in suitable culture medium；
(3) be separated from culture medium or cell, protein purification.
In the present invention, VEGFR polynucleotide sequence be can be plugged into recombinant expression carrier.As long as in short, can be in host
Interior duplication and stabilization, any plasmid and carrier can be used.One important feature of expression vector be usually contain replication orgin,
Promoter, marker gene and translation control element.
Method well-known to those having ordinary skill in the art can be used to construct DNA sequences encoding containing VEGFR and suitably transcribe/turn over
Translate the expression vector of control signal.These methods include recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc..
The DNA sequence dna can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to synthesize.Expression vector also wraps
Include the ribosome bind site and transcription terminator of translation initiation.
In addition, expression vector preferably includes one or more selected markers, to provide for selecting conversion
The phenotypic character of host cell, such as the dihyrofolate reductase of eukaryotic culture, neomycin resistance and green fluorescence egg
White (GFP), or tetracycline or amicillin resistance for Escherichia coli.
Carrier comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence, can be used for converting suitable
When host cell, allow it to expression protein.
Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high
Equal eukaryocytes, such as mammalian cell.Representative example has: Escherichia coli, the bacterial cell of streptomyces；Fungal cell is such as
Yeast；Plant cell；Insect cell；Zooblast etc..
It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original
When core biology such as Escherichia coli, the competent cell that can absorb DNA can harvest after exponential phase of growth, use CaCl2Method processing, institute
With the step of it is generally well-known in the art.Another method is using MgCl2.If desired, conversion can also use the side of electroporation
Method carries out.When host is eucaryote, following DNA transfection method can be selected: calcium phosphate precipitation, conventional mechanical methods are such as
Microinjection, electroporation, liposome packaging etc..
The transformant of acquisition can use conventional method culture, express the polypeptide of coded by said gene of the invention.According to used
Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of being suitable for host cell growth
It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction)
Cell is further cultured for a period of time by the promoter for inducing selection.
Recombinant polypeptide in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.Such as
Fruit needs, and can be separated by various separation methods and purify the albumen of recombination using its physics, chemical and other characteristics.This
A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to: conventional renaturation process is used
Protein precipitant handles (salting-out method), centrifugation, permeates broken bacterium, super processing, ultracentrifugation, sieve chromatography (gel filtration), inhales
The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods.
Adenovirus
Adenovirus (Adenovirus, ADV) is a kind of particle for not having tunicary diameter for 70~90nm, by 252 shells
Grain is arranged to make up in twenty face body.The diameter of each capsomere is 7~9nm.It is linear double chain DNA molecule in capsid, containing about 4.7kb,
Respectively there is the inverted repeats for being about 100bp at both ends.Since the end 5' of every DNA chain is 55X10 with relative molecular mass3Da's
The cyclic structure of double-stranded DNA can occur in protein molecule covalent bond.
Human body adenovirus is respectively designated as adl~ad52 it has been known that there is 52 kinds, studies most to be in detail ad2.Adenoviral gene
Group transcription generate mRNA, it is known that at least area 5: E I, transcript unit be located at viral genome left side, E I A and E I can be separated into
B, it is related with cell transformation；II area's coding DNA binding protein of E participates in the duplication of virus；IIIth area E coding appears in host cell
A kind of glycoprotein on surface；IVth area E is located at ad2 genome right end, and the DNA binding protein dna encoded by IIth area E regulates and controls；5th
Transcript unit synthesizes ad2 protein IV in virus infection mid-term.
Adenovirus has carciongenic potency to rodent, or can convert the rodent zooblast of in vitro culture.Make cell
Conversion only needs a part of adenoviral gene group, these genes are located at the left end of genome, account for about whole gene group 7%~
10%.Although adenovirus distribution is very wide, there is not carcinogenicity to human body.Human body cell is a kind of permission cell
(permissive cell), i.e., this kind of cell allows to infect the virus invaded, and duplication proliferation, last cell cracking are dead in the cell
It dies and releases a large amount of progeny virus.Do not find adenovirus particles in a variety of human body tumour cells cultivated in vitro, but
There is the integration site of adl2 on No. 1 chromosome of people, it means that human body cell is also likely to be nonpermissive cell for adenovirus,
After the virus infection, virus cannot replicate proliferation to i.e. this kind of cell in the cell, but can be incorporated into the genome of infected cell
It is interior.These cells are changed by virus Transformation, phenotype, and can indefinitely subculture in vitro.
