CN105943548A - Retinal degeneration model and drug screening method thereof - Google Patents
Retinal degeneration model and drug screening method thereof Download PDFInfo
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- CN105943548A CN105943548A CN201610394919.2A CN201610394919A CN105943548A CN 105943548 A CN105943548 A CN 105943548A CN 201610394919 A CN201610394919 A CN 201610394919A CN 105943548 A CN105943548 A CN 105943548A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- 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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
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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 invention relates to a retinal degeneration model. Model animal retinal inferior vena injection is performed by using a slow virus mediated antisense mir-24 virus. Compared with the prior art, the slow virus mediated antisense mir-24 virus is adopted for model animal retinal inferior vena injection for the first time, the retinal degeneration model is established, RPE cells are selectively destructed, secondary retinal degeneration is caused, and the retinal degeneration model has the characteristics of cases of retinal degeneration. The retinal degeneration model can be used for researching a retinal degeneration mechanism and drug screening. The retinal degeneration model is one of important miRNA-mir-24 for closing one RPE cell, and the epidemiological characteristics of idiopathic AMD can be also simulated according to the action mechanism (one miRNA acts on multiple target genes) of miRNA.
Description
Technical field
The present invention relates to a kind of animal model, especially relate to a kind of retinal degeneration model and drug screening side thereof
Method.
Background technology
The model of retinal degeneration is divided into heritability and chemical two kinds, a kind of typical model of chemical model at present
To utilize sodium iodate to induce, for sodium iodate induction memory retinal degeneration model due to the course of disease very anxious, iodic acid
Sodium is as a strong oxidizer, although destroy retinal pigment epithelium (RPE), also destroys light sensation simultaneously
Receiver cell, thus while there is report (the such as Chinese patent of relevant sodium iodate induction monkey retinal degeneration model
CN 102198278 A i.e. discloses a kind of Rhesus Macacus retinal degeneration model and drug screening method thereof), but its
Repeatability is poor, is difficult to repeat out.For heritability model, there is the heritability age phase that single gene mutation causes
Closing property degeneration of macula (AMD) rat model (RCS rat), thin because of its RPE due to Mertk gene mutation
Endocytosis bites hypofunction, causes secondary retinal degeneration.
Summary of the invention
Defect that the purpose of the present invention is contemplated to overcome above-mentioned prior art to exist and provide one can simulate spy
The retinal degeneration model of the property sent out AMD pathogeneticing characteristic and drug screening method thereof.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of retinal degeneration model, carries out animal pattern view by the virus of the antisense mir-24 of lentivirus mediated
Hypostegal cavity is injected.
The virus of the antisense mir-24 of described lentivirus mediated specifically refers to be cloned into antisense miR-24 sequence slowly
The virus obtained on viral vector, described miR-24 base sequence is as shown in SEQ ID NO.1.
The injection dosage of the virus of the antisense mir-24 of described lentivirus mediated is 3ul, and virus titer is
5-10*10^7tu/ml。
Described animal pattern includes rat and monkey, preferably SD rat or machin.
A kind of method of medicine screening treatment retinal degeneration, comprises the steps:
A, virus with the antisense mir-24 of lentivirus mediated carry out animal pattern subretinal space injection, and foundation regards
Nethike embrane fibrosis models；
B, drug candidate is applied to fibrosis models；
C, observe drug candidate to retinal degeneration, the situation that affects of the quantizating index of degree of injury, and comment
Point, evaluate the medicine of potential treatment retinal degeneration.
In step a, the injection dosage of the virus of the antisense mir-24 of described lentivirus mediated is 3ul, and virus is dripped
Degree is 5-10*10^7tu/ml.
Described animal pattern includes rat and monkey, preferably SD rat or machin.
