CN106636330B - The diagnosis of Alzheimer disease and medicine - Google Patents
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Abstract
Disclosed herein is a kind of diagnosis for being related to Alzheimer disease, treatment method and corresponding kit.Specifically, present invention discover that the expression of TREM1 albumen directly affect phagocytosis Scavenging activity of the microglia to amyloid beta and and then cause amyloid beta normally to be removed, the diagnosis and therapy target that it can be as Alzheimer disease.
Description
Technical field
The invention belongs to neuroscience field.A kind of in particular it relates to Alzheimer disease (Alzheimer ' s
Disease, AD) diagnosis and therapy target, and corresponding diagnosis, treatment method and related kit.
Background technology
AD is clinically most common nervous system degenerative disease, its memory being in progress with progressive, visual space and is held
Row dysfunction is typical clinical manifestations[1].In recent years, as the quickening of aging degree, China AD patient populations significantly increase
Add, thus bring very white elephant to patient, family and society[2].Up to now, the cause of disease is still not clear,
And without effective treatment means.
According to having studied, one of AD major pathologic features be intracerebral accumulate a large amount of amyloid betas (Amyloid- β,
A β), and then a series of neurotoxicities are induced, ultimately result in cognition dysfunction[3].Therefore, A β are suitable one at this stage
Divide the AD main research target spots for the treatment of.A large amount of evidences show that brain tissue declines to A β Scavenging activity, is to cause course of disease early stage A β
The main reason for accumulation[4].Newly enter evidence prompting, after onset early stage removes the A β of AD model mice intracerebrals, the cognition work(of mouse
It can recover, prompt that the progress of the AD courses of disease can be delayed for the early stage application of A β therapies[5]。
However, the shortage of AD early diagnosis means is the maximum obstruction using such therapy.At present, carry out in AD patient
Brain Biopsy is more difficult, and diagnoses AD, its sensitiveness and specificity by medical history and scale Evaluation Method and extremely limited.Cause
This, verifies a kind of more easy-operating, and specificity and the higher AD diagnostic methods of sensitiveness also turn into the task of top priority.
Summary of the invention
It is an object of the invention to provide a kind of method for early diagnosing and treating AD.
Specifically, present invention firstly discovers that TREM1 albumen is an important ring in AD pathogenesis, it is thin in monokaryon-macrophage
Expression in born of the same parents' system, which is lowered, directly affects Scavenging activity of the system to A β, so as to cause A β to be accumulated in intracerebral.One
In individual embodiment, the invention provides a kind of early diagnosis AD method, and its key point is in detection TREM1 outside Zhou Danhe
Expression.In a specific embodiment, TREM1 gene transcription levels are detected by qRT-PCR to assess TREM1
Expression.In another specific embodiment, directly detected by ELISA, Western blotting or ImmunohistochemistryMethods Methods
TREM1 protein expression levels.
In addition, in another embodiment, the invention provides a kind of method for treating AD, its key point is up-regulation
TREM1 is horizontal or activates TREM1 paths to recover phagocytosis energy of the intracerebral monocytes/macrophages system (i.e. microglia) to A β
Power.In a specific embodiment, by being transferred to exogenous TREM1 genes to intracerebral microglia to improve its expression
It is horizontal.Its carrier can be plasmid, slow virus or other feasible nucleic acids.In another specific embodiment, to
The transcriptional control material of TREM1 gene expression doses can be improved by being introduced in body.In another specific embodiment, to
Intracerebral introduces predetermined substance with targeted activation TREM1 paths, the predetermined substance can be agonistic antibody and its active fragment,
The activator of Agonistic ligands or passage downstream molecule.
Brief description of the drawings
Fig. 1 .Trem1 can promote phagocytosis of the microglia of WT children mouse to A β.To understand fully under physiological condition
Effects of the TREM1 in microglia phagocytosis A β, we are isolated primary using the manipulation of slow virus mechanism from WT children mouse
Trem1 expression in microglia.After the 72h that transduces, it is utilized respectively qRT-PCR and elisa technique confirms Trem1
MRNA expression (a) and the change of Trem1 protein levels (b).Afterwards, the isolated test provided using Griciuc et al.
