CN108434454B - Application of the ADAR1 in terms of alleviating cognition dysfunction - Google Patents
Application of the ADAR1 in terms of alleviating cognition dysfunction Download PDFInfo
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Abstract
The present invention discloses application of the ADAR1 as intervention target spot in terms of alleviating cognition dysfunction, the application including ADAR1 in terms of alleviating cognition dysfunction；The application includes application of the ADAR1 as intervention target spot in terms of alleviating cognition dysfunction, the specifically application in terms of cognition dysfunction drug is treated in preparation.The application includes the application of ADAR1 inducer or ADAR1 inhibitor in terms of cognition dysfunction drug is treated in preparation.Further, the ADAR1 inducer is IFN-γ, and the ADAR1 inhibitor is EHNA.The application includes promoting patient's intracerebral ADAR1 to be restored to normal level in human brain using ADAR1 inducer or ADAR1 inhibitor.The present invention is for the pathogenesis of announcement cognition dysfunction class disease, and discovery is directed to the drug target of such disease, is effectively prevented, with important theory significance and application value.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to ADAR1 is alleviating cognitive function barrier as target spot is intervened
Hinder the application of aspect.
Background technique
Cognitive function refer to brain to the ability of the processing of information, storage and extraction, including study, memory, consciousness, attention,
The abilities such as mind and imagination.Cognition dysfunction, that is, Cognitive deficiency or exception cannot normally process information, be stored
And extraction, it is mainly shown as disturbance of perception, the disturbance of thought and memory disorders.Society isolation stress stimulation is decrease of cognitive function
Risk factors, can caused by cognition dysfunction, the neurotransmitter and its associated receptor function of pathogenesis and hippocampus and cortex
Disorder, endocrine system and the Immune system imbalance of energy etc. are related.
Since the pathogenesis of cognition dysfunction not yet illustrates completely, at present treatment can only with suit the medicine to the illness and neuroprotective
Based on treatment, including applying different nerve cell-protective agents, such as Brain circlulation improver, energetic supersession activator, neurotransmitter
With nerve growth factor protective agent, Ca2+Antagonist, glutamate receptor antagonist, antioxidant, spongiocyte regulator and non-
Steroidal anti-inflammatory agent etc.；By intervening recovery and the normal level of neurotransmitter, including drug being maintained to supplement its precursor L-3,4 dihydroxyphenylalanine
The enzyme gene of amine, various cell transplantations to substitute dopaminergic neuron, gene therapy implantation promotes dopamine to synthesize, to promote
Generation or implantation neurotrophic factor gene into Dopamine In Striatum, improve nervous system second using anticholinesterase
The content etc. of phatidylcholine；Traditional operative treatment includes pallidectomy, Thalamectomy and stereoscopic localized heeling-in brain thorn
Swash device etc..Since above-mentioned treatment is for the downstream links and symptomatic treatment of cognitive function morbidity, therapeutic effect is extremely undesirable,
With property drug-dependent, traumatic and easy to recur.
Summary of the invention
In order to solve the above technical problems, the present invention is intervened, Jin Erfa from epigenetics angle in post-transcriptional level
The crucial target spot of the upstream link of existing cognition dysfunction.It determines technical solution of the present invention, is to protect ADAR1 as intervention
Application of the target spot in terms of alleviating cognition dysfunction.
The present invention further uses new object identification by preparing cognition dysfunction mouse model caused by stress stimulation
The Cognitive function change of experiment detection mouse, immunohistochemistry and the ADAR1 expression in western blot detection mouse brain change
Become, the ADAR1 inhibitor confirmed in cellular level or inducer intervention is given, to confirm that ADAR1 is answered in society isolation
Swash the mechanism of action in BALB/c mouse abnormal behaviour caused by stimulating.
For technique described above scheme, further, the application includes ADAR1 as target spot is intervened and exists
Application in terms of preparation treatment cognition dysfunction drug.
For technique described above scheme, further, the application includes ADAR1 in preparation treatment cognition
Application in terms of dysfunction drug.
For technique described above scheme, further, the application includes ADAR1 inducer or ADAR1
Application of the inhibitor in terms of cognition dysfunction drug is treated in preparation.
For technique described above scheme, further, the ADAR1 inducer is IFN
(Interferon)-γ, gamma interferon；The ADAR1 inhibitor is EHNA (erythro-9- (2-hydroxy-3-
Nonyl) adenine), pentostatin Pentostatin.
For technique described above scheme, further, the described application include using ADAR1 inducer or
ADAR1 inhibitor promotes patient's intracerebral ADAR1 to be restored to normal level in human brain.
For technique described above scheme, further, the application is induced including the use of the ADAR1
Agent promotes the expression of the mRNA and albumen of the ADAR1 of intracerebral, or promotes the active increase of ADAR1.Research discovery of the invention
After 4 weeks society isolation stress stimulations, there is non-space cognitive function and is obviously reduced in BALB/c mouse；Society isolation stress pierce within 4 weeks
After swashing, BALB/c mouse prefrontal cortex, hippocampus ADAR1 protein expression are substantially reduced；ADAR1 inhibitor enhancing society every
The non-space cognition dysfunction occurred from stress stimulation BALB/c mouse；ADAR1 inducer mitigates society isolation stress stimulation
The non-space cognition dysfunction of BALB/c mouse；ADAR1 inducer reverses society isolation stress stimulation BALB/c mouse forehead
The low expression of leaf skin layer, hippocampus ADAR1.This pathogenesis for disclosing such disease, discovery are directed to the medicine of such disease
Object target spot, is effectively prevented, and has important theory significance and application value.
For technique described above scheme, further, the application is induced including the use of the ADAR1
Agent promotes the expression of the mRNA and albumen of the ADAR1 of intracerebral prefrontal cortex and hippocampus, or promotes the active increasing of ADAR1
Add.