Compared with prior art, the virus of the antisense mir-24 of present invention employing lentivirus mediated first carries out mould
Type animal retina cavity of resorption is injected, and sets up retinal degeneration model, selective destruction RPE cell, causes secondary
Property retina film degeneration, has the case feature of retinal degeneration.This model that the present invention provides can be used for studying
The mechanism of retinal degeneration and drug screening.Model of the present invention is that one RPE cell of closing is important
MiRNA-mir-24, according to the mechanism of action (a multiple target gene of miRNA effect) of miRNA, more can
The feature of simulation idiopathic AMD morbidity.
Accompanying drawing explanation
Fig. 1 is that the visual function that the injection of anti-miR-24 rat retina cavity of resorption causes reduces and retina related gene
The change expressed, opsin, rhodopsin and recoverin are that the labelled protein of photoreceptor cell is significantly lowered；
Bim, bax, bak etc. promote apoptogene and have no.
Fig. 2 is that the retina stratum nucleare that the injection of anti-miR-24 rat retina cavity of resorption causes is thinning, Muller cell
Activation.
Fig. 3 is that anti-miR-24 rat retina cavity of resorption injects the proinflammatory factor CHI3L1 up-regulated caused,
RPE65 down-regulated expression, points out RPE miopragia.
Fig. 4 is that the CHI3L1 that ELISA detection anti-miR-24 rat retina cavity of resorption injection causes produces increasing
Many.
Fig. 5 is the RPE cytoclasis that anti-miR-24 rat retina cavity of resorption injection retina causes, RPE65
Immunofluorescence reduces (RBCC tile).
Fig. 6 is that anti-miR-24 machin subretinal space injects the seepage (FFA and fundus photography) caused.
Fig. 7 is machin retina normal OCT testing result；Anti-miR-24 machin subretinal space is noted
Penetrate 7 weeks OCT detection, find that retinal structure is disorderly, RPE once with outer nuclear layer adhesion.
Detailed description of the invention
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1
Carry out animal pattern subretinal space injection by the virus of the antisense mir-24 of lentivirus mediated, obtain view
Film fibrosis models.
The antisense sequences that antisense mir-24 is as shown in SEQ ID NO.1, for
CTGTTCCTGCTGAACTGAGCCA.Antisense miR-24 sequence is cloned on slow virus carrier and obtains
The virus of the antisense mir-24 of lentivirus mediated is this area conventional technique means, it is also possible to buy, i.e. antisense
Mir-24 virus is also commercialization.After obtaining virus, subpackage is frozen in-80%.Take normal adult SD rat,
About body weight 200g, it is placed under Stereo microscope, and on rat eye, places concavees lens；Use 30gauge pin
Head stings a pin hole after corneoscleral junction at 2mm, then with the microsyringe equipped with 33gauge syringe needle from pin
At hole, inserting needle is to subretinal space, injects 3 μ l (containing 5-10*10^7tu/ml) viruses.Under Second eye retina
Chamber equal-volume injection equivalent comparison virus.2w, 5w and 7w after injecting virus carries out retina electro physiology
(ERG) detection.
ERG method: APS full-automatic vision electrophysiologic study instrument (APS-2000) is purchased from Chongqing Kang Hua science and technology to be had
Limit company.Do visual electrophysiology functional check the previous day, DM rat is transferred to dark place, carries out dark adaptation.
