CN109182355B - The construction method of retinal neovascularization disease model and application - Google Patents
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Abstract
The invention discloses a kind of construction method of retinal neovascularization disease model and applications, are related to field of biotechnology.The construction method makes target animal show typical retinal neovascularization genius morbi by knocking out the Tmem30a gene order in the genome in target animal vascular endothelial cell.The target animal can not only be used for retinal neovascularization disease model, for the research of retinal neovascularization disease, can help the pathogenic process and mechanism that illustrate retinal neovascularization disease, and provide new target for the treatment or prevention of the disease.
Description
Technical field
The present invention relates to field of biotechnology, in particular to a kind of building of retinal neovascularization disease model
Methods and applications.
Background technique
Retinal neovascularization (Retinal neovascularization, RNV) correlation eye disease seriously endangers
Human eyesight health, such disease mainly include retinopathy of prematurity (retinopathy of prematurity,
ROP), proliferative diabetic retinopathy (proliferative diabetic retinopathy, PDR), retina are quiet
Arteries and veins blocks (central retinal vein occlusion, RVO) etc..The generation of pathologic retinal neovascularization is this kind of disease
The major pathologic features of disease.In the world, retina neovascular diseases disease incidence height, end-stage disease therapeutic effect
Difference, blind rate are high.
Currently, laser photocoagulation and intraocular injection anti-vascular endothelial cell growth factor (vascular endothelial
Growth factor, VEGF) drug be clinical treatment RNV main means.But a large number of studies show that anti-vegf treatment is simultaneously
It is non-effective to all patients, and anti-vegf treatment there are it is expensive, need to inject that increase patient's infection risk etc. many repeatedly
Disadvantage causes its clinical application limited.
Therefore, the pathogenesis for fully stating RNV is sought other effective treatment means or target spot, is had become urgently
The difficult medical problem of solution.
And currently, lacking the relevant animal model that can be used for retinal neovascularization disease research.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of construction methods of retinal neovascularization disease model, using the building side
The available retinal neovascularization disease model of method, the model can express retinal neovascularization genius morbi, the model
It can be used for the research of retinal neovascularization disease, the pathogenic process and mechanism that illustrate RNV, and controlling for the disease can be helped
It treats or prevention provides new target.
Another object of the present invention is to provide the retinal neovascularization disease models obtained by above-mentioned construction method to exist
Application in retinal neovascularization disease research.Using the model, the pathogenic process and mechanism that illustrate RNV can be helped, and
New target is provided for the treatment or prevention of the disease.
The present invention is implemented as follows:
On the one hand, the present invention provides a kind of construction methods of retinal neovascularization disease model comprising: knock out mesh
Mark the Tmem30a gene order in the genome in animal blood vessels endothelial cell.
Tmem30a gene is the β subunit of phosphatidylserine varus enzyme, it for the fermentoid stability and its function extremely
It closes important.When lacking Tmem30a, phosphatidylserine varus enzyme correctly cannot be folded and be positioned, and can not play its function, in turn
Cause the asymmetry of biological film phospholipid to be destroyed, leads to a series of pathological characters.
The present inventor has found for the first time, and it is i.e. heavy that Tmem30a gene is knocked out in the vascular endothelial cell of target animal
The expression of silent Tmem30a gene, can make target animal show the relevant feature of retinal neovascularization disease and for example regard
Retinal vasculature hypoevolutism, vessel density reduce, blood vessel teloblast number reduces and shows the end of aneurysmal, teloblast
Sufficient number is touched to reduce.Therefore, the animal that the Tmem30a gene in vascular endothelial cell is knocked can be used as retinal neovascularization
Disease model, in the fields such as retinal neovascularization disease research, for the disease research such as pathogenic process, mechanism with
And the screening of related drugs provides a kind of new model.
Further, in some embodiments of the present invention, it knocks out Tmem30a gene order and refers to knockout Tmem30a
The exon sequence of gene.