For technique described above scheme, further, the application inhibits including the use of the ADAR1
Agent inhibits the expression of the mRNA and albumen of the ADAR1 of intracerebral, or inhibits the activity of ADAR1.
For technique described above scheme, further, the application inhibits including the use of the ADAR1
Agent inhibits the expression of the mRNA and albumen of the ADAR1 of intracerebral prefrontal cortex and hippocampus, or inhibits the activity of ADAR1.
The study find that using ADAR1 (rna editing enzyme) as the novel targets intervened, from the angle of epigenetics, discovery
The crucial target spot of cognition dysfunction morbidity upstream link, preferably annotates how body adapts to body caused by environmental stress stimulation
The destruction of homeostasis.The present invention uses behaviouristics, morphology and Protocols in Molecular Biology detection and analysis ADAR1 (adenosine
Deaminase acting on RNA) inducer (IFN (Interferon)-γ, gamma interferon) and inhibitor [EHNA
(erythro-9- (2-hydroxy-3-nonyl) adenine), pentostatin Pentostatin] to society isolation mouse
The influence of cognitive function finds that ADAR1 inducer or ADAR1 inhibitor promote patient's intracerebral ADAR1 to be restored in human brain normally
Level can effectively alleviate the abnormal cognitive function of society isolation mouse.This pathogenesis for disclosing such disease, hair
It is now directed to the drug target of such disease, is effectively prevented, there is important theory significance and application value.
Detailed description of the invention
Fig. 1 is that experimental group and society isolation stress stimulation model prepare schematic diagram；
Fig. 2 is that schematic diagram is tested in new object identification；(ORT) index of discrimination is tested in new object identification；EHNA and IFN-γ point
It Wei not ADAR1 inhibitor and ADAR1 inducer；Data are indicated with mean ± standard deviation；*P<0.05,**P<0.01；(n=10
Only/group).
Fig. 3 is the new object identification experimental result that ADAR1 inhibitor and inducer intervene society isolation BALB/c mouse；
Fig. 4 is the ImmunohistochemistryResults Results that the society isolation BALB/c mouse prefrontal cortex ADAR1 of pharmaceutical intervention is expressed；
(a) ImmunohistochemistryResults Results of prefrontal cortex ADAR1 expression；(b) the brain area schematic diagram of prefrontal cortex；(c) prefrontal cortex
The OD value statistical result of the positive neuron of ADAR1 expression.EHNA and IFN-γ are respectively ADAR1 inhibitor and ADAR1
Inducer；Data are indicated with mean ± standard deviation；**P<0.01；(n=5/group).
Fig. 5 is the ImmunohistochemistryResults Results that the society isolation BALB/c mouse hippocampus ADAR1 of pharmaceutical intervention is expressed；(a) extra large
The ImmunohistochemistryResults Results of horse CA1 area ADAR1 expression；(b) ImmunohistochemistryResults Results of the area hippocampus Hilus ADAR1 expression；(c) hippocampus
The brain area schematic diagram in the area CA1 and Hilus；(d) the OD value statistical result of the positive neuron of Hippocampal CA 1 ADAR1 expression；
(e) the OD value statistical result of the positive neuron of the area hippocampus Hilus ADAR1 expression.EHNA and IFN-γ are respectively ADAR1
Inhibitor and ADAR1 inducer；Data are indicated with mean ± standard deviation；**P<0.01；(n=5/group).
Fig. 6 is the western blot result of the society isolation BALB/c mouse intracerebral ADAR1 protein expression of pharmaceutical intervention.
(a) the western blot result and gray analysis statistical result of prefrontal cortex ADAR1 albumen；(b) society of pharmaceutical intervention
The western blot result and gray analysis statistical result of BALB/c mouse hippocampus ADAR1 protein expression is isolated.Statistics knot
Fruit is the gray level ratio result of purpose albumen and internal reference；EHNA and IFN-γ are respectively ADAR1 inhibitor and ADAR1 inducer；
Data are indicated with mean ± standard deviation；**P<0.01；(n=5/group).
Specific embodiment
Following non-limiting embodiments can with a person of ordinary skill in the art will more fully understand the present invention, but not with
Any mode limits the present invention.
Experimental animal used in the present invention and experiment reagent are as follows:
Experimental animal is using healthy BALB/c mouse 60 of birth 21 days, and SPF grades, (Dalian medical courses in general are big by 12~15g of weight
It learns major disease genetic engineering model animal research institute to provide).
Immunohistochemistry reagent:
Chloraldurate (auxiliary reagent factory Yangzhou Ao Xin)
Paraformaldehyde (Beijing Suo Laibao Science and Technology Ltd)
Sucrose (U.S. Biosharp)
OCT frozen section embeds liquid (U.S. SAKURA)
PBS phosphate buffer (pulvis) (Beijing Suo Laibao Science and Technology Ltd)
Immunohistochemical staining kit SP-9000 (Beijing Bioisystech Co., Ltd, Zhong Shan Golden Bridge)
Concentrated type DAB colour reagent box ZLI-9032 (Beijing Bioisystech Co., Ltd, Zhong Shan Golden Bridge)
Dehydrated alcohol (Tianjin Kermel Chemical Reagent Co., Ltd.)
Hydrochloric acid (Tianjin Kermel Chemical Reagent Co., Ltd.)
Dimethylbenzene (Tianjin Kermel Chemical Reagent Co., Ltd.)
Neutral gum (Beijing Suo Laibao Science and Technology Ltd)
Western blot reagent:
Holoprotein extracts kit KGP250/KGP2100 (Jiangsu Kai Ji Biotechnology Ltd.)
BCA protein content detection kit KGPBCA (Jiangsu Kai Ji Biotechnology Ltd.)
