CN109381478A - A kind of miRNA inhibits new vessels to generate and inhibit the application in the reagent of VEGF-A factor expression in preparation - Google Patents
A kind of miRNA inhibits new vessels to generate and inhibit the application in the reagent of VEGF-A factor expression in preparation Download PDFInfo
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- CN109381478A CN109381478A CN201811284351.4A CN201811284351A CN109381478A CN 109381478 A CN109381478 A CN 109381478A CN 201811284351 A CN201811284351 A CN 201811284351A CN 109381478 A CN109381478 A CN 109381478A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A61P27/00—Drugs for disorders of the senses
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Abstract
The present invention provides a kind of application of miRNA in the reagent that preparation inhibits new vessels to generate, and belong to field of biomedicine technology, and the miRNA is miR-204, and the nucleotide sequence of the miR-204 is as shown in SEQ ID NO.1.MiR204 provided by the invention can significantly inhibit VEGF-A factor expression, to inhibit the proliferation of vascular endothelial cell, migration and at pipe, inhibit the generation of new vessels.
Description
Technical field
The invention belongs to fields of biomedicine more particularly to a kind of miRNA to inhibit new vessels to generate and inhibit in preparation
Application in the reagent of VEGF-A factor expression.
Background technique
Normal cornea it is extremely important for immune privilege and dioptric without vascularization, under some pathologic conditions, as eye traumas,
Infection, inflammation, limbal stem cell deficiency etc., various inflammatory cells and immunocyte infiltration in cornea, corneal edema, on cornea
Skin continues defect, and Corneal transparency reduces, and cornea rebirth blood vessel is caused to grow, and leads to visual impairment even blinding.
Classical angiogenesis theory thinks that the blood vessel of damage or tumour granulation tissue is generated by following three kinds of modes
: new branch is issued from previous existing blood vessel；Endothelial cell budding growth；The telescopiform of capilary is grown.But
2009, Kilarski etc. proposed that a kind of completely new angiogenesis is theoretical, they think that new vessels can be independent of
The extension of existing blood vessel and generate, the generation of tissue tension energy mediate vascular.
In normal vascular development and pathologic vessels forming process, vascular endothelial growth factor (VEGF) family is played
Essential effect, VEGF be a kind of rush vascular endothelial growth factor of high degree of specificity, have that increase blood vessel logical
The effects of permeability and vascularization, wherein VEGF-A is most important blood vessel regulatory factor, is widely present in body, in cornea
In, it is mainly expressed in epithelium layer, VEGF-A promotes intravascular and in conjunction with its receptor VEGFR-1 and VEGFR-2
Epithelial cell proliferation, migration.
At present still without a kind of reagent and method for effectively inhibiting corneal vessels growth.
Summary of the invention
New vessels are inhibited to generate and inhibit in preparation in view of this, the purpose of the present invention is to provide a kind of miRNA
Effective inhibition of application and a kind of non-disease diagnosing and treating purpose in the reagent of VEGF-A factor expression is answered by tissue
The method of the growth of corneal vessels caused by power.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical schemes:
A kind of application of miRNA in the reagent that preparation inhibits new vessels to generate, the miRNA are miR-204, institute
The nucleotide sequence of miR-204 is stated as shown in SEQ ID NO.1.
Preferably, the miR-204 inhibits the proliferation of vascular endothelial cell, migrates and at pipe.
The present invention provides application of the miRNA in the reagent that preparation inhibits VEGF-A factor expression.
Preferably, the miR-204 inhibits the expression of the VEGF-A factor induced by tissue tensile stress.
Preferably, the miR-204 includes miR-204agomir；The miR-204agomir is dense in the reagent
Degree is 150~250nmol/l.
The present invention also provides a kind of methods of the VEGF-A factor expression of induction, comprising the following steps: will be between mechanical force
Having a rest property acts on corneal epithelial cell, and the mechanical force is 4~6% in the degree of drawing of corneal epithelial cell, the machinery
The frequency that power acts on corneal epithelial cell is 0.8~1.2Hz, and the mechanical force is in the time of corneal epithelial cell
5.5~6.5h.
Preferably, the mechanical force is 5% in the degree of drawing of corneal epithelial cell, and the mechanical force is in cornea
The frequency of epithelial cell is 1.9~1.1Hz, and the mechanical force is 5.8~6.3h in the time of corneal epithelial cell.
Preferably, the mechanical force is tissue tensile stress.
Beneficial effects of the present invention: the present invention provides miRNA answering in the reagent that preparation inhibits new vessels to generate
With miR204 provided by the invention can significantly inhibit VEGF-A factor expression, to inhibit the proliferation of vascular endothelial cell, move
It moves and at pipe, inhibits the generation of new vessels.