Knocking out Tmem30a gene order can be knockout Tmem30a full length gene sequence, be also possible to knock out Tmem30a
No matter Gene Partial sequence such as partial exon sequences knock out the sequence of that type (part or overall length), as long as can
In the blood vessels in chrotoplast silencing Tmem30a gene expression, so that animal is shown retinal neovascularization disease individual features
Fall into protection scope of the present invention.
Further, in some embodiments of the present invention, the exon sequence for knocking out Tmem30a gene refers to knockout
Any one or more exons on Tmem30a gene in the 1st exon, exon 2, the 3rd exon and the 4th exon
Sequence.
Further, in some embodiments of the present invention, the 3rd exon sequence on Tmem30a gene is knocked out.
Further, in some embodiments of the present invention, the construction method includes: by Tmem30a gene condition
Property knock out homozygous animals with turn PDGF β-Cre ER genetic animal and mate, obtain vascular endothelial cell conditionity and knock out
Tmem30a genetic animal can be used as retinal neovascularization disease model.
Wherein, Tmem30a gene conditionity knock out homozygous animals acquisition can refer to application No. is
2017103803265, entitled beta Cell of islet conditionity knocks out construction method and the application of Tmem30a genetic mouse model
Chinese patent application.
Turn PDGF β-Cre ER genetic animal purchased from London University College London.This transgenosis
The special platelet derived growth factor of animal expression vascular endothelial cell (Platelet-derived growth factor β, PDGF
β) the Cre gene expression of the special transformation of promoter driving.It is positioned in cytoplasm in the case of the Cre protein normal of transformation,
In conjunction with nucleus can be entered after tamoxifen (Tamoxifen), identifies the site LoxP on genome, realize gene knockout.
Above-mentioned knockout strategy is to knock out Tmem30a gene using Cre-loxP knockout technology.But in other examples,
Realize that the technological means of gene knockout has very much, such as the Zinc finger nuclease skill that CRISPR/Cas9 technology, artificial nuclease mediate
Art (zinc-finger nucleases, ZFN), activating transcription factor sample effector nucleic acid zymotechnic (transceription
Activator-like effector nucleases, TALEN) etc..Therefore, CRISPR/ is used in other examples
Cas9 technology or other technological means knock out Tmem30a gene, and belong to the scope of protection of the present invention.
Further, in some embodiments of the present invention, the target animal is selected from mouse, rat, dog, pig, monkey
And any one in ape.
It should be noted that target animal of the present invention is not limited to above-mentioned animal, it is also possible to other kinds of
Animal such as rabbit, ox, horse, sheep etc..No matter which kind of animal is selected, as long as the animal with Tmem30a gene, can be used as this
The target animal in the construction method is invented, Tmem30a gene is knocked out in its vascular endothelial cell, it is made to show view
Film neovascular disease feature, as retinal neovascularization disease model, for being led to retinal neovascularization disease research
In domain, all belong to the scope of protection of the present invention.
Further, in some embodiments of the present invention, above-mentioned retinal neovascularization disease includes premature
Retinopathy, proliferative diabetic retinopathy and retinal vein obstruction.
On the other hand, the present invention provides the construction method by above-mentioned retinal neovascularization disease model is obtained
Application of the retinal neovascularization disease model in retinal neovascularization disease research.
Further, in some embodiments of the present invention, the research is for the purpose of the treatment of non-disease.
Further, in some embodiments of the present invention, above-mentioned retinal neovascularization disease includes premature
Retinopathy, proliferative diabetic retinopathy and retinal vein obstruction.
It has typical retinal neovascularization genius morbi to the animal model that construction method through the invention obtains,
With boundless application prospect, such as it is used for the pathogenic process of research retinal neovascularization disease, pathogenesis,
Basis is provided to understand research retinal neovascularization disease in depth.Or screening is used for for preventing or treating view
Film neovascular disease drug, the curative effect for evaluating drug or prognosis etc..
On the other hand, the present invention provides the construction method by above-mentioned retinal neovascularization disease model is obtained
Retinal neovascularization disease model is in screening for preventing or treating the application in retinal neovascularization disease medicament.Screening
Drug can promote blood vessel normal development.