Method detects microglia to A β1-42Phagocytosis and degradation.(c) mouse primary microglia is placed in 5 μM of A β1-42
Cultivate 6h.A β using elisa technique to internalization1-42Quantity is detected (t=0h).(d) then, will be remaining in culture medium
Aβ1-42Clear out, primary microglia is further cultivated into 6h to cause A β1-42Fully degraded.Utilize elisa technique pair
Intracellular A β1-42Level is measured (t=6h).(e) by A β1-42Surplus (t=6h) and total A β1-42Internalization amount (t=0h)
Ratio be considered as A β1-42Degradation index.Degradation index is higher, represents degraded A β1-42Ability it is lower.Utilize single factor test variance point
Analysis and Tukey ' s post hoc, which examine, to be analyzed data.Block diagram represents average ± standard deviation (n=4, a formula three
Part), * P ＜ 0.05.
Fig. 2 .Trem1 can promote phagocytosis of the microglia of adult APP/PSEN1 mouse to A β.In order to enter one
Walk confirms adjustment effects of the TREM1 to A β phagocytosis under the related backgrounds of AD, and we manipulated for 7 monthly ages by slow virus mechanism
Trem1 expression in APP/PSEN1 mouse microglias.After the 72h that transduces, qRT-PCR and elisa technique card are utilized respectively
The real change of Trem1 mRNA expression (a) and Trem1 protein levels (b).Afterwards, provided using Griciuc et al.
Isolated test method detect microglia to A β1-42Phagocytosis and degradation.(c) mouse primary microglia is put
In 5 μM of A β1-42Cultivate 6h.A β using elisa technique to internalization1-42Quantity is detected (t=0h).(d) then, will cultivate
Remaining A β in base1-42Clear out, primary microglia is further cultivated into 6h to cause A β1-42Fully degraded.Utilize
Elisa technique is to intracellular A β1-42Level is measured (t=6h).(e) by A β1-42Surplus (t=6h) and total A β1-42It is interior
The ratio of change amount (t=0h) is considered as A β1-42Degradation index.Degradation index is higher, represents degraded A β1-42Ability it is lower.Utilize list
Analysis of variance and Tukey ' s post hoc, which examine, to be analyzed data.Block diagram represents average ± standard deviation (n=
4, triplicate), * P ＜ 0.05.
Fig. 3, which knock out Trem1, can promote A β europathologies in APP/PSEN1 mouse to occur.The slow of Trem1shRNA will be contained
Virus or its control group slow virus inject the bilateral cerebral of 7 monthly age APP/PSEN1 mouse and the WT mouse to match with its age
In cortex and hippocampus.(a) after injecting 2 months, confirmed by qRT-PCR in APP/PSEN1 mouse brains in microglia
The change that Trem1mRNA expression occurs.(b) after injecting 2 months, confirmed by ELISA small in APP/PSEN1 mouse brains
The change that the expression of Trem1 albumen occurs in spongiocyte.(c) represent and receive carrier, control group is viral or contains
The amyloid plaques photo of the APP/PSEN1 Cerebral Cortexes of Trem1shRNA slow virus injection.According to immuno-chemical method,
Amyloid plaques are detected using anti-amyloid beta antibodies.In × 200 micro- Microscopic observation these patches.Engineer's scale=200 μm.(d) noting
Penetrate after 2 months, utilize the A β of soluble in ELISA method detection APP/PSEN1 Cerebral Cortexes and insoluble form1-42。
(e) after injection 2 months, soluble in ELISA method detection APP/PSEN1 Hippocampus of Mice and insoluble form is utilized
Aβ1-42.Examined by one-way analysis of variance and Tukey ' s post hoc and data are analyzed.Block diagram represents average
± standard deviation.Every group of n=12, * P ＜ 0.05.