Adeno-associated virus
It, can because of adeno-associated virus (Adeno-associated virus, AAV), no pathogenicity small compared with other viral vectors
Transfection is being divided and the characteristics such as the cell that does not divide, based on the gene therapy method for genetic disease of AAV carrier by
Extensive concern is arrived.
Adeno-associated virus (adeno-associated virus, AAV), also referred to as adeno-associated virus, belong to Parvoviridae
Dependovirus is the simplest single stranded DNA defective virus of a presently found class formation, needs helper virus (usually
Adenovirus) participate in duplication.It encodes cap the and rep gene in the inverted repeats (ITR) of two ends.ITRs is for disease
The duplication of poison and packaging have decisive role.Cap gene encoding virus coat proteins, rep gene participate in virus duplication and
Integration.AAV can infect various kinds of cell.
Recombined glandulae correlation viral vectors (rAAV) be derived from nonpathogenic wild type adeno-associated virus, due to its safety is good,
The features such as host cell range wide (division and non-dividing cell), immunogenicity are low, and the expression alien gene time is long in vivo, quilt
It is considered as one of most promising gene transfer vector, is answered extensively in gene therapy worldwide and vaccine research
With.By the research in more than 10 years, oneself is understood in depth for the biological characteristics of recombinant adeno-associated virus, and especially it is various thin
Many data have been had accumulated in terms of application effect in born of the same parents, tissue and experiment in vivo.In medical research, rAAV is used for more
The research (including In vitroandin vivotrial) of the gene therapy of kind disease；It is used as a kind of characteristic gene transfer vector simultaneously, also
Be widely used in gene functional research, construct disease model, prepare clpp gene deratization etc..
In a preferred embodiment of the invention, carrier is recombination AAV carrier.AAV is relatively small DNA virus,
It, which can stablize, is integrated into the genome for the cell that they are infected with site-specific fashion.They can infect a big system
The cell of column is without growing cell, form or differentiation generate any influence, and they seem to be not related to Human pathology.
AAV genome oneself be cloned, be sequenced and characterize.AAV is in the inverted terminal repeat that each end includes about 145 bases
(ITR) region, the replication orgin as virus.Remaining of the genome is divided into two important areas for having capsidation function
Domain: comprising being related to the genome left-hand component of the rep gene of virus replication and viral gene expression；And comprising encoding viral clothing
The genome right-hand component of the cap gene of glutelin.
The standard method preparation of this field can be used in AAV carrier.The adeno-associated virus of any serotype is suitable.With
Such as United States Patent (USP) No.6566118,6989264 and 6995006, disclosures of which are found in the method for cmy vector
Entirety is herein incorporated by reference.The preparation of Hybrid Vector in such as PCT application No.PCT/US2005/027091
Description, the disclosure of the application are integrally herein incorporated by reference.AAV is derived from vitro and in vivo transporter gene
Carrier be described (see, for example, international application published No.WO91/18088 and WO93/09239 using oneself；The U.S. is special
Sharp No.4,797,368,6,596,535 and 5,139,941 and European patent No.0488528, they are whole with the side of reference
Formula is incorporated herein).These patent disclosures describe wherein rep and/or cap gene delection and by each of gene replacement of interest
Kind (enters in the cell of culture) in vitro from the construct of AAV and these constructs or (is directly entered biology in vivo
Body) transhipment gene of interest purposes.Replication defective recombinates AAV can be by assisting disease by the mankind for following plasmid co-transfection
The cell line of malicious (such as adenovirus) infection and prepare: the flank of contained nucleic acid sequence of interest is two AAV opposing ends
The plasmid in the region repetitive sequence (ITR), and carry the plasmid of AAV capsidation gene (rep and cap gene).Then pass through standard
Technology purifies generated AAV recombinant.