SDS-PAGE albumen sample-loading buffer (5X) (the green skies Bioisystech Co., Ltd in Shanghai)
30%Acr-Bis (29:1) (the green skies Bioisystech Co., Ltd in Shanghai)
Lower layer's glue buffer (4X) (the green skies Bioisystech Co., Ltd in Shanghai)
Upper layer glue buffer (4X) (the green skies Bioisystech Co., Ltd in Shanghai)
Ammonium persulfate (the green skies Bioisystech Co., Ltd in Shanghai)
TEMED (the green skies Bioisystech Co., Ltd in Shanghai)
Dehydrated alcohol (Tianjin Kermel Chemical Reagent Co., Ltd.)
PageRuler prest Protein Ladder (U.S. Thermo Scientific)
SDS-PAGE electrophoresis liquid (the green skies Bioisystech Co., Ltd in Shanghai)
Western transferring film liquid (the green skies Bioisystech Co., Ltd in Shanghai)
Western cleaning solution (10X) (the green skies Bioisystech Co., Ltd in Shanghai)
Methanol (Tianjin Kermel Chemical Reagent Co., Ltd.)
Skimmed milk power (P1622 Puli's lema gene Technology Co., Ltd.)
Bovine serum albumin(BSA) (BSA) (U.S. D009Sigma)
Special super quick ECL chemical luminescence reagent kit (the green skies Bioisystech Co., Ltd in Shanghai)
Drug and antibody:
EHNA hydrochloride E114 (U.S. Sigma)
Recombinant murine IFN-γ (U.S. Peprotech)
ADAR1 polyclonal antibody (U.S. Proteintech)
GAPDH monoclonal antibody (U.S. ImmunoWay)
Horseradish enzyme marks goat anti-rabbit igg/HRP (U.S. Arigobio)
Horseradish enzyme marks goat anti-mouse IgG/HRP (Beijing Bo Aosen Bioisystech Co., Ltd)
Other reagents:
2,4,6- trinitrophenol (picric acid) (reagent plastics Co., Ltd is resided abroad in Taizhou plain Guangdong)
Dimethyl diaminophenazine chloride (Tianjin Kermel Chemical Reagent Co., Ltd.)
The following embodiment the methods of the present invention be all made of GraphPad Prism 5.0 (San Diego, CA, USA) and
SPSS 21.0 (Aramonk, NY, USA) carries out statistical analysis.All data use mean ± standard deviation to indicate, are examined using T
It tests and detects conspicuousness with one-way analysis of variance.P < 0.05 is to have significant difference.
Embodiment 1
1. immunohistochemical experiment main solution is prepared:
(1)0.01M PBS
It takes one bag of PBS powder to be placed in beaker (specification: 1L), tri-distilled water is added and is sufficiently dissolved to 1L, then in graduated cylinder
It is settled to 2L.Room temperature preservation is spare.
(2) 4% chloraldurates
Chloraldurate crystallization 0.6g is weighed with electronic balance, tri-distilled water 10ml dissolution is added, 15ml is settled in graduated cylinder.
Room temperature is kept in dark place spare.
(3) paraformaldehyde
4% paraformaldehyde: weighing 40g paraformaldehyde powder with electronic balance and be placed in beaker (specification: 1L), is added three and steams
Water places the beaker and is heated to 55 DEG C on magnetic stirring apparatus, pays attention to ventilation, and continue stirring until paraformaldehyde and be completely dissolved,
1L is settled in graduated cylinder.Room temperature preservation is spare.
1% paraformaldehyde: measuring 4% paraformaldehyde of 200ml with graduated cylinder, and tri-distilled water is added and is settled to 800ml.Room temperature is protected
It deposits spare.
(4) sucrose
30% sucrose solution: weighing 150g sucrose with electronic balance, is placed in beaker (specification: 500ml), and 300ml is added
Tri-distilled water sufficiently dissolves, and 500ml is then settled in graduated cylinder.4 DEG C save backup.
20% sucrose solution: measuring 30% sucrose solution of 100ml with graduated cylinder, and tri-distilled water is added and is settled to 150ml.4 DEG C of guarantors
It deposits spare.
10% sucrose solution: measuring 30% sucrose solution of 50ml with graduated cylinder, and tri-distilled water is added and is settled to 150ml.4 DEG C of guarantors
It deposits spare.
2.Western blot tests main solution and prepares:
(1) 10% ammonium persulfate solution (Ammonium Persulfate, APS)
0.5g ammonium persulfate is taken, tri-distilled water is added to dissolve and is settled to 5ml, is dispensed into 0.5ml centrifuge tube, -20 DEG C of preservations
It is spare.
(2) preparation (2 plate) of SDS-PAGE running gel
5% concentration glue prepares (4ml): tri-distilled water 2.33ml, 30%Arc-Bis (29:1) 0.67ml, upper layer glue buffering
Liquid (4X) (PH6.8) 1ml, 10%APS 0.04ml, TEMED 0.004ml.
10% separation gel prepares (15ml): tri-distilled water 6.1ml, 30%Arc-Bis (29:1) 5ml, lower layer's glue buffer
(4X) (PH8.8) 3.75ml, 10%APS 0.15ml, TEMED 0.006ml.
(3) SDS-PAGE electrophoresis liquid
One bottle of pulvis that can prepare 1LSDS-PAGE electrophoresis liquid is taken, is poured into clean beaker (specification 1L), is added
Tri-distilled water is sufficiently dissolved to about 900ml.Constant volume can be used after mixing to 1L in graduated cylinder.It can room temperature preservation.
(4) Western transferring film liquid
One bottle of pulvis that can prepare 1L Western transferring film liquid is taken, is poured into clean beaker (specification 1L), is added
Tri-distilled water is sufficiently dissolved to about 700ml.200ml dehydrated alcohol or 95% ethyl alcohol of 210ml is then added, mixes.In graduated cylinder
It is settled to 1L with tri-distilled water, mixing can be used.It can room temperature preservation.