Further, in some embodiments of the present invention, above-mentioned retinal neovascularization disease includes premature
Retinopathy, proliferative diabetic retinopathy and retinal vein obstruction.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1: expression of the detection TMEM30A gene in three kinds of different blood vessel endothelial cells；
In figure:
A:RT-PCR detects TMEM30A gene in the expression of results of three kinds of different blood vessel endothelial cells；
B:Western Blot detects TMEM30A albumen in the expression of three kinds of different blood vessel endothelial cells.
The identification of Fig. 2: TMEM30A knock out mice；
In figure:
The route of A:TMEM30A gene knockout.
The genotype identification result of B:TMEM30A knock out mice.
C: the inner nuclear layer retina coloration result figure of the knock out mice with TdTomato reporter gene.
Fig. 3: gene knockout efficiency in immunoblotting analysis experimental analysis Tmem30a knock-out mice retina.
The detection of the inner nuclear layer retina of Fig. 4: Tmem30a knock out mice；
In figure:
A: vascular endothelial cell specifically knock out Tmem30a DNA murine inner nuclear layer retina coloration result (above) and
Isolectin dyes blood vessel result (following figure).
B: vascular endothelial cell specifically knocks out the statistics of Tmem30a DNA murine inner nuclear layer retina blood vessel density and length
Analysis.
Fig. 5: vascular endothelial cell specifically knocks out Tmem30a DNA murine retinal slice immunohistochemical staining result.
The inner nuclear layer retina teloblast of Fig. 6: Tmem30a knock out mice and the detection for touching sufficient number；
In figure:
A: vascular endothelial cell specifically knocks out Tmem30a DNA murine inner nuclear layer retina dyeing teloblast observation result.
B: vascular endothelial cell specifically knocks out Tmem30a DNA murine inner nuclear layer retina dyeing touching foot observation result.
C: the number statistical analysis chart of teloblast and touching foot.
The proliferation experiment of the vascular endothelial cell of Fig. 7: Tmem30a knock out mice；
A: vascular endothelial cell specifically knocks out Tmem30a DNA murine vascular endothelial cell proliferation experimental result.
B: vascular endothelial cell specifically knocks out Tmem30a DNA murine vascular endothelial cell proliferation number statistical result.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment is using mouse as target animal, to the building side of retinal neovascularization disease model provided by the invention
Method is illustrated, and the route of TMEM30A gene knockout is as shown in Figure 2 A, and concrete operations are as follows:
1. referring to application No. is 2017103803265, entitled beta Cell of islet conditionitys to knock out Tmem30a DNA murine
The method of the Chinese patent application of the construction method and application of model obtains Tmem30a gene (the 3rd exon) conditionity and strikes
Except homozygote mouse；
2. Tmem30a gene conditionity is knocked out homozygote mouse to mate with PDGF β-Cre ER DNA murine is turned, obtain
Vascular endothelial cell conditionity knocks out Tmem30a DNA murine.
Turn PDGF β-Cre ER DNA murine and Tmem30a geneware knocks out the mating of homozygote mouse, offspring has half
Animal simultaneously carry PDGF β-Cre ER and Tmem30a conditionity knockout.This animal and Tmem30a gene conditionity knock out pure
The mating of zygote mouse, birth generation mice continuously injected 100 microlitres of tamoxifen by second day to the 4th day after birth
The mouse that vascular endothelial cell specifically knocks out Tmem30a gene can be obtained in corn oil (concentration 10mg/ml).
Embodiment 2
1 detects TMEM30A gene in the expression of three kinds of vascular endothelial cells using RT-PCR method.
Method: three kinds of vascular endothelial cell human retina vascular endothelial cells (HREC), human umbilical veins are extracted respectively
The total serum IgE of endothelial cell (HUVEC) and human microvascular endothelial cell (mvec) (HMVEC) then uses cDNA synthetic agent box
(Invitrogen, Waltham, MA, USA) synthesizes cDNA.According to the cDNA sequence design primer of TMEM30A:
TMEM30A-H-F:5 '-TCGGTCTCATCTTCATTCCC-3'；
TMEM30A-H-R:5 '-GGAAGGCTCTGTTCCGGTAT-3'.