In some embodiments, recombinant vector by capsidation to virion (for example including but be not limited to AAV1,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and
The AAV virion of AAV16) in.Therefore, the disclosure include containing any carrier as described herein recombinant virus particle (because
It is recombination it includes recombination of polynucleotide).The method for generating such particle is that this field is known, and in United States Patent (USP)
It is described in No.6,596,535.
Modeling reagent
The present invention also provides a kind of modeling reagents, it contains a effective amount of (a) VEGF sustained release preparation；(b) VEGFR,
Or its expression vector.
In general, can be by (a) VEGF sustained release preparation；(b) VEGFR or its expression vector be formulated in it is nontoxic, inert and
In pharmaceutically acceptable aqueous carrier medium, in physiological saline, wherein pH is usually about 5-8, preferably, pH is about 7-8.
As used herein, term " effective quantity " or " effective dose " can give full expression to after referring to transfection in iris tissue,
And the adenovirus amount of animal dead will not be caused；The fraction of particle (please supplement) of sustained release VEGF can be stablized after injection.At this
In the preferred embodiment of invention, the effective quantity are as follows: VEGF microsome, 10ng/ μ l -100ng/ μ l, preferably, 30ng/ μ
L-60ng/ μ l, more preferably, 50ng/ μ l-55ng/ μ l.VEGFR adenovirus, 1 × 107A/l -6 × 10 μ7A/μ l, preferably, 2
×107A/l-5 × 10 μ7A/μ l, more preferably, 3 × 107A/l-3.5 × 10 μ7A/μ l.Preferably, the effective quantity one
Secondary property injection finishes.
As used herein, the ingredient of " pharmaceutically acceptable " is suitable for people and/or mammal and without excessively bad
Side reaction (such as toxicity, stimulation and allergy), i.e., with the substance of reasonable benefit/risk ratio.Term " can pharmaceutically connect
The carrier received " refers to the carrier for Therapeutic Administration, including.In the present invention, the pharmaceutically acceptable carrier that can be used
It is not particularly limited, can be one or more biocompatible solids or liquid filler or gelatinous mass, they are suitable for people
It uses and it is necessary to have enough purity and sufficiently low toxicity.In " compatibility " referred to herein as composition each component can and
Fat mesenchymal progenitor cells of the invention are mutually admixed, and significantly reduce its therapeutic effect.The present invention can pharmaceutically receive
Carrier part example have physiologically acceptable sterile, aqueous or anhydrous solution, dispersion liquid, suspension or a lotion, and for weight
Newly be dissolved into the aseptic powdery of sterile Injectable solution or dispersion liquid, suitable aqueous and nonaqueous carrier, diluent, solvent or
Excipient includes water, ethyl alcohol, polyalcohol and its suitable mixture.It, can also be according to fat other than above-mentioned conventional carrier
The carrier of the property design optimization of mesenchymal stem/progenitor cells.The carrier preferably infusion agent carrier and/or injection agent carrier.
Modeling reagent of the invention contains a effective amount of (a) VEGF sustained release preparation；(b) VEGFR or its expression vector.
Usual pharmaceutical preparation should match with administration mode, and medicine modeling reagent of the invention can be made into injection form, such as with giving birth to
Reason salt water or the aqueous solution containing glucose and other adjuvants are prepared by conventional method.The pharmaceutical composition preferably exists
It is manufactured under aseptic condition.The dosage of active constituent is therapeutically effective amount.Modeling reagent of the invention may also be fabricated which sustained release preparation.