(5) Western cleaning solution (TBST)
One bottle of Western cleaning solution (5X) is taken, is poured into clean beaker (specification 1L), tri-distilled water is added and constant volume arrives
1L, mixing is Western cleaning solution (1X), can be used for the washing of Western.It can room temperature preservation.
(6) 5%BSA (confining liquid)
2.5gBSA powder is weighed, is placed in 50ml centrifuge tube, 30ml TBST is measured with graduated cylinder and is added thereto, it is sufficiently molten
Solution, is settled to 50ml.- 20 DEG C of preservations.
(7) 5% skimmed milk powers (confining liquid)
It weighs 2.5g milk powder to be placed in 50ml centrifuge tube, measures 30ml TBST with graduated cylinder and be added thereto, sufficiently dissolve, it is fixed
Hold to 50ml.Matching while using.
3. the preparation of drug:
(1)EHNA hydrochloride(0.5mg/ml)
60.0mg EHNA powder is weighed with semimicro-analy-tical balance to be placed in beaker (100ml), and physiological saline 100ml is added and fills
Divide dissolution, 120ml is settled in graduated cylinder, mixes packing into 10ml centrifuge tube.- 20 DEG C of preservations, avoid multigelation.
(2) recombinant murine IFN-γ (0.5ug/ml)
0.1g BSA is weighed, the BSA buffer of 100ml 0.1% is made into.By recombinant murine IFN-γ with palm centrifuge from
It is opened after the heart, with normal saline dilution at 1.0mg/ml, is then further diluted to 0.5ug/ml with 0.1% BSA buffer,
Packing is mixed into 10ml centrifuge tube.- 80 DEG C of preservations, avoid multigelation.
Embodiment 2
1 experimental group
The male BALB/c mouse of the health being born 21 days is chosen, single cage is raised 4 weeks, and society isolation stress stimulation mould is established
Type, the gregarious raising of the identical male BALB/c mouse of week old health is as a control group.According to random group forming criterion, it is divided into isolation group
(SI), gregarious control group (GH), pharmaceutical intervention group [ADAR1 inhibitor isolation group (SI+EHNA) and ADAR1 inducer isolation group
(SI+IFN- γ)] and pharmaceutical intervention control group (the gregarious group of ADAR1 inhibitor and the gregarious group of ADAR1 inducer).Every group 10.
Schematic diagram is prepared such as Fig. 1 experimental group and society isolation stress stimulation model.
The preparation of 2 society isolation stress stimulation models
Isolation group: the male BALB/c mouse of the health of birth 21 days (wean), after adapting to one day, single cage (29cm ×
The cm of 17.8cm × 16) raising 4 weeks.Feeding environment is suitable for temperature: 22 ± 1 DEG C, humidity: and 60%~70%, drinking-water and food fill
Foot adjusts circadian rhythm (12h:12h) with light.Gregarious control group: the male BALB/c mouse of the health of birth 21 days (wean),
After adapting to one day, raising 4 weeks of living in groups, 5 every cages (29cm × 17.8cm × 16cm).Feeding environment is suitable for temperature: 22 ± 1 DEG C,
Humidity: 60%~70%, drinking-water and food are sufficient, adjust circadian rhythm (12h:12h) with light.
3 pharmaceutical interventions
After model foundation success, ADAR1 inhibitor isolation group and the gregarious group of ADAR1 inhibitor give ADAR1 inhibitor
EHNA (10mg/kg, 20ml/kg, ip) is intervened, and ADAR1 inducer isolation group and the gregarious group of ADAR1 inducer are given
ADAR1 inducer IFN-γ (5.0 × 104U/kg, 20ml/kg, ip) intervened, the dosage of pharmaceutical intervention and administration route according to
According to seminar's preliminary result early period.Isolation group and gregarious control group then give physiological saline (20ml/kg) as pharmaceutical intervention
Control treatment carries out 8 pharmaceutical interventions in total.After the 4th gives pharmaceutical intervention, behaviouristics detection is carried out, detection time is medicine
After object intervenes 30min.
The detection of 4 behaviouristics
New object identification experiment (Object Recognition Test, ORT)
New object identification experiment is the non-space cognitive ability for test experience animal, such as the new object identification experiment of Fig. 2
Schematic diagram.Testing tool is transparent opening plastic box (40cm × 40cm × 40cm), and cabinet is separated with curtain and ambient enviroment, is put
It sets in the test room of sound insulation, keeping intensity of illumination is about 40LX, avoids generating shade.The object of identification has an A, and B, C tri-
The special cube of size identical (5cm × 5cm × 5cm), the weight of cube cannot be pushed by experiment mice, wherein A, B
Object (white) is just the same, and C object (chequered with black and white) and A, B object are entirely different.New object identification experiment includes two parts:
Sample experiment and test experiments.3 days before being explored and being tested, experiment mice 1min is stroked daily, is avoided stimulation mouse, is made
It, which is eliminated, is placed on animal in the room of test 24 hours before being explored or being tested with the jamais vu of tester, adapts to survey
Test ring border.As shown in Fig. 2, by A, two objects of B be placed on plastic box two opposite side walls centre (object and side wall at a distance of 8 centimetres,
At a distance of 14 centimetres between two objects).In sample experiment, experiment mice is put into place facing away from two articles, and mouse nose
The length of sharp two object of distance is consistent.Mouse is put into 10min, opens video recording equipment after being put into immediately, and experimenter leaves survey immediately
Room is tried, the case where mouse contacts with the two objects is recorded, touches the number of object including nose or mouth and apart from object
(fore paw, which rides on object, nose smells object, licks object etc. belonged to and probed into object, posing or climbs the time probed within the scope of 2cm
It is motionless on to object that new object cannot be probed at last).After 10min, mouse is put back to the mouse cage raised originally immediately
It is interior, (mouse still stays in test room during this) is tested again after it is rested 1 hour.In test experiments, by place
Interior B object, which changes, makees C object, will mouse backwards to two articles be put into experimental site, nose according to two articles apart from identical, such as Fig. 1
It is shown, the 5min time is observed, is equally recorded a video with video recording equipment, observer leaves test room, the same explorative experiment of observation index.It is real
In order to avoid experiment mice smell or excreta interfere with each other in testing, all with 75% after each explorative experiment or test experiments
Alcohol wipe disinfection.Mouse is sent back in original feeding room to the end of all experimentss.