Using the cDNA of extraction as template, RT-PCR is carried out.Electrophoresis, the result is shown in Figure 1 are carried out after amplification.
The result is shown in Figure 1 A, it can be seen that it is thin in human retina blood vessel endothelium that TMEM30A gene is detected by RT-PCR method
Expression in born of the same parents (HREC), human umbilical vein endothelial cell (HUVEC) and human microvascular endothelial cell (mvec), as a result, it has been found that this three
In kind of cell, TMEM30A has expression, illustrates that the gene may play critical function in chrotoplast in the blood vessels.
2 detect TMEM30A gene in three kinds of vascular endothelial cells using the method for immunoblotting (western blot)
Expression.
Method:
(1) HMREC, HUVEC, HMVEC cell are cultivated respectively, and 200 μ l protein lysate RIPA are added after cell covers with.
(2) after sound smudge cells, 20min is cracked on ice.
(3) 4 DEG C, after 16000g is centrifuged 10min, supernatant is taken to be transferred to another clean centrifuge tube, be added on the albumen of 50 μ l
Sample liquid, 95 DEG C of heating 5min after mixing.
(4) after sample is cooling, take respectively 20 μ l, 160V voltages carry out polyacrylamide gel electrophoresis (SDS-PAGE) with
Protein isolate.
(5) after SDS-PAGE, as needed, cut appropriately sized nitrocellulose filter, spread in order filter paper,
Glue, nitrocellulose filter and filter paper, and bubble of rushing, transferring film slot are put into ice-water bath, and the electric current using constant current 0.28A is turned
Film, transferring film 2h.
(6) it after transferring film, pure water rinsing nitrocellulose filter one time, dries and marks.Then with 8% skim milk
Close 2h.
(7) after the completion of closing, addition is a certain amount of to be diluted in confining liquid (according to antibody operation instructions) by a certain percentage
Primary antibody, 4 DEG C overnight incubation.
(8) primary antibody is recycled, 1 × TBST buffer is washed film 4 times, and each 10min selects suitable secondary antibody according to primary antibody source,
With the secondary antibody of 1 × TBST dilution horseradish peroxidase (HRP) label, room temperature is in being incubated for 2h on shaking table.
(9) it after secondary antibody is incubated for, is washed film 3 times, each 10min with 1 × TBST, with the ELC luminescence reagent box of Thermo
Albumen is detected, instrument is the chemiluminescence gel imaging system of Bio-Rad.
The result is shown in Figure 1 B, it can be seen that TMEM30A albumen is demonstrated by the method for immunoblotting (western blot)
There is expression in HREC, HMVEC and HUVEC.
Embodiment 3
The murine genes type identification of Tmem30a gene is specifically knocked out to the vascular endothelial cell in embodiment 1.
Method:
(1) a little tissue samples of mouse end pin are cut, are placed in clean 1.5ml centrifuge tube；
(2) 100 μ l lysates (40mM NaOH, 0.2mM EDTA solution) is added in centrifuge tube, and in metal bath 100
DEG C heating 1h；(3) centrifuge tube is taken out, after being cooled to room temperature, the neutralizer (40mM Tris-HCl, pH5.5) of 100 μ l is added,
After 10000g is centrifuged 2min, supernatant is taken to identify for murine genes type.
(4) PCR amplification: according to following system configurations PCR reaction system
Primer sequence is as follows:
Tmem30a-Forward sequence: 5 '-ATTCCCCTTCAAGATAGCTAC-3 '；
Tmem30a-Reverse sequence: 5 '-AATGATCAACTGTAATTCCCC-3 '；
PDGF β-Forward sequence: 5 '-GCCGCCGGGATCACTCTCG-3 '；
PDGF β-Reverse sequence: 5 '-CCAGCCGCCGTCGCAACTC-3 '.