Modeling reagent of the invention is preferably ejection preparation, more preferably puncture of anterior chamber ejection preparation.In another preference
In, in the modeling reagent (such as puncture of anterior chamber injection reagent), the concentration of VEGF sustained release preparation is 10ng/ μ l -100ng/ μ
L, preferably, 30ng/ μ l-60ng/ μ l, more preferably, 50ng/ μ l-55ng/ μ l, the concentration of the expression vector of the VEGFR is 1
×107A/l -6 × 10 μ7A/μ l, preferably, 2 × 107A/l-5 × 10 μ7A/μ l, more preferably, 3 × 107A/μ l-3.5 ×
107A/μ l.The injection system of the modeling reagent is not particularly limited, and can be single injection preparation, is also possible to repeatedly
The formulation compositions of injection.In one preferred embodiment of the invention, the modeling reagent is single injection agent.
In the present invention, the modeling reagent is preferably ejection preparation, more preferably puncture of anterior chamber ejection preparation.
Animal model
In the present invention, a kind of non-human mammal model of very effective research eye disease is provided.
In the present invention, the example of non-human mammal includes (but being not limited to): mouse, rat, rabbit, monkey etc., more preferably
Ground is rat and mouse.
Drug candidate or therapeutic agent
In the present invention, additionally provide a kind of using animal model of the invention, screening mitigates or treatment eye disease
The method of drug candidate or therapeutic agent.
In the present invention, drug candidate or therapeutic agent refer to it is known with certain pharmacological activity or be detected can
Can have the substance of certain pharmacological activity, including but not limited to nucleic acid, albumen, chemically synthesized small molecule or macromolecular chemical combination
Object, cell etc..The administration mode of drug candidate or therapeutic agent can be it is oral, intravenous injection, intraperitoneal injection, subcutaneous injection or
Canalis spinalis administration.
Main advantages of the present invention include:
(a) present invention firstly discovers that, (a) VEGF sustained release preparation and (b) VEGFR or its expression vector are applied in combination and can have
Effect building eye disease animal model.
(b) present invention firstly discovers that, (a) VEGF sustained release preparation and (b) VEGFR or its expression vector are simultaneously or successively infused
Injecting in ocular tissue (such as anterior chamber) can effectively construct eye disease animal model, and animal model of the invention can be used for studying
Eye disease, and can be used for screening and the testing experiment of certain drug.
(c) present invention firstly discovers that, (a) VEGF sustained release preparation and (b) VEGFR or its expression vector are applied in combination and can have
Effect building eye disease animal model, and compared with iris neovascularization classics modeling method (laser closing retinal vessel),
Anterior ocular segment side reaction (corneal edema, aqueous flare, hemorrhagic aqueous humor etc.) significantly reduces.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor
Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no
Then percentage and number are weight percent and parts by weight.
The material used in embodiment is commercial product unless otherwise specified.
The intraocular VEGFR of 1 experimental mouse of embodiment is overexpressed situation
Rat injected into anterior chambers VEGFR is overexpressed adenovirus (3.5 × 107A/μ l), after 3 days, from Normal group, ADV-
Experimental mouse iris tissue is extracted in VEGFR group and ADV- empty vectors group at random, carries out Western blot experiment, and carry out egg
White differential expression analysis.
The result is shown in Figure 1.Fig. 1 shows the protein expression and statistical result of the VEGFR in tissue samples of the present invention, ADV-
The expression of VEGFR group VEGFR obviously raises (P < 0.05) compared with ADV- empty vectors group, ADV- empty vectors group and blank control group
Between VEGFR expression indifference (P > 0.05).
2 intraocular pressure analysis on monitoring result of embodiment
Verifying VEGFR be overexpressed after injected into anterior chambers VEGF microsome (50ng/ μ l, the 10 μ g of total drugloading rate of microsome, directly
Diameter 150nm), influence of the nanoparticle to aqueous humor efferent tract is excluded with Blank Microparticles.Utilize tonometer same period daily
Each group rat intraocular pressure is monitored, and carries out intraocular pressure result statistical analysis.