Second day starts to do.The preparation of rat: enter to rats by intraperitoneal injection 2% pentobarbital sodium (1mL/500g body weight)
Row anesthesia, 1 × Su Mian Xin (0.1ml/200g) allows exophthalmos, then gives a 0.5% tropicamide mydriasis
(Wuxi Shanhe Group, Jiangsu, China), a 0.4% Oxybuprocaine topical anesthesia (Eisai Co
Ltd, Tokyo, Japan), every eyes are coated with some conductive pastes.Intercalative electrode: ground wire connects on rat tail, negative
Pole connects between rat two ear, and positive pole connects on two cornea eye, is careful not to contact on eyelid and sclera.Open soft
Part " visual electrophysiology figure ", point " FERG ", then put 1,2 passages, one is left eye channel, and another is
Right eye channel, point " is arranged ", and stimulating number of times is 2 times, and stimulus frequency is 0.05Hz；Click on successively and stimulate by force
Degree (1)-0.0006325 (cd*s/m), (5)-0.006325 (cd*s/m), (9)-0.06325 (cd*s/m),
Intensity is at least spaced 2min every time.Point " oscillography ", when the baseline of ripple is steady, clicks on " collection ", waits and listen
After a sound of " ticking ", gather complete, click on " preservation ".Click on again " setting ", amendment number of documents and thorn
Swash intensity, the like.After finishing etc. all intensity, change a rat.After all rats finish, open " literary composition
Part ", to demarcate, double-click curve, curve becomes white, clicks on " demarcation ".After having demarcated a ripple, press
Space bar, demarcates b ripple.All demarcation are complete, click on " printing ", save as .PDF form.Click on lower-left
Angle button, exits software, closes computer, closes amplifier.
Fluorescence quantitative PCR detection:
Extracting the method that RNA uses Trizol cracking, key step is as follows:
1. cell PBS is washed 1-2 time, be proportionally added into Trizol lysate (such as six orifice plates
One hole correspondence 1mL lysate).
2. cell is after wall, transfers to the centrifuge tube of 1.5mL, adds the chloroform of 1/5th volumes, acutely mixes
After even, it is centrifuged 15 minutes with the rotating speed 4 DEG C of 12000rpm.
3. after being centrifuged, supernatant is transferred in new centrifuge tube, be careful not to get the albumin layer of centre, add
Isopyknic isopropanol, ice bath 20 minutes.
Centrifugal 15 minutes of the rotating speed of 4.12000rpm 4 DEG C, supernatant discarded, precipitation is washed 1-2 with the ethanol of 75%
Secondary.
5. by dried, with appropriate DEPC water dissolution for precipitation room temperature.Survey concentration.
RNA reverse transcription, cDNA Article 1 chain is to be obtained by the M-MLV reverse transcription of Promega company
, key step is as follows:
First taking oligo d (T) mixing of the RNA and 2 μ L of 1-2 μ g, 72 DEG C of water-baths are placed 5 minutes,
Ice bath is after 2 minutes immediately, makes the poly-A tail of oligo d (T) and RNA be combined, gentle centrifugation.
2. add the dNTP (10 μMs) of 1.25 μ L, 1 μ L M-MLV reverse transcription and 0.5 μ L RNase to press down
Preparation, is adjusted to 25 μ L with DEPC water by reaction volume.42 DEG C of water-baths are placed 1 hour.
3.70 DEG C of placements make reverse transcription inactivate in 10 minutes.The cDNA strand obtained is stored in-20 DEG C of refrigerators.
Primer see table 1.
Table 1 is for detecting the primer of gene
| Name | forward | reverse |
| qRo-GAPDH | CCCCTTCATTGACCTCAACTACA | TCCCATTCTCAGCCTTGACTGT |
| qRo-Revn | CGGCAGGAGTTCGAAAGTATCT | TGCTGGGCGTAGGCCTTA |
| qRo-RHO | CAGAGGGCCCCAATTTTTATG | GGTAGTACTGCGGCTGCTCAA |
| qRo-OPN | GAAGGCTACATTGTCTCACT | AGAAGACGATTCCCACAG |
| qRo_bim | ATGAGACTTACACGAGGAG | ACCAGACGGAAGATGAATC |
| qRo_bak | AGAGTTCCAGACCATGCCGC | GTAGCCGAAGCCCAGAAG |
| qRo_bax | GCAAACTGGTGCTCAAGG | GGTCCCGAAGTAGGAAAGG |
| qRo_caspase3 | CTGGACTGCGGTATTGAG | GGGTGCGGTAGAGTAAGC |
Reverse transcription system such as table 2:
Table 2 reverse transcription system
Reverse transcription program: 16 DEG C 30 minutes, 42 DEG C 30 minutes, 85 DEG C are immediately placed on ice 5 after 10 minutes
Minute.Then can be standby in-20 DEG C of Refrigerator stores.