The present invention also provides the methods of the VEGF-A factor expression of tissue tensile stress induction, utilize side of the present invention
Tissue tensile stress intermittence is acted on corneal epithelial cell, can cause significantly improving for VEGF-A factor expression amount by method.
Detailed description of the invention
Fig. 1 grows for cornea rebirth blood vessel and miR-204 expression, wherein A, 10 days, 20 after normal cornea and suture
Its cornea rebirth blood vessel growing state, with CD31 label vascular；B, in situ hybridization detection miR-204 are mainly expressed on cornea
Skin；C, PCR detect the expression of miR-204 in cornea, Ctrl, normal control；*p<0.05；
Fig. 2 is that miR-204agomir inhibits corneal neovascularization, wherein left, 10 days cornea rebirth blood vessels after suture
Distribution；In, cornea rebirth blood vessel is distributed after injecting NTC；Cornea vascular distribution after miR-204 agomir is injected on the right side；
Fig. 3 is the expression that miR-204 inhibits cornea VEGF-A and VEGFR2, wherein A, Immunohistochemical Method detect VEGF-A and
VEGFR2 expression；B, western blot method analyze VEGF-A and VEGFR2 expression；
Fig. 4 causes the high VEGF expression of Human glioma, low expression miR-204 for periodical tensile stress stimulation, wherein
A, PCR detect VEGF-A expression；B, PCR detect miR-204 expression；C, western blot detect VEGF-A protein expression；D,
VEGF-A protein expression statistical chart, p < 0.05 *)；
Fig. 5 is that MiR-204 inhibits the migration of human microvascular endothelial cell (mvec) and at pipe, wherein A, cell migration figure；B, cell
Migration area statistical chart；C, cell Cheng Guantu；D, cell is at pipe statistical chart, p < 0.05 *.
Specific embodiment
The present invention provides a kind of application of miRNA in the reagent that preparation inhibits new vessels to generate, the miRNA is
The nucleotide sequence of miR-204, the miR-204 are as shown in SEQ ID NO.1, specially
uucccuuugucauccuaugccu.Heretofore described miR-204 is located at human chromosome 9q21.12；The miR-204 energy
Enough inhibit the generation of new vessels, the miR-204 is able to suppress the generation of new vessels caused by mechanical force, additionally it is possible to inhibit
The new vessels of alkali burn induction generate；The mechanical force is preferably tissue tensile stress.In the present invention, the miR-204 can
VEGF-A and VEGFR2 factor expression is significantly inhibited, to inhibit the proliferation of vascular endothelial cell, migration and at pipe, inhibits new
The generation of angiogenic.
In the present invention, the miR-204 preferably includes miR-204agomir；The miR-204 agomir is in institute
Stating the concentration in reagent is preferably 150~250nmol/l, more preferably 180~220nmol/l, most preferably 200nmol/l.
In the present invention, the miR-204agomir is preferably purchased from Guangzhou Rui Bo company, is powdered object.The miR-204
Agomir when in use, is preferably diluted with no RNA enzyme deionized water dissolving, is configured to above-mentioned preferred concentration.
The present invention also provides a kind of methods of the VEGF-A factor expression of induction, comprising the following steps: will be between mechanical force
Having a rest property acts on corneal epithelial cell, and the mechanical force is 4~6% in the degree of drawing of corneal epithelial cell, the machinery
The frequency that power acts on corneal epithelial cell is 0.8~1.2Hz, and the mechanical force is in the time of corneal epithelial cell
5.5~6.5h.In the present invention, the mechanical force is preferably 5% in the degree of drawing of corneal epithelial cell, the mechanical force
The frequency for acting on corneal epithelial cell is preferably 1.0Hz；The mechanical force is preferably in the time of corneal epithelial cell
6h.In the present invention, the mechanical force is preferably tissue tensile stress, and the tissue tensile stress is provided preferably through reinforcing instrument, more
Preferably periodical tensile stress loading device, the stress loading system that the periodicity tensile stress loading device is controlled by computer
It is constituted with round stress loading platform, the rubber gasket on the weighted platform makes row between platform base and tissue culture plate
At seal chamber, when being sucked by vacuum to this seal chamber, negative pressure is generated, elastic membrane is stretched, to provide biaxially for cell
Stress.In specific implementation process of the present invention, preferably the following steps are included: 1) by corneal epithelial cell, with 0.25wt% pancreas
Be inoculated in tissue culture plate after enzyme -0.02wt% EDTA digestion, adhere-wall culture, 2) tissue culture plate is placed in plus
On the weighted platform of power instrument, setting reinforcing parameter collects cell after being stretched.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
Organize influence of the tensile stress to mouse cornea new vessels
1. the influence of the foundation and tissue tensile stress of mouse cornea neovascularization model to cornea rebirth blood vessel
After 12 adult BALB/C mice general anesthesia successes, fixed mouse right eye ball, with the corneal ring of diameter 1.5mm
Brill is gently pressed in Central corneal makees corneal indentation, and 11-0 line is sewed up corneal stroma perpendicular to impression is radial, is knotted, knot is sudden and violent
Dew sutures 2 needles.Cornea rebirth blood vessel growing state and miR-204 expression are as shown in Figure 1, the obvious drop of miR-204 expression
It is low, new vessels vigorous growth.Mouse is randomly divided into treatment group and control group, is performed the operation under the same day, postoperative 3,6 days difference conjunctivas
Injection miR-204 is treatment group's (PBS, NTC are control group), and cornea neovascularization growth situation is observed after treatment, as a result as schemed
There are vigorous new vessels in 10 days corneas after modeling shown in 2, and cornea rebirth blood vessel obviously subtracts after subconjunctival injection miR-204
It is few.