As a result see Fig. 2.Fig. 2 shows after modeling the 7th day, VEGF particle group rat intraocular pressure start to increase (14.97 ±
0.9207mmHg), the 14th day intraocular pressure is significantly raised (24.23 ± 0.9333mmHg) compared with blank microparticles group, and intraocular pressure is down within the 21st day
Normal level (9.533 ± 0.4055mmHg)；Blank control group, with ADV- empty carrier injection group, ADV- empty carrier injection group with
Rat intraocular pressure is unchanged between ADV-VEGFR injection group, ADV-VEGFR injection group and blank microparticles group.
The photograph interpretation of result of 3 prosthomere of embodiment
1 after modeling, slit-lamp observation anterior ocular segment variation on the 2,4,7,14,21st, 10% chloraldurate intraperitoneal injection of anesthesia
Rat notices that ciliary congestion, corneal edema, keratic precipitates, the scintillation of aqueous humor muddiness or hemorrhagic aqueous humor, iris tissue color become
Change, whether iris tissue visible new vessels under mirror occurs, pupil shape changes.
As a result see Fig. 3.Experimental mouse prosthomere does not occur corneal edema, keratic precipitates, aqueous flare after Fig. 3 shows modeling
Equal side reactions.
4 iris angiography interpretation of result of embodiment
7 after modeling, row iris fluoroscopic visualization on the 14th, 21,10% chloraldurate intraperitoneal injection of anesthesia rat, through tail vein
It injects and has been infused in 20% Sodium fluorescein 0.04ml, l~3s, injection, which starts simultaneously at, takes pictures, until fluorescence subsides.
As a result see Fig. 4.Fig. 4 shows after modeling that laser closes iris and retinal vessel joint is overexpressed VEGFR the 14th day
Adenovirus (3.5 × 107A/μ l) injection+VEGF microsome (concentration is 50ng/ μ l, total drugloading rate 10ul, diameter 150nm) group
The visible a large amount of fluorescence leakages of rat iris, iris neovascularization generate.
Embodiment 5
Injection concentration is 3.5 × 10 in rat anterior chamber7The ADV-VEGFR of a/μ l totally 5 μ l, injected into anterior chambers concentration after 3 days
For 50ng/ μ l, the 5 μ l of VEGF microsome of total drugloading rate 10ul, diameter 150nm.Iris blood was carried out to rat in the 14th day after modeling
Pipe radiography, laser closes iris and retinal vessel joint is overexpressed VEGFR adenoviral injection+VEGF microsome group rat iris
It can be seen that a large amount of fluorescence leakages, it was demonstrated that iris neovascularization generates.As a result see Fig. 4.
Comparative example 1
With embodiment 5, difference is method, injection of VEGF.
As a result it shows, rat intraocular pressure does not rise, and iris angiography has no obvious Fluorescein Leakage, the results showed that, iris is new
The unstructured success of angiogenic animal models.
Comparative example 2
With embodiment 5, difference is method, injection of VEGF R or its expression vector.
The results show that Adenovirus Transfection success in rat iris tissue, VEGFR is successfully overexpressed, but rat intraocular pressure does not rise
Height, iris angiography have no obvious Fluorescein Leakage, the results showed that, the unstructured success of iris neovascularization animal model.
Comparative example 3
With embodiment 5, difference is method, and the microsome of injection is blank nanoparticle body (being free of VEGF).
The results show that rat intraocular pressure does not increase, iris angiography has no obvious Fluorescein Leakage, the results showed that, iris
The unstructured success of new vessels animal model.As a result see Fig. 4.
Comparative example 4
With embodiment 5, difference is method, and the VEGF microsome diameter of injection is respectively 10nm and 500nm.
The results show that rat intraocular pressure does not increase, iris angiography has no obvious Fluorescein Leakage, the results showed that, iris
The unstructured success of new vessels animal model.Microsome diameter is too small, can flow out through aqueous humor efferent tract from preceding room with aqueous humor, can not
VEGF is discharged to anterior chamber；Diameter is excessive, and VEGF release is excessively slow, it is difficult to reach VEGF effective concentration within effective time.Knot
Fruit sees Fig. 5.