Quantitative PCR
Using cDNA the first chain of obtaining after RNA reverse transcription as template, design primer.Utilize Tian Gen company
SYBR Green real-time fluorescence quantitative PCR detection kit, the expression of testing goal gene.PCR expands
Increasing condition is as follows: 94 DEG C of degeneration 10 minutes, enters circulation (95 DEG C of 5sec, 60 DEG C of 60sec), 40 altogether
Circulation, and collect solubility curve.
Result sees Fig. 1, anti-mir-24 and injects the 5th week at subretinal space, finds b wave-wave width and contrast ratio
Significance declines, and by the 7th week, the decline of b ripple became apparent from (p < 0.05), and prompting retinal function declines；At note
Penetrate latter 7th week, in mRNA level in-site, use real-time fluorescence quantitative PCR to find, the labelling of photoreceptor cell
Thing is expressed, and opsin, rhodospin and recoverin significantly lower, and promote expression of apoptotic gene and do not have significance poor
Different, prompting apoptosis does not mediate the death of photoreceptor cell.
ERG detection b ripple occurs and declines (having significant difference) when, think that retina there occurs degeneration,
Now experimentally can intervene for cell transplantation, gene therapy etc. with this model.
Immunofluorescence and stratum nucleare Thickness Analysis is carried out after the injection of embodiment 2 subretinal space.
After subretinal space injection, by carry out immunofluorescence dyeing carry out stratum nucleare Thickness Analysis and
The detection of Muller cell colloid label.
Immunofluorescence:
The preparation of tissue slice: DM rat carefully extracts eyeball (as far as possible with the presence of optic nerve), in 4% poly first
Aldehyde is fixed three hours.Under anatomic microscope, clip at 2mm above corneoscleral junction, corneal scissors
Fall, more carefully remove crystalline lens and iris.Remaining eyeball is put into the sucrose of 30% and is dehydrated 3 hours.Eye
Organization embedding agent embedding put into by ball, and optic nerve is placed on side, has been careful not to bubble, and 4 DEG C of refrigerators balance overnight.
Within second day, moving to-80 DEG C of refrigerators standby, a labelling is done in the place of papilla of optic nerve.The eyeball of embedding from-80 DEG C of ice
After case is taken out, carrying out serial section with freezing microtome through labelling and the papilla of optic nerve done, slice thickness is
10μm。
Immunofluorescence test: retinal slice PBS moistens 10 minutes, 0.25%tritonx-100 permeable membrane 10 points
Zhong Hou, with PBS 3 times, each 5 minutes.After closing 30 minutes with 1%BSA under room temperature, with one
The anti-GS of anti-mouse (1:200), little mouse-anti CRALBP (1:50), the anti-Recoverin of rabbit (1:500), rabbit resist
GMFB (1:200), the anti-GFAP of rabbit (1:200) (resist with little mouse-anti mCherry respectively 4 DEG C of night incubation
Body (1:1000-1:2000) dye altogether), it is not added with one anti-group as negative control.Continue next day to use PBS 3 times,
5 minutes/time, under room temperature lucifuge respectively with two anti-anti-mouse FITC (1:100) or anti-rabbit FITC
(1:100) one hour is hatched.After reject two is anti-, hatches 30 seconds with the DAPI of 0.5 μ g/mL, use PBS
Clean 3 times, 5 minutes/time.Use DAKO mounting, observe glimmering after adding coverslip under inverted fluorescence microscope
Light result.
Stratum nucleare thickness measure: fix eyeball with 4% paraformaldehyde, dissects and saccharose gradient dehydration, then uses OCT
Embedding eyecup.With freezing microtome, eyecup is cut into by papilla of optic nerve the section of 8 μm.By survival light sensation
The amphiblestroid damage of statistical analysis of the number of receiver cell, respectively in nasal side and the equidistant setting of temporo branch hole ball 10
Individual point.The thickness of each ONL and INL is obtained by measurement.