(5) gel electrophoresis
It weighs 1g agarose to be put in 100ml TAE buffer, 1% agarose gel is made after melting in micro-wave oven.It takes
In well, 120V constant pressure agarose electrophoresis 15min. is imaged 10ul PCR product with gel imaging system.
The left side Fig. 2 B is that Tmem30a conditionity knocks out qualification result, and -/- indicates wild type control, stripe size is
179bp；Loxp/- indicates heterozygote, and there are two band 179bp and 214bp；Loxp/loxp indicates homozygote, and stripe size is
214bp.It is DGF β-cre qualification result on the right of Fig. 2 B.DGF β-cre size is 500bp.According to fig. 2 B's as a result, display institute
The identification method of use can the genotype to newborn mice effectively identified, to carry out follow-up study.
(6) building has the mouse model of tdTomato reporter gene
TdTomato is a transgenosis red fluorescent protein Tomato reporter gene, in the initial close of red fluorescent protein
There is terminator sequence before numeral；Terminator both ends have the site LoxP being collectively aligned, termination when having Cre expression of enzymes
Son can be deleted, and red fluorescent protein can express, and Cre positive cell is marked as red.In order to determine the loxP of Cre mediation
Site recombination efficiency is introduced when building vascular endothelial cell specifically knocks out Tmem30a gene positioned at ROSA26 genome position
Point tdTomato reporter gene.In Cre missing, two sides can prevent downstream with the STOP box gene of loxP recombination site
The expression of red fluorescent protein tdTomato.In the presence of Cre, STOP box gene is removed in the specifically expressed tissue of Cre,
Lead to the expression of tdTomato, therefore the fluorescence that tdTomato expression generates can be observed by inner nuclear layer retina to determine
The expression position of Cre recombinase and expression efficiency.
Inner nuclear layer retina dyeing:
The vascular endothelial cell for taking building to obtain specifically knocks out Tmem30a gene and with transgenosis red fluorescent protein
The report base mouse of Tomato is broken after neck execution, quickly takes eyeball, and be put into 4% PFA, after room temperature is protected from light fixed 20min,
It is put into PBS buffer solution.Cornea is cut off under anatomical lens after 20min, entire sclera is torn with tweezers, leaves inner retina,
And it is cut into bunge bedstraw herb shape, it paves and removes excess tissue.It is put into 4 DEG C of fixations in 4%PFA and overnight, is transferred to 4 DEG C of guarantors in 0.4%PFA
It deposits.
Retina creep plate is taken out, PBS is washed three times 5 minutes every time.5% NDS (containing 0.25%Triton) closing is penetrating
1h is incubated for primary antibody, and 4 DEG C overnight.Second day, after PBS cleaning three times, it is incubated for corresponding fluorescence secondary antibody, then cleans three with PBS again
It is secondary, mounting, observation.
As a result see Fig. 2 C.
Blood vessel is dyed by inner nuclear layer retina and Isolectin, the results showed that vascular endothelial cell has red fluorescence
Protein expression, and only vascular endothelial cell expresses red fluorescent protein, shows that this Cre can specifically chrotoplast strikes in the blood vessels
Except Tmem30a.The efficiency and specificity of Cre are indicated by tdTomato.As the result is shown PDGF β-Cre can specificity in the blood vessels
Chrotoplast expression, it was demonstrated that the Cre be it is effective, special, can carry out follow-up study.
(7) gene knockout efficiency in immunoblotting analysis experimental analysis Tmem30a knock-out mice retina.
Immunoblotting analysis experimental method:
(1) wild type and Mutant Mice retinal tissue are separated respectively, are placed in 1.5ml centrifuge tube, and 200 μ l eggs are added
White lysate RIPA.
(2) after ultrasonication retinal tissue, 20min is cracked on ice.
(3) 4 DEG C, after 16000g is centrifuged 10min, supernatant is taken to be transferred to another clean centrifuge tube, be added on the albumen of 50 μ l
Sample liquid, 95 DEG C of heating 5min after mixing.