Fig. 4, which are overexpressed Trem1, can weaken A β Pathological levels in APP/PSEN1 mouse.The slow of Trem1 cDNA will be contained
Virus or its control group slow virus inject the bilateral cerebral of 7 monthly age APP/PSEN1 mouse and the WT mouse to match with its age
In cortex and hippocampus.(a) after injecting 2 months, confirmed by qRT-PCR in APP/PSEN1 mouse brains in microglia
The change that Trem1mRNA expression occurs.(b) after injecting 2 months, confirmed by ELISA small in APP/PSEN1 mouse brains
The change that the expression of Trem1 albumen occurs in spongiocyte.(c) represent and receive carrier, control group is viral or contains Trem1
The amyloid plaques photo of the APP/PSEN1 Cerebral Cortexes of shRNA slow virus injection.According to immuno-chemical method, use
Anti-amyloid beta antibodies detect amyloid plaques.In × 200 micro- Microscopic observation these patches.Engineer's scale=200 μm.(d) in injection 2
After month, the A β of soluble in ELISA method detection APP/PSEN1 Cerebral Cortexes and insoluble form are utilized1-42.(e) exist
After injection 2 months, the A of soluble in ELISA method detection APP/PSEN1 Hippocampus of Mice and insoluble form is utilized
β1-42.Examined using one-way analysis of variance and Tukey ' s post hoc and data are analyzed.Block diagram represents average
± standard deviation.Every group of n=12, * P ＜ 0.05.
Fig. 5 expression Trem1 can save the spatial cognition infringement of the AD correlations of APP/PSEN1 mouse.Trem1 will be contained
CDNA slow virus or its control group slow virus inject 7 monthly age APP/PSEN1 mouse and the WT mouse to match with its age
In bilateral cerebral cortex and hippocampus.After slow virus is injected 2 months, it is overexpressed using Y- maze tests detection Trem1 short to mouse
The influence of phase spatial memory.In exposure period, mouse is placed on " starting " arm end, and allow it freely to be explored in 5 minutes
First arm section (being defined as " originating " arm) in Y- labyrinths and the second arm section (being defined as " other " arm).The stage, we will
Towards the entrance Basilar artery of the arm section of labyrinth the 3rd (being defined as " new " arm).We are calculated relative to " starting " arm, mouse
The time (a) spent in " other " arm region.Using single-sample t-test (compared with 50% chance, with red dotted line table
Show) or one-way analysis of variance and Tukey ' s post hoc examine (group difference) to analyze data.Afterwards, mouse quilt
It is transferred to from labyrinth in the cage of its inhabitation and treats upper 2min.In experimental stage, mouse is placed again into " starting " arm in labyrinth
It is interior.Meanwhile " new " arm entrance is opened, and mouse is allowed freely to explore 1min in labyrinth.(b) for experimental period, Wo Menji
An index of discrimination=[new arm time/(new arm time+other arms time)] × 100 are calculated.Using one-way analysis of variance and
Tukey ' s post hoc are examined and data are analyzed.Block diagram represents average ± standard deviation, * P ＜ 0.05.It is small putting to death
Last 5 days before mouse, the spatial cognition function of mouse is measured by Morris water maze tests.(c) represent 5 days and test
The swimming rate of middle each group.Examined using one-way analysis of variance and Tukey ' s post hoc and data are analyzed.(d) generation
Path length of the table each group in water maze test.Using two factor duplicate measurements variance analyses and Bonferroni Multiple range tests
Inspection is analyzed path length data.# is relative to control group WT mouse, P ＜ 0.05；* it is viral relative to control group is received
The APP/PSEN1 mouse of injection, P ＜ 0.05.(e) appearance form of exploratory experiment the data obtained is：Relative at other three
The time spent in quadrant (A.O.), mouse is in the time scale spent in target quadrant (Q1).Using independent samples t test
Data are analyzed.* relative to the time spent in other three quadrants (A.O.), P ＜ 0.05.Column diagram represents average
Number ± standard deviation.Every group of n=24.