By Fig. 2 finding, subretinal space injection anti-mir-24 is after 7 weeks, and injection side outer nuclear layer thickness is the most thinning,
GFAP immunofluorescence display Muller substantially activates.
Embodiment 3
The injection of Anti-mir-24 subretinal space causes the change of RPE marker protein and the change of the target protein of mir-24
Change.Subretinal space injection anti-mir-24, after 7 weeks, separates RPE-bruch film-choroid under body formula anatomical lens
Complex, carries out wetsern blot, expresses at protein level detection RPE65 and Chil3
Western Blot method is as follows
1. preparation gel used by SDS-PAGE, first preparation lower floor separation gel, after preparing, the most slowly
Join in the layer glass plate being equipped with, add appropriate distilled water and close.
2., after lower floor's separation gel solidification, start to prepare upper strata and concentrate glue.Upper water in 1st step is outwelled,
Adding and concentrate after glue, slowly plugged by comb, standing etc. is to be solidified.
3. by extract albumen denatured by boiling after, join gel pore according to protein concentration according to identical sample size
In, under the voltage of 100V, stop electrophoresis, the method using wet turn after certain time, protein delivery is arrived
On the pvdf membrane of Millipore company.
4. after transferring film terminates, being taken out by pvdf membrane, film is closed by defatted milk powder or BSA with 3% in room temperature
1-2 hour.
5. close after terminating, pvdf membrane taken out from skim milk or BSA, clean 2 times with PBST,
After blotting, adding the anti-diluent prepared in right amount, room temperature shakes up 2 hours, makes an anti-and destination protein knot
Close.Used one is anti-to be included: anti-RPE65 (Abcam), anti-Chi3L1 (Proteintech) and anti-actin
(Proteintech)。
The ELISA kit of detection Chi3L1 is purchased from CUSABIO company.Require to carry out according to test kit.
As it is shown on figure 3, anti-miR-24 rat retina cavity of resorption is injected in the proinflammatory factor CHI3L1 expression caused
Adjust, RPE65 down-regulated expression, point out RPE miopragia.After subretinal space injection anti-mir-24 7 weeks,
Separate RPE-bruch film-choroid complex under body formula anatomical lens, extract total protein, and carry out ELISA, inspection
Survey Chi3l1 to express, as shown in Figure 4, find that Chi3l1 expresses notable rising in injection anti-mir-24 group.
Embodiment 4
Subretinal space injection anti-mir-24, after 7 weeks, carries out RBCC tile, by immunofluorescence, detection
The immunoreactivity of RPE65.Inner nuclear layer retina is as follows:
After being anaesthetized by rat, extremity are fixed, and cut off thoracic cavity, carry out left ventricle perfusion, first irrigate with PBS,
Then Reperfu-sion 4%PFA.After perfusion, take off eyeball, 4 DEG C can be steeped in PBS and deposit, it is also possible at once
Under stereomicroscope, remove anterior ocular segment, carefully take out retina, microscope slide is cut into 6 lobes, then uses
Add the mountant mounting of anti-quencher, take pictures.Once inner nuclear layer retina completes, and carries out the immunofluorescence of RPE65.
Immunofluorescence is with embodiment 2.
As seen from Figure 5: subretinal space injection anti-mir-24 is after 7 weeks, and RPE obscure boundary, change is big, RPE65
Immune fluorescence intensity weakens, and points out RPE miopragia.
Embodiment 5
Use above-mentioned same procedure to carry out the injection of anti-miR-24 machin subretinal space, and investigate result.As
Shown in Fig. 6, the injection of anti-miR-24 machin subretinal space causes seepage (FFA and fundus photography), as
Shown in Fig. 7, machin retina normal OCT testing result is noted with anti-miR-24 machin subretinal space
Penetrate 7 weeks OCT testing results, find that retinal structure is disorderly, RPE once with outer nuclear layer adhesion.