The influence that 2.miR-204 expresses mouse cornea suture neovascularization model VEGF-A, VEGFR2
BALB/C mice eyeball after taking treatment, makes paraffin sample, and paraffin slicing machine cuts 6 μm of thickness paraffin organizations
Slice, the influence with immunohistochemical method detection miR-204 to VEGF-A, VEGFR2.Specifically includes the following steps:
1) paraffin section bakes piece, dewaxing, Gradient elution using ethanol；
2) antigen retrieval: slice is placed in antigen retrieval buffers (1:100), is boiled 3 minutes, and room temperature is cooling；PBS is washed 5 minutes,
It dries；
3) 3%H is dripped on slide2O2, 20 minutes；PBS is washed 3 times, 5 minutes/time；
4) 5%BSA is closed 1 hour；
5) primary antibody is added dropwise, 4 DEG C, stays overnight；PBS is washed 3 times, 5 minutes/time；
6) reinforcing agent is dripped, 37 DEG C of water-baths, 15 minutes；PBS is washed 3 times, 5 minutes/time；
7) plus secondary antibody, 37 DEG C, 37 DEG C, 1 hour；PBS is washed 3 times, 5 minutes/time；
8) DAB colour developing (1ml H2O+1 drop A+1 drop B+1 drop C), haematoxylin is redyed 20 seconds；Tap water rinses 30 seconds；Drying,
Neutral gum mounting；Optical microphotograph is taken a picture under the microscope.
As a result as shown in Figure 3A, miR-204 treatment group can significantly reduce the expression of VEGF-A, VEGFR2 in cornea.In addition
The cornea for the treatment of group and control group is collected, albumen is extracted, feelings are expressed with the analysis of western blot method VEGF-A, VEGFR2
Condition, specifically includes the following steps:
Western blot method:
1) albumen sample extraction: 2+60 μ l RIPA of cornea (contain 1%PMSF), on ice Ultrasonic Pulverization tissue；Centrifugation,
12000 turns, 5 minutes；Supernatant is taken, adds 4 × loading buffer, 95 DEG C, 5 minutes；- 80 DEG C of refrigerators save.
2) match glue: taking separation gel buffer and each 2.7ml of separation sol solution to mix, add the modified form ammonium persulfate of 60 μ l molten
Liquid mixes；Solution is injected in glue glass plate；After gelling to be separated is solid, take concentration glue buffer and concentration sol solution each
0.75ml is mixed, and the modified form ammonium persulfate solution of 15 μ l is added, and is mixed；It injects in glue glass plate, is inserted into comb teeth；
3) electrophoresis: 10 holes μ l/ of sample-adding；80V, 0.5 hour；120V, 1.5 hours.
4) transferring film: 100mA, 2~3 hours on ice.
5) it closes, add antibody: 5% skimmed milk power is closed 1 hour；Add+1% skimmed milk power of primary antibody (1:1000), it is 4 DEG C light
It shakes overnight；TBST washes film, adds+2.5% skimmed milk power of secondary antibody (1:2000~1:3000), and room temperature yawing 1 hour；TBST washes film；
6) it chemiluminescence: draws suitable luminescent solution and drops on film, be placed in instrument and develop.
As a result such as Fig. 3 B, miR-204 treatment group VEGF-A, VEGFR2 expression are substantially reduced, and show that miR-204 can inhibit
Cornea rebirth blood vessel.