Fig. 6 excitability Trem1 antibody activation Trem1 signal pathways can weaken in APP/PSEN1 mouse brains A β pathology and
Save the infringement of its spatial cognition.In order to further confirm protective effects of the TREM1 in AD progressive stages, we use osmotic minipumps
Pump continues 6 circumferential 7 monthly age APP/PSEN1 mouse brain diacele dorsal parts and pours into a kind of excitability Trem1 antibody to activate
Trem1 signal pathways.Afterwards, we determine influence of the activation of Trem1 signal pathways to A β pathology.(a) ELISA method is utilized
Detect soluble in APP/PSEN1 Cerebral Cortexes and insoluble form A β1-42.Using one-way analysis of variance and
Tukey ' s post hoc are examined and data are analyzed, every group of n=12 of P ＜ 0.05..(b) ELISA method detection APP/ is utilized
Soluble and insoluble form A β in PSEN1 Hippocampus of Mice1-42.Using one-way analysis of variance and Tukey ' s post
Hoc is examined and data is analyzed, every group of n=12 of P ＜ 0.05..Then, the short-term sky of mouse is assessed using Y- maze tests
Between memory function.(c) for experimental period, we calculate an index of discrimination=[new arm time/(during new arm time+other arms
Between)] × 100.Using single-sample t-test (compared with 50% chance, being represented with red dotted line) or one-way analysis of variance and
Tukey ' s post hoc examine (group difference) to analyze data, every group of n=24.(d) for experimental period, we calculate
One index of discrimination=[new arm time/(new arm time+other arms time)] × 100.Using one-way analysis of variance and
Tukey ' s post hoc are examined and data are analyzed.Block diagram represents average ± standard deviation, * P ＜ 0.05, every group of n=
24.Just put to death last 5 days before mouse, the spatial cognition function of mouse is measured by Morris water maze tests.(e)
Represent the swimming rate of each group in test in 5 days.Examine to enter data using one-way analysis of variance and Tukey ' s post hoc
Row analysis, every group of n=24.(f) path length of each group in water maze test is represented.Using two factor duplicate measurements variances point
Analysis and Bonferroni multiple comparative tests are analyzed path length data.# is relative to control group WT mouse, P ＜
0.05；* relative to the APP/PSEN1 mouse for receiving control group virus injection, P ＜ 0.05, every group of n=24.(g) exploratory experiment institute
The appearance forms of data is：Relative to the time spent in other three quadrants (A.O.), mouse is at target quadrant (Q1)
Interior spent time scale.Data are analyzed using independent samples t test.* relative in other three quadrants
(A.O.) time spent in, P ＜ 0.05.Column diagram represents average ± standard deviation.Every group of n=24.
Embodiment
Signified TREM1 in the present invention, English full name are triggering receptor expressed on
Myeloid cells-1, i.e. 1 type myeloid cell trigger acceptor, be a kind of acceptor for being expressed in mononuclear macrophage surface, its with
Receptor tyrosine protein kinase is coupled, and then regulates and controls immune response.
The present invention is found that the TREM1 on microglia film surface swallows A β key rush for microglia first
Enter effect.Downward TREM1 expression directly results in microglia phagocytic activity decline, even in some A β formation speed simultaneously
Under the physiology situation do not accelerated, still A β can not be removed so that the latter largely accumulates in central nervous system, and then produces god
Through toxic action, Alzheimer disease (AD) occurs to a certain extent for progress.Although the cause of disease of Alzheimer disease is very multiple
It is miscellaneous, it is difficult to single reason or inducement it is general in it, but if detection monocyte in TREM1 expression quantity significantly lower, this
The possible Ahl tribulus sea silent sickness of one situation has very strong association.
Meanwhile the present invention is, it was also found that the situation accumulated for A β, raises TREM1 expression quantity or activate its path, energy
Microglia phagocytosis A β ability is enough significantly increased, accelerates to remove the A β of accumulation, eliminates its toxic action to nervous system,
And the symptom occurred may be improved.Therefore, new discovery of the present invention TREM1 be Alzheimer disease diagnosis and
The target spot for the treatment of.
Signified detection TREM1 of the invention expression quantity, is referred to according to detection technique commonly used in the art, including but not
The methods of being limited to qRT-PCR, ELISA, Western blotting, immunohistochemistry etc., detect TREM1 mRNA and/or the table of albumen
Up to level, its specific primer/probe/antibody/conjugate etc. can be designed, designed or commercialization.