(4) after sample is cooling, take respectively 20 μ l, 160V voltages carry out polyacrylamide gel electrophoresis (SDS-PAGE) with
Protein isolate.
(5) after SDS-PAGE, as needed, cut appropriately sized nitrocellulose filter, spread in order filter paper,
Glue, nitrocellulose filter and filter paper, and bubble of rushing, transferring film slot are put into ice-water bath, and the electric current using constant current 0.28A is turned
Film, transferring film 2h.
(6) it after transferring film, pure water rinsing nitrocellulose filter one time, dries and marks.Then with 8% skim milk
Close 2h.
(7) after the completion of closing, addition is a certain amount of to be diluted in confining liquid (according to antibody operation instructions) by a certain percentage
Primary antibody, 4 DEG C overnight incubation.
(8) primary antibody is recycled, 1 × TBST buffer is washed film 4 times, and each 10min selects suitable secondary antibody according to primary antibody source,
With the secondary antibody of 1 × TBST dilution horseradish peroxidase (HRP) label, room temperature is in being incubated for 2h on shaking table.
(9) it after secondary antibody is incubated for, is washed film 3 times, each 10min with 1 × TBST, with the ELC luminescence reagent box of Thermo
Albumen is detected, instrument is the chemiluminescence gel imaging system of Bio-Rad.
Immunoblot experiment shows that Tmem30a vascular endothelial cell knocks out Tmem30a expression in retina and is reduced to
60% or so (Fig. 3) of wild type.Ctrl refers to that wild type control, cKO refer to that vascular endothelial cell knocks out in Fig. 3.Tmem30a
Expression, which refers to that expression is opposite, to be changed, with wild type expression level for 1.WT is wild control；KO is gene knockout
Mouse.In view of vascular endothelial cell only accounts for retina cell's a part, it is relatively high that this knocks out efficiency.
Embodiment 4
Inner nuclear layer retina dyeing: Example 1 constructs obtained vascular endothelial cell and specifically knocks out Tmem30a DNA murine
After disconnected neck is put to death, eyeball is quickly taken, and be put into 4% PFA, after the fixed 20min of room temperature, is put into PBS buffer solution.20min
Cornea is cut off under anatomical lens afterwards, entire sclera is torn with tweezers, inner retina is left, and be cut into bunge bedstraw herb shape, paves
Fall excess tissue.It is put into 4 DEG C of fixations in 4%PFA and overnight, is transferred to 4 DEG C of preservations in 0.4%PFA.
Retina creep plate is taken out, PBS is washed three times 5 minutes every time.5% NDS (containing 0.25%Triton) closing is penetrating
1h is incubated for primary antibody, and 4 DEG C overnight.Second day, after PBS cleaning three times, it is incubated for corresponding fluorescence secondary antibody, then cleans three with PBS again
It is secondary, mounting, observation.
As a result see Fig. 4 A and Fig. 4 B.
Blood vessel is dyed by inner nuclear layer retina and Isolectin, the results showed that vascular endothelial cell specific knockdown
After Tmem30a gene, retinal vessels development is slow, and vessel density and blood vessel radiation radius have aobvious compared to wild-type mice
Writing reduces.
Embodiment 5
The dyeing of retina frozen section: the embodiment 1 taken out raw 9th day constructs obtained vascular endothelial cell and specifically knocks out
Tmem30a DNA murine breaks after neck execution, quickly takes eyeball, and be put into 4% PFA, after fixing 15min on ice, in cornea
On cut an openning, then proceed to fix on ice.After 2h, PBS buffer solution is rinsed 3 times, and it is molten that eyeball is then placed in 30% sucrose
It is dehydrated 2h in liquid, cornea and crystal are then cut off under anatomical lens, OCT is embedded and is immediately placed in -80 DEG C of refrigerator freezings.About
After 10min, the eyeball of OCT embedding is taken out, can be sliced after being placed in -25 DEG C of freezing microtome balance about 30min.Slice thickness is
12μm。
After the completion of slice, higher son of quality is chosen in 37 DEG C of baking ovens and places 30min, then immunohistochemistry pen is having view
The place of omental organization is drawn a circle, and PBS washes three times to remove OCT, and then 5% NDS (containing 0.25%Triton) closing is penetrating
2h is incubated for primary antibody, and 4 DEG C overnight.Second day, after PBS cleaning three times, it is incubated for corresponding fluorescence secondary antibody, then cleans three with PBS again
It is secondary, mounting, observation.