Comparative example 5
With embodiment 5, difference is method, and the VEGF microsome drug concentration of injection is respectively 10ng/ μ l and 100ng/ μ
l。
The results show that rat intraocular pressure does not increase, iris angiography has no obvious Fluorescein Leakage, the results showed that, iris
The unstructured success of new vessels animal model.Drug concentration is too low, not up to the effective concentration of new vessels generation, can not induce
New vessels are formed；Drug concentration is excessively high, is more than animal to VEGF tolerance, experimental animal mortality.As a result see Fig. 6.
It discusses
Neovascular glaucoma (neovascular glaucoma, NVG) is a kind of scarce secondary to popularity retina
The Refractory Glaucoma of blood is a kind of serious blinding eye disease, clinical treatment with the characteristics of iris and room angle new vessels
Difficult and unsatisfactory curative effect.The formation of NVG is the process of an active development, and the premise occurred is the formation of NVI, therefore, structure
It builds ideal animal model and NVI model is for the further investigation NVG key for having great significance, and constructing NVG model
Foundation.
The construction method of traditional NVI model mainly includes Model of Retinal Vein Occlusion, diabetes model, oxygen induction view
Film disease model, anterior eye segment ischemia model, lens cortex re-injection model, transgenic mice are overexpressed insulin-like growth factor-i mould
Type etc..These traditional model building methods there are animal origins it is limited, with high costs, complicated for operation, complication is more the deficiencies of,
It is often difficult to successfully construct NVI model.
With progress of research, present invention discover that effect of the VEGF in new vessels should not be underestimated.In NVI eyes of patients
In aqueous humor, the content of VEGF, which has, significantly to be increased, and in aqueous humor the content of VEGF and iris surface new vessels hyperplasia degree
There is more close connection.Therefore, the present invention is constructed using the method for being overexpressed VEGFR joint VEGF particle injected into anterior chambers
NVI animal model.NVI model is constructed using this method, time of origin morning simple and easy with production method, duration can
Control, side reaction is small and the series of advantages such as success rate is high, relative to traditional NVI model, with certain challenge and new
Newness.In addition, constructing NVI using method of the invention, the Forming Mechanism of NVI is clear and single, can be used for different growth factors,
The research that inflammatory cytokine and adhesion molecule induction NVI are formed.Related intervention, which is carried out, in NVG early stage for research creates item
Part provides platform for the screening of related drugs.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (10)
1. a kind of modeling reagent characterized by comprising
(a) VEGF sustained release preparation；
(b) VEGFR or its expression vector.
2. modeling reagent as described in claim 1, which is characterized in that the VEGF sustained release preparation is selected from the group: VEGF particle
Body, VEGF suspension, or combinations thereof.
3. modeling reagent as claimed in claim 2, which is characterized in that the microsome includes nanoparticle.
4. modeling reagent as claimed in claim 3, which is characterized in that the diameter of the microsome is 30-300nm, preferably
50-200nm, more preferably 100~150nm.
5. modeling reagent as claimed in claim 1 or 2, which is characterized in that in the modeling reagent, the medicine of the microsome
Object concentration is 20-90ng/ μ l, preferably, 30-70ng/ μ l, more preferably, 40-55ng/ μ l.
6. modeling reagent as described in claim 1, which is characterized in that in the modeling reagent, the concentration of component (b) is 1 ×
107A/l -6 × 10 μ7A/μ l, preferably, 2 × 107A/l-5 × 10 μ7A/μ l, more preferably, 3 × 107A/μ l-3.5 ×
107A/μ l.
7. modeling reagent as described in claim 1, which is characterized in that the modeling reagent is preparation eye disease disease animal model
Reagent.
8. modeling reagent as claimed in claim 7, which is characterized in that the eye disease disease includes iris neovascularization correlation disease
Disease.