The non-space that experimental animal is assessed in experiment using index of discrimination (discrimination index, DI) recognizes energy
Power, when calculation formula is that DI=(Tn-Tf)/(Tn+Tf), Tn and Tf respectively represent the exploration for exploring new and known things
Between.
Behaviouristics result
Influence of the pharmaceutical intervention to society isolation BALB/c mouse cognitive function
Fig. 3 is the new object identification experimental result that ADAR1 inhibitor and inducer intervene society isolation BALB/c mouse；Newly
(ORT) index of discrimination is tested in object identification；EHNA and IFN-γ are respectively ADAR1 inhibitor and ADAR1 inducer；Data are with equal
Number ± standard deviation indicates；*P<0.05,**P<0.01；(n=10/group).In new object identification experiment (Fig. 3, a and b), with
Gregarious control group is compared, index of discrimination (GH:0.41 ± 0.17 of isolation group mouse；SI:-0.16 ± 0.09, p < 0.01) it is obvious
It reduces；Compared with isolation group, index of discrimination (SI:-0.16 ± 0.09 of ADAR1 inducer isolation group mouse；SI+IFN-γ:
0.20 ± 0.09, p < 0.01) it is remarkably reinforced, and ADAR1 inhibitor isolation group index of discrimination (SI:-0.16 compared with isolation group
±0.09；SI+EHNA:-0.29 ± 0.12, p < 0.05) it is substantially reduced.These results prompt society isolation stress stimulation can be led
Cause BALB/c mouse non-space cognition dysfunction, cognition of the ADAR1 inducer to society isolation stress stimulation BALB/c mouse
Dysfunction has certain alleviation, and ADAR1 inhibitor can aggravate cognition dysfunction.3 morphologic detection of embodiment
1 perfusion
(1) it anaesthetizes: 4% chloraldurate, 0.01ml/g, intraperitoneal injection.Inserting needle near groin, is walked to ventrimeson direction
Row.If anaesthetic effect is bad, it can suitably increase dosage；
(2) fixed: mouse four limbs being fixed on cystosepiment with syringe needle；By cystosepiment as in pallet, wherein
Tray edge is ridden on one side, tilts cystosepiment, and perfusate is facilitated to flow out；
(3) perfusion liquid prepares: extracting 1% paraformaldehyde of 30ml, 4% paraformaldehyde of 30ml, 1% paraformaldehyde respectively
Front connector sword-shaped needle connects conventional needle before 4% paraformaldehyde, empties air, it is ensured that syringe needle is unobstructed, spare；
(4) perfusion: cutting off abdominal cavity first, then cut off diaphram, cuts off thorax, exposure heart on a small quantity.With skin pincers or haemostatic clamp
Fixed skin and thorax etc., cut off pericardium.By the apex of the heart into scalp acupuncture, it is to note that inserting needle is unsuitable too deep, otherwise can puncture heart.Into
Discovery has blood backflow to scalp acupuncture after needle, usually indicates that position is accurate, cuts off right auricle of heart at this time, fixes scalp acupuncture.Slowly
Emitter is injected, if there is dark red solution outflow in heart, inserting needle is accurate.The speed of 30ml/10min or so is advisable.First inject
1% paraformaldehyde, reinjects 4% paraformaldehyde, after the completion of the two equal perfusions of pipe liquid, mouse is placed at room temperature for 30min, makes its brain
Tissue is sufficiently fixed；
(5) it removes brain tissue: carefully removing brain tissue with haemostatic clamp, avoid damaging；
(6) fixed: brain tissue being placed in the centrifuge tube containing 4% paraformaldehyde, fixes 3 days；
(7) serial dehydration: the paraformaldehyde in the centrifuge tube equipped with brain tissue is discarded, it is molten to inject prepared sucrose
Liquid carries out serial dehydration according to 10%, 20% and 30% (twice) three concentration.Each concentration sucrose is dehydrated 1 day, to brain tissue
It thoroughly sinks to the bottom to be dehydrated completely, i.e., the sucrose of replaceable high concentration continues to be dehydrated, until dehydration is complete.
2 brain tissue frozen sections
First 3 hours debugging freezing microtome temperature of slice are extremely pre-chilled for -22 DEG C in advance.The complete Mice brain tissues of dehydration are taken,
Sucrose solution is discarded, with knife equating brain tissue bottom.Brain tissue is placed on sample carrier and guarantees its state in a vertical shape, on surface
Add appropriate OTC embedding liquid, is put into the freezing microtome of pre-cooling about 90min, after brain tissue all frost (or liquid nitrogen speed
Freeze), it is sliced.The slice thickness that this experiment uses is 16 μm.Each group slice marks, and -20 DEG C save backup.