Embodiment 2
Study influence of the tissue tensile stress to Human glioma vegf expression
With 2.4U/ml Dispase (being dissolved in DMEM culture medium) digest corneal ring, 37 DEG C, 2 hours；Take corneal epithelium
Layer is digested, 37 DEG C, 15 minutes with 0.25wt% pancreatin+0.02wt%EDTA；By primary Human glioma CMC model
Base weight is outstanding, is inoculated in culture plate, is P0 generation, by P2-P4 for cell inoculation (1 × 10 in flexcell tissue culture plate5
Cells/well), the flexcell tissue culture plate is elastic 6 porocyte culture plates, the flexible silicon glue film flexibility of bottom is good,
Transparency is high；Silica Surface is coated with by type i collagen, to increase the adherent ability of cell.
Grouping: blank control group, reinforcing+200nmol/l negative control (NTC) group, reinforcing+200nmol/l miR-
204agomir group；Flexcell tissue culture plate is placed on the weighted platform of reinforcing instrument, setting reinforcing parameter (degree of drawing
5%, frequency 1Hz), cell is collected after 6 hours with PCR, western blot method detection VEGF-A expression of results sees Fig. 4,
As shown in figure 4, periodical tensile stress stimulation causes the vegf expression in Human glioma to rise, miR-204 low expression, and
After exogenous increase miR-204, this high expression is obvious to be suppressed.Exogenous miR-204 moves human microvascular endothelial cell (mvec)
Move and at pipe influences result as shown in figure 5, the migration of cell and being all decreased obviously at pipe ability after increase miR-204.
As can be seen from the above embodiments, miR204 provided by the invention can significantly inhibit VEGF-A factor expression, to press down
The proliferation of vascular endothelial cell processed migrates and at pipe, inhibits the generation of new vessels.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>affiliated hospital, University Of Qingdao
<120>a kind of miRNA inhibits new vessels to generate and inhibit the application in the reagent of VEGF-A factor expression in preparation
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 1
uucccuuugu cauccuaugc cu 22
Claims (8)
1. a kind of application of miRNA in the reagent that preparation inhibits new vessels to generate, the miRNA is miR-204, described
The nucleotide sequence of miR-204 is as shown in SEQ ID NO.1.
2. application according to claim 1, which is characterized in that the miR-204 inhibits the proliferation of vascular endothelial cell, moves
It moves and at pipe.
3. application according to claim 1 or 2, which is characterized in that the miR-204 inhibits VEGF-A factor expression.
4. application according to claim 3, which is characterized in that the miR-204 inhibition was induced by tissue tensile stress
The expression of the VEGF-A factor.
5. application according to claim 3 or 4, which is characterized in that the miR-204 includes miR-204agomir；It is described
Concentration of the miR-204agomir in the reagent is 150~250nmol/L.
6. a kind of method of the VEGF-A factor expression of induction, comprising the following steps: act on mechanical force intermittence on cornea
Chrotoplast, the mechanical force are 4~6% in the degree of drawing of corneal epithelial cell, and the mechanical force is in corneal epithelium
The frequency of cell is 0.8~1.2Hz, and the mechanical force is 5.5~6.5h in the time of corneal epithelial cell.
7. according to the method described in claim 6, it is characterized in that, the mechanical force is in the degree of drawing of corneal epithelial cell
It is 5%, the mechanical force is 1.9~1.1Hz in the frequency of corneal epithelial cell, and the mechanical force is in corneal epithelium
The time of cell is 5.8~6.3h.
8. method according to claim 6 or 7, which is characterized in that the mechanical force is tissue tensile stress.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112094808A (en) * | 2020-09-16 | 2020-12-18 | 中山大学中山眼科中心 | MiR-204-containing exosome and preparation method and application thereof |
| CN116286628A (en) * | 2023-05-15 | 2023-06-23 | 四川大学华西医院 | Dental pulp mesenchymal stem cell culture medium additive, culture medium and application thereof |
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| CN1460024A (en) * | 1999-07-14 | 2003-12-03 | 诺沃挪第克公司 | Use of FVIIa or tissue factor antagonist for regulating gene expression and cell migration or chemotaxis |
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| CN108618753A (en) * | 2017-03-23 | 2018-10-09 | 王继宏 | A method of continuing Tensile stress stimulation and repairs rabbit rupture of achilles tendon |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112094808A (en) * | 2020-09-16 | 2020-12-18 | 中山大学中山眼科中心 | MiR-204-containing exosome and preparation method and application thereof |
| CN116286628A (en) * | 2023-05-15 | 2023-06-23 | 四川大学华西医院 | Dental pulp mesenchymal stem cell culture medium additive, culture medium and application thereof |
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