9. a kind of purposes of modeling reagent described in claim 1, which is characterized in that be used to prepare eye disease animal model
Drug or preparation.
10. purposes as claimed in claim 9, which is characterized in that the eye disease disease includes iris neovascularization related disease.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114365717A (en) * | 2022-01-14 | 2022-04-19 | 华中科技大学同济医学院附属同济医院 | Construction method of animal model of glaucomatous ciliitis syndrome and animal model |
| CN114480490A (en) * | 2021-12-31 | 2022-05-13 | 四川省医学科学院·四川省人民医院 | Method for constructing animal model of retinal neovascular diseases |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020194630A1 (en) * | 1999-03-15 | 2002-12-19 | Manning William C. | Use of recombinant gene delivery vectors for treating or preventing diseases of the eye |
| US20080268051A1 (en) * | 2007-04-30 | 2008-10-30 | Allergan, Inc. | High viscosity macromolecular compositions for treating ocular conditions |
| CN103007291A (en) * | 2011-09-26 | 2013-04-03 | 苏州圣诺生物医药技术有限公司 | Composition for treating eye diseases by double-target/multi-target small nucleic acid and applications of composition |
| CN104906084A (en) * | 2014-03-11 | 2015-09-16 | 中山大学中山眼科中心 | Application of artemisinin and derivatives thereof in preparation of ophthalmic vascular disease prevention and treatment medicines, and medicinal composition |
| CN107970436A (en) * | 2017-12-07 | 2018-05-01 | 山东大学齐鲁医院 | The modeling method of new vessels in new zealand white rabbit atherosclerotic plaque in abdominal aorta in model |
-
2019
- 2019-06-03 CN CN201910476999.XA patent/CN110025768B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020194630A1 (en) * | 1999-03-15 | 2002-12-19 | Manning William C. | Use of recombinant gene delivery vectors for treating or preventing diseases of the eye |
| US20080268051A1 (en) * | 2007-04-30 | 2008-10-30 | Allergan, Inc. | High viscosity macromolecular compositions for treating ocular conditions |
| CN103007291A (en) * | 2011-09-26 | 2013-04-03 | 苏州圣诺生物医药技术有限公司 | Composition for treating eye diseases by double-target/multi-target small nucleic acid and applications of composition |
| CN104906084A (en) * | 2014-03-11 | 2015-09-16 | 中山大学中山眼科中心 | Application of artemisinin and derivatives thereof in preparation of ophthalmic vascular disease prevention and treatment medicines, and medicinal composition |
| CN107970436A (en) * | 2017-12-07 | 2018-05-01 | 山东大学齐鲁医院 | The modeling method of new vessels in new zealand white rabbit atherosclerotic plaque in abdominal aorta in model |
Non-Patent Citations (4)
| Title |
|---|
| MICHAEL J. TOLENTINO等: "Vascular Endothelial Growth Factor Is Sufficient to Produce Iris Neovascularization and Neovascular Glaucoma in a Nonhuman Primate", 《ARCH OPHTHALMOL》 * |
| YUJI OSHIMA等: "Intraocular gutless adenoviral-vectored VEGF stimulates anterior segment but not retinal neovascularization", 《JOURNAL OF CELLULAR PHYSIOLOGY 》 * |
| 刘敬花等: "虹膜新生血管动物模型", 《国际眼科纵览》 * |
| 张俊杰等: "眼科缓释新剂型――微粒体眼用给药***", 《眼科研究》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480490A (en) * | 2021-12-31 | 2022-05-13 | 四川省医学科学院·四川省人民医院 | Method for constructing animal model of retinal neovascular diseases |
| CN114365717A (en) * | 2022-01-14 | 2022-04-19 | 华中科技大学同济医学院附属同济医院 | Construction method of animal model of glaucomatous ciliitis syndrome and animal model |
| CN114365717B (en) * | 2022-01-14 | 2023-09-12 | 华中科技大学同济医学院附属同济医院 | Method for constructing glaucoma ciliary body inflammation syndrome animal model and animal model |
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