3 immunohistochemical experiments
(1) it from -20 DEG C of taking-up frozen sections, is put into spare in wet box.Slice drying is placed on shaking table, washes 3 with PBS
It is secondary, each 5min；
(2) it takes out slice to be put into wet box, the liquid (keeping tissue in moisture state) on surface is blotted with filter paper.Use liquid relief
Hydrogen peroxide (3%H is added dropwise in device2O2Deionized water) closing endogenous peroxydase, wet box lid is covered, is protected from light, room temperature 5-
10min, it is unsuitable too long, in order to avoid rotten；
(3) it is placed on shaking table, is washed 3 times with PBS, each 5min；
(4) it takes out slice to be put into wet box, blots the liquid (keeping tissue in moisture state) on surface.Reagent A (mountain is added dropwise
Sheep blood serum working solution), cover wet box lid, 37 DEG C of incubation 30min, reclaim reagent A are not washed；
(5) diluted primary antibody [ADAR1 (1:100)] is added dropwise, has to guarantee that tissue surface and periphery are covered completely by primary antibody
Lid, 4 DEG C overnight；
(6) slide is tilted, primary antibody is recycled.It is placed on shaking table, is washed 3 times with PBS, each 5min；
(7) it takes out slice to be put into wet box, blots the liquid (keeping tissue in moisture state) on surface.It is (raw that reagent B is added dropwise
Object element secondary antibody working solution), 37 DEG C of incubation 40min incline；
(8) it is placed on shaking table, is washed 3 times with PBS, each 5min；
(9) it takes out slice to be put into wet box, blots the liquid (keeping tissue in moisture state) on surface.It is (peppery that reagent C is added dropwise
Root enzyme marks streptomysin albumen working solution), cover wet box lid, 37 DEG C of incubation 40min；
(10) it is placed on shaking table, is washed 3 times with PBS, each 5min；
(11) DAB colour developing is carried out, DAB is ready-to-use, uses, is kept in dark place in 30min.DAB developing solution, Jing Xiaguan is added dropwise
It examines, terminates (PBS buffer solution is dipped in) in due course, terminated in general 8min-15min；
(12) carry out Gradient elution using ethanol, respectively by slice develop the color be dipped in gradient for 70%, 80%, 90%, 95%,
100%, in 100% ethyl alcohol, each 3min.It takes out, dimethylbenzene is transparent twice, each 5min；
(13) neutral gum mounting is added dropwise rapidly after taking out in dimethylbenzene and (prevents from organizing after doing dry and cracked for mounting, slice；If
Mounting is ineffective, can put back in dimethylbenzene mounting again after dissolving).Bubble is careful not to when mounting, natural gum uses appropriate.
Naturally dry, room temperature preservation；
(14) it staining conditions and is taken pictures using just setting optical electron microscope observation, with Image-Pro Plus 6.0 to exempting from
Epidemic disease group picture brain area carries out photodensitometry.
Immunohistochemical experiment result
The influence that pharmaceutical intervention expresses society isolation BALB/c mouse prefrontal cortex ADAR1
Fig. 4 is the ImmunohistochemistryResults Results (a) that the society isolation BALB/c mouse prefrontal cortex ADAR1 of pharmaceutical intervention is expressed
The ImmunohistochemistryResults Results of prefrontal cortex ADAR1 expression；(b) the brain area schematic diagram of prefrontal cortex；(c) prefrontal cortex
The OD value statistical result of the positive neuron of ADAR1 expression.EHNA and IFN-γ are respectively ADAR1 inhibitor and ADAR1
Inducer；Data are indicated with mean ± standard deviation；**P<0.01；(n=5/group).
In the immunohistochemical experiment of prefrontal cortex ADAR1 expression (such as Fig. 4), compared with gregarious control group, isolation group
The positive neuron OD value of the prefrontal cortex ADAR1 expression of mouse (GH:0.023 ± 0.003, SI:0.008 ±
0.001；P < 0.01) it is substantially reduced；Compared with isolation group, the prefrontal cortex ADAR1 of inducer isolation group mouse expresses positive
Neuron OD value (SI:0.008 ± 0.001, SI+IFN- γ: 0.015 ± 0.003；P < 0.01) obviously increase, and press down
Preparation isolation group no significant difference.OD value statistical result such as (Fig. 4, c).
Fig. 5 is the ImmunohistochemistryResults Results that the society isolation BALB/c mouse hippocampus ADAR1 of pharmaceutical intervention is expressed；Hippocampus
The ImmunohistochemistryResults Results of the area CA1 ADAR1 expression；(b) ImmunohistochemistryResults Results of the area hippocampus Hilus ADAR1 expression；(c) hippocampus
The brain area schematic diagram in the area CA1 and Hilus；(d) the OD value statistical result of the positive neuron of Hippocampal CA 1 ADAR1 expression；
(e) the OD value statistical result of the positive neuron of the area hippocampus Hilus ADAR1 expression.EHNA and IFN-γ are respectively ADAR1
Inhibitor and ADAR1 inducer；Data are indicated with mean ± standard deviation；**P<0.01；(n=5/group).
The influence that pharmaceutical intervention expresses society isolation BALB/c mouse hippocampus ADAR1
In the immunohistochemical experiment of hippocampus ADAR1 expression (such as Fig. 5), compared with gregarious control group, isolation group mouse
Hippocampus CA1 and positive neuron OD value (CA1, GH:0.047 ± 0.004, SI:0.021 of the area Hilus ADAR1 expression
± 0.005；p<0.01；Hilus,GH:0.021±0.002,SI:0.013±0.003；P < 0.01) it is substantially reduced；Be isolated
Group is compared, the positive neuron OD value of hippocampus CA1 and the area Hilus the ADAR1 expression of inducer isolation group mouse (CA1,
SI:0.021 ±0.005,SI+IFN-γ:0.040±0.005；p<0.01；Hilus,SI:0.013±0.003,SI+IFN-
γ:0.023±0.004；P < 0.01) obviously increase, and inhibitor isolation group no significant difference.OD value statistical result is such as
(Fig. 5, d and e).