As a result see Fig. 5.
The 9th day after mouse birth, found after dyeing Isolectin by retina frozen tissue section, knockout type is small
Mouse vascular development is limited only to blood vessel surface, and compared to wild-type mice, knockout type mouse vascular development is obviously slow.
Embodiment 6
Detect the retinal vessel teloblast number of the knockout type mouse of embodiment 1.Method is the same as embodiment 4.
As a result see Fig. 6 A Fig. 6 B and Fig. 6 C.
After inner nuclear layer retina dyeing, by carrying out detailed analysis to blood vessel teloblast, knockout type Mouse Retina blood is found
Pipe end cell number is significantly reduced than wild-type mice, and the touching foot number on each teloblast also significantly reduces.
Embodiment 7
EdU (5-ethynyl-2 '-deoxyuridine, 5- acetylene -2 '-BrdU) dyeing: of the right age mouse, abdominal cavity are taken
Inner nuclear layer retina is taken after tri- hours of EdU 50uL of 2 μM of injection, EDU secondary antibody dye is carried out according to the method for inner nuclear layer retina dyeing
Color compares the knockout type Mouse Endothelial proliferative conditions of wild-type mice and embodiment 1.
As a result see Fig. 7 A and Fig. 7 B.
By EdU Coloration experiment, the vascular endothelial cell in proliferation period in Mutant Mice retinal vessel is found
Number is substantially reduced compared to wild-type mice.Demonstrate the retinal endothelial cell proliferation slowed down of knockout type mouse.
To sum up, it can be seen that the embodiment of the present invention knocks out technology in its blood vessel endothelium by taking mouse as an example, through Cre-loxP
Specific knockdown Tmem30a gene in cell, showing mouse, retinal vessels development is slow, vessel density reduces, blood vessel
Teloblast number reduces and shows the end of aneurysmal, teloblast touches sufficient number and reduces.These features are all retina new lives
The characteristic feature of vascular diseases.Thus it absolutely proves, chrotoplast conditionity knocks out Tmem30a gene in the blood vessels, can make mesh
Mark animal shows retinal neovascularization disease.Vascular endothelial cell conditionity knocks out the animal of Tmem30a gene, Ke Yizuo
For retinal neovascularization disease model.The disease model can be used in the fields such as retinal neovascularization disease research, be
The screening of the research such as pathogenic process, mechanism and related drugs of the disease provides a kind of new model.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (4)
1. a kind of construction method of retinal neovascularization disease model, characterized in that it comprises: knock out target animal blood vessel
Tmem30a gene order in genome in endothelial cell；
It knocks out Tmem30a gene order and refers to the exon sequence for knocking out Tmem30a gene；
The exon sequence for knocking out Tmem30a gene refers to the 3rd exon sequence knocked out on Tmem30a gene；
The construction method includes: that Tmem30a gene conditionity is knocked out homozygous animals to move with PDGF β-Cre ER gene is turned
Object mating obtains vascular endothelial cell conditionity and knocks out Tmem30a genetic animal, can be used as retinal neovascularization disease mould
Type.
2. construction method according to claim 1, which is characterized in that the target animal be selected from mouse, rat, dog, pig,
Any one in monkey and ape.
3. by the obtained view of construction method of retinal neovascularization disease model of any of claims 1-2
Application of the film neovascular disease model in retinal neovascularization disease research, the research be with the diagnosis of non-disease or
For the purpose for the treatment of.
4. by the obtained view of construction method of retinal neovascularization disease model of any of claims 1-2
Film neovascular disease model is in screening for preventing or treating the application in retinal neovascularization disease medicament.
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