Signified up-regulation TREM1 of the invention expression quantity, is referred to by being transferred to TREM1 gene sheets to mononuclear macrophage
Body, or its upstream strong promoter is transferred to, or corresponding transcription factor, or the factor for suppressing its expression is eliminated, or other can reach
To similar effect, known other means or scheme, the increased effect of TREM1 developed by molecule quantity is actually reached.At one
In specific embodiment, by being realized to live body intracerebral injection TREM1- carrier granulars, described carrier is preferably virus and carried
Body, more preferably slow virus carrier.
Signified activation TREM1 paths of the invention, refer to living by Agonistic ligands, agonistic antibody or model molecule
Change TREM1, and and then activation downstream signal transduction.Signified activation TREM1 paths of the invention, also including well known in the art
, direct activation TREM1 downstream molecules, such as DAP12 etc..
1 experiment material of the present invention of embodiment and method
Human subjects
This research follows Declaration of Helsinki and passed through through the examination ＆ approval of Ethics Committee of Nanjing Medical University.Research object source
In Nanjing No.1 Hospital is on-job and retired employee in healthy young people and the elderly, all participation objects be China of Han nationality
People, and each other without any genetic connection.Each participant endorsed written consent book.
The purifying and culture of monocyte in human circulation blood
The method described according to Zondler and its colleague[6], the isolated monocyte from human vein whole blood.Greatly
Body process is：Use(Sigma-Aldrich companies) instrument, using density gradient centrifugation and CD-14 positive magnetic
Pearl isolation technics (Miltenyi Biotec companies) realizes that cell separates.Adding 10% hyclone (FBS) and 1% disk Buddhist nun
The settling flux of cell is carried out in the culture mediums of RPMI 1640 in XiLin/streptomysin (P/S).Controlled by immunocytochemistry
The purity of isolated monocyte.Then, by monocyte with 1 × 106Cell/mL density is transferred in naked 96 orifice plate,
24 hours are placed in case functional analysis.
Animal
Applicant from Nanjing Medical University's Experimental Animal Center obtain male APP/PSEN1 transgenic mices and with its year
Wild type (WT) mouse that age matches.Whole research approach is ratified through Nanjing Medical University's animal care and administration committee.
All experiments that this research institute is carried out are observed NIH experimental animal and shown loving care for and instruction opinion.
The separation of mouse microglia
The method the previously described according to applicant[7], small colloid is isolated from wild type children's murine brain in 1 day postpartum
Cell.The purity of microglia is confirmed using immunocytochemistry method.
Applicant is to Bliederhaeuser and its method for colleague's description[8]After being finely tuned, using it from APP/
Microglia is isolated in PSEN1 adult mices and wild-type mice brain tissue.General procedure is：Mouse is by deep anaesthesia
Afterwards, the Ringer solution of ice is poured into through orifice of the stomach.Mouse brain is shredded with scalpel, in 37 DEG C of temperature and 5%CO2The bar of concentration
Under part, brain tissue is placed in the enzyme solutions containing papain and is incubated 60min.Then Hanks of the addition containing 20%FBS is put down
The salting liquid (HBSS) that weighs carries out acid-base neutralization to enzyme solutions, and centrifuges 4min under 200g (rotating speed).Toward the graininess being centrifuged out
The HBSS solution for adding 2ml enzymes containing 0.5mg/mlDNA in thing again realizes settling flux, and continues to be incubated 5min at room temperature.Profit
Light damage is carried out to brain tissue with the pasteur pipet polished through fire, then filters (Thermo Fisher through 70 μm of cell sieves
Scientific companies), and centrifuge 4min under 200g (rotating speed).Then resulting granular substance is contained 20% in 20mlSettling flux is realized in the HBBS isotonic solution of (Sigma-Aldrich companies).Carefully pure HBSS (20ml) is put
In the top of percoll layers, then 20min is centrifuged under 200g (rotating speed).Abandon the boundary containing myelin and cell fragment
Layer, will containing mixing spongiocyte group granular material be eluted with HBSS, and contain 10%FBS, 1%P/S, 1 ×
Acetonate, 1 × GlutaMAXTM (Thermo Fisher Scientific companies) and the carrier-free mouse of 5ng/mL
Recombinate in the Du Shi improved culture mediums of granulocyte and macrophage colony stimulatory factor (GM-CSF, R＆D systems companies) again
Suspend.In 37 DEG C of temperature, 5%