4 Western blot of embodiment detection
The extraction of 1 mouse brain partitioned organization holoprotein
(1) holoprotein extracts kit is used, 10 μ l inhibitors of phosphatases, 1 μ l is added in every cold Lysis Buffer of 1ml
Protease inhibitors and 10 μ l 100mM PMSF are mixed.Several minutes of preservation on ice spare；
(2) the brain partitioned organization that will have been taken is placed in pre-cooling centrifuge tube, and it is cold that 0.5ml is added according to every 100mg solid tissue
Cold Lysis Buffer is added in the standard of Lysis Buffer.With homogenate 30~50 times up and down manually of high-speed homogenization machine, pay attention to low
Temperature operation；
(3) centrifuge is opened, 4 DEG C of pre-coolings are adjusted the temperature to.Tissue homogenate is transferred to the centrifuge tube of 1.5ml pre-cooling
In, it is centrifuged according to 12000g, 4 DEG C of centrifugation 5min；
(4) supernatant is taken to be transferred in the centrifuge tube of new pre-cooling, as holoprotein extract；
(5) sample packing be stored in -80 DEG C of refrigerators (volume of Care Mark protein sample, it is slow plus sample convenient for the later period
Fliud flushing), avoid multigelation.
The measurement of 2 protein concentrations
(1) it prepares BCA working solution: according to sample size, adding 1 volume BCA reagent B (50:1) by 50 volume BCA reagent As
Suitable BCA reagent is configured, mixes well and is placed in ice chest for use；
(2) Protein standards are sequentially added to the standard sample wells of 96 orifice plates by the sequence of 0,1,2,4,8,12,16,20 μ l
In, add deionized water to supply to 20 μ l, then plus 200 μ l BCA working solutions；
(3) testing protein sample is taken, suitable concentration is diluted to；
(4) protein sample that 20 μ l have diluted is added in each hole, adds prepared 200 μ l of BCA working solution.Enzyme mark version
It is placed on concussion instrument vibration 30sec, mixes well, is placed on 30min in 37 DEG C of incubators；
(5) colorimetric estimation under 562nm wavelength.With protein content (ug) for abscissa, light absorption value is ordinate, draws mark
Directrix curve；
(6) according to the light absorption value of institute's sample, corresponding protein content (ug) can be calculated to obtain on standard curve, divided by sample
Product dilution total volume (20uL) is sample actual concentrations (unit: ug/uL) multiplied by sample extension rate.
3 SDS- polyacrylamide gel electrophoresis
(1) determine that resolving gel concentration is 8% according to detected molecular weight of albumen, concentration gum concentration is 5%；
(2) clean glass plate: glass plate is rinsed with tap water, then after being rinsed well with tri-distilled water, is placed on glue frame and is dried；
(3) it encapsulating: is poured into tri-distilled water wait 10min first, it is determined whether leak.Separation is first prepared according to gum concentration
Glue is slowly added to dehydrated alcohol until glass plate, drives bubble away to comb bottom end 1cm or so after pouring into insertion comb.
It is placed at room temperature for 40min, after being gelled admittedly, dehydrated alcohol is poured out.Concentration glue is prepared, is mixed, implantation glass plate, until glass plate top
End, careful insertion comb, avoids generating bubble, is placed at room temperature for 40min rapidly, polymerize separation gel and concentration glue sufficiently；
(4) prepare protein sample: by 6 × SDS is added according to protein sample volume in the albumen taking-up of concentration after measured
Sample-loading buffer to final concentration of 1 ×, be put into water-bath, 96 DEG C~98 DEG C heating 5min make albuminous degeneration, pay attention to preventing quick-fried
Pipe；
(5) electrophoresis: prepared glue is placed in electrophoresis tank, and the sufficient electrophoresis liquid of injection is loaded to not crossing inside glue
The conductor wire that electrophoresis liquid there was not bottom is also added in hole, periphery, guarantees to be powered.It is vertical to extract comb, remove the bubble in loading hole
And with indenting ball righting.With the adherent addition protein sample of micropipettor, albumen applied sample amount is 30 μ g.It is inserted into electrophoresis apparatus power supply,
Modulation voltage is constant 80V, and voltage is adjusted to constant 120V after 15min, until bromophenol blue moves to separation gel bottom, closes electricity
Gel is taken out in source；
(6) transferring film: gel is cut in the container for being put into and filling transferring film liquid with glue device is cut, and according to the big of cut gel
It is small to cut pvdf membrane of corresponding size, it is placed in anhydrous methanol and impregnates 30sec activation, place into transferring film liquid and wash for use.
Transferring film folder, sponge, filter paper are put into the disk added with transferring film liquid.Downward by the part of transferring film folder black, sponge, 3 are sequentially placed
Layer filter paper, gel, pvdf membrane, 3 layers of filter paper, sponge, every step are all gently pressed through above with cutting glue device, drive bubble away.By transferring film
It folds up in transferring film slot, makes the black flour of folder to the black flour of slot, the red face of the fine flour of folder to slot.Use constant current 300mA transferring film
90min.Electrophoresis tank need to be placed in the basin for being placed with sufficient mixture of ice and water when transferring film, avoid heat production from destroying transferring film, become band
Shape；
(7) it closes: pvdf membrane being taken out from clip rapidly after to transferring film, is face-up put into the appearance equipped with TBST
Washing, is then transferred in the incubation box of prepared 5% skimmed milk power (or 5%BSA), is placed on shaking table in device, room temperature envelope
Close 2h.Notice that skimmed milk power is ready-to-use, BSA recoverable 2-3 times, -20 DEG C of preservations；
(8) primary antibody is incubated for: being outwelled skimmed milk power (recycling BSA), is washed film 3 times with TBST, each 10min.Be separately added by
The prepared primary antibody of ratio [ADAR1 (1:1000) and GAPDH (1:5000)], 4 DEG C of shaking tables are stayed overnight；
(9) secondary antibody is incubated for: recycling primary antibody.TBST washes film 3 times, each 10min.It is incubated for prepared secondary antibody [goat antirabbit
IgG/HRP (1:5000), goat anti-mouse IgG/HRP (1:5000)], after being incubated for 20min on shaking table, move to 37 DEG C of insulating boxs
Middle incubation 30min, is incubated for 10min on shaking table again later；
(10) chemiluminescence and development: recycling secondary antibody.TBST washes film 3 times, each 10min.It is prepared according to the ratio of 1:1 aobvious
Shadow liquid is added drop-wise on pvdf membrane with pipettor and is developed and (be protected from light), is imaged using gel imager；
(11) gray analysis is carried out with Image Lab 3.0.