The above-mentioned description to embodiment is to be understood that for ease of those skilled in the art and use to send out
Bright.These embodiments obviously easily can be made various amendment by person skilled in the art, and at this
The General Principle illustrated is applied in other embodiments without through performing creative labour.Therefore, the present invention does not limits
In above-described embodiment, those skilled in the art are according to the announcement of the present invention, without departing from changing that scope is made
Entering and revise all should be within protection scope of the present invention.
Claims (7)
1. a retinal degeneration model, it is characterised in that enter by the virus of the antisense mir-24 of lentivirus mediated
Row animal pattern subretinal space is injected.
A kind of retinal degeneration model the most according to claim 1, it is characterised in that described slow virus
The virus of antisense mir-24 of mediation specifically refers to be cloned into antisense miR-24 sequence and obtains on slow virus carrier
Virus, described miR-24 base sequence is as shown in SEQ ID NO.1.
A kind of retinal degeneration model the most according to claim 1, it is characterised in that described slow virus
The injection dosage of the virus of the antisense mir-24 of mediation is 3ul, and virus titer is 5-10*10^7tu/ml.
A kind of retinal degeneration model the most according to claim 1, it is characterised in that described model moves
Thing includes rat and monkey, preferably SD rat or machin.
5. the method for the medicine screening treatment retinal degeneration, it is characterised in that comprise the steps:
A, virus with the antisense mir-24 of lentivirus mediated carry out animal pattern subretinal space injection, and foundation regards
Nethike embrane fibrosis models；
B, drug candidate is applied to fibrosis models；
C, observe drug candidate to retinal degeneration, the situation that affects of the quantizating index of degree of injury, and comment
Point, evaluate the medicine of potential treatment retinal degeneration.
The method of a kind of medicine screening treatment retinal degeneration the most according to claim 5, its feature exists
In, in step a, the injection dosage of the virus of the antisense mir-24 of described lentivirus mediated is 3ul, and virus is dripped
Degree is 5-10*10^7tu/ml.
The method of a kind of medicine screening treatment retinal degeneration the most according to claim 5, its feature exists
In, described animal pattern includes rat and monkey, preferably SD rat or machin.
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| CN108721327A (en) * | 2018-08-24 | 2018-11-02 | 苏州药明康德新药开发股份有限公司 | The preparation method of experimental animal retinal pigment degeneration test model |
| CN112043833A (en) * | 2020-08-31 | 2020-12-08 | 同济大学 | Autophagy and apoptosis inhibitor of retinal pigment cells (RPE) and application thereof |
| CN113226020A (en) * | 2018-11-14 | 2021-08-06 | 珠海岐微生物科技有限公司 | Animal models, screening methods and treatment methods for intraocular diseases or disorders |
| CN113329773A (en) * | 2018-11-14 | 2021-08-31 | 珠海岐微生物科技有限公司 | Animal models and screening methods for intraocular diseases or disorders |
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| CN108721327A (en) * | 2018-08-24 | 2018-11-02 | 苏州药明康德新药开发股份有限公司 | The preparation method of experimental animal retinal pigment degeneration test model |
| CN113226020A (en) * | 2018-11-14 | 2021-08-06 | 珠海岐微生物科技有限公司 | Animal models, screening methods and treatment methods for intraocular diseases or disorders |
| CN113329773A (en) * | 2018-11-14 | 2021-08-31 | 珠海岐微生物科技有限公司 | Animal models and screening methods for intraocular diseases or disorders |
| CN112043833A (en) * | 2020-08-31 | 2020-12-08 | 同济大学 | Autophagy and apoptosis inhibitor of retinal pigment cells (RPE) and application thereof |
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| Publication number | Publication date |
|---|---|
| CN105943548B (en) | 2019-01-25 |
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