Western Blot result
Influence of the pharmaceutical intervention to society isolation BALB/c mouse intracerebral ADAR1 protein expression
In the western blot knot of the society isolation BALB/c mouse prefrontal cortex ADAR1 protein expression of pharmaceutical intervention
In fruit (such as Fig. 6, a), compared with gregarious control group, the expression of isolation group mouse prefrontal cortex ADAR1 albumen (ADAR1, GH:
1.00±0.00；SI:0.44 ± 0.08, p < 0.01) it is substantially reduced；Compared with isolation group, inducer isolation group mouse forehead
Expression (ADAR1, SI:0.44 ± 0.08 of leaf skin layer ADAR1 albumen；SI+IFN- γ: 0.84 ± 0.05, p < 0.01) it is obvious
Increase, and inhibitor isolation group then no significant difference.Gray value statistical result see (Fig. 6, a).
Fig. 6 is the western blot result of the society isolation BALB/c mouse intracerebral ADAR1 protein expression of pharmaceutical intervention；
The western blot result and gray analysis statistical result of prefrontal cortex ADAR1 albumen；(b) society isolation of pharmaceutical intervention
The western blot result and gray analysis statistical result of BALB/c mouse hippocampus ADAR1 protein expression.Statistical result is
The gray level ratio result of destination protein and internal reference；EHNA and IFN-γ are respectively ADAR1 inhibitor and ADAR1 inducer；Data
It is indicated with mean ± standard deviation；**P<0.01；(n=5/group).
In the western blot result of the society isolation BALB/c mouse horse sea area ADAR1 protein expression of pharmaceutical intervention
(such as Fig. 6, b), compared with gregarious control group, the expression of isolation group mouse horse sea area ADAR1 albumen (ADAR1, GH:1.00 ±
0.00；SI:0.48 ± 0.07, p < 0.01) significantly reduce；Compared with isolation group, inducer isolation group mouse horse sea area
Expression (ADAR1, SI:0.48 ± 0.07 of ADAR1 albumen；SI+IFN- γ: 0.82 ± 0.04, p < 0.01) obviously increase.Ash
Angle value statistical result is shown in (Fig. 6, b).
For any person skilled in the art, without departing from the scope of the technical proposal of the invention, all
Many possible changes and modifications are made to technical solution of the present invention using the technology contents of the disclosure above, or are revised as equivalent
The equivalent embodiment of variation.Therefore, anything that does not depart from the technical scheme of the invention, according to the technical essence of the invention to
Any simple modifications, equivalents, and modifications that upper embodiment is done should all still fall within the range of technical solution of the present invention protection
It is interior.
Claims (2)
- Application of the 1.ADAR1 as intervention target spot in terms of cognition dysfunction drug is treated in preparation, which is characterized in that described Using the application for ADAR1 inducer in terms of cognition dysfunction drug is treated in preparation, the cognition dysfunction drug is Non-space cognition dysfunction drug, the ADAR1 inducer is IFN-γ, and the application is to use ADAR1 inducer Patient's intracerebral ADAR1 is promoted to be restored to normal level in human brain, the application promotes to utilize the ADAR1 inducer The expression of the mRNA and albumen of the ADAR1 of intracerebral, or promote the active increase of ADAR1, the application is described to utilize ADAR1 inducer promotes the expression of the mRNA and albumen of the ADAR1 of intracerebral prefrontal cortex and hippocampus, or promotes ADAR1 Active increase.
- 2. application according to claim 1, it is characterised in that: the application includes ADAR1 in preparation treatment cognition function Application in terms of energy disorder remedies.
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| WO2016200690A1 (en) * | 2015-06-08 | 2016-12-15 | The Regents Of The University Of California | USE OF H3K9me3 MODULATION FOR ENHANCING COGNITIVE FUNCTION |
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| Title |
|---|
| 5-HT2CR antagonist/5-HT2CR inverse agonist recovered the increased isolation-induced aggressive behavior of BALB/c mice mediated by ADAR1 (p110) expression and Htr2c RNA editing;Yu Weizhi et al;《BRAIN AND BEHAVIOR》;20180207;第8卷(第3期);第1-13页 * |
| Antiviral Actions of Interferons;Samuel et al;《Clinical Microbiology Reviews》;20011031;第14卷(第4期);第789页左栏第1段 * |
| Effects of social isolation and re-socialization on cognition and ADAR1 (p110) expression in mice;Wei Chen et al;《Peer J》;20160818;第4卷;第1-18页 * |
| Effects of social isolation, re-socialization and age on cognitive and aggressive behaviors of Kunming mice and BALB/c mice;An Dong et al;《Animal Science Journal 》;20170531;第88卷(第5期);第798-806页 * |
| 社会隔离对BABL/c小鼠认知功能和海马5-HT2CR、ADAR1表达的影响;徐红，等;《中华行为医学与脑科学杂志》;20170531;第26卷(第5期);摘要、第387页表1、第388页右栏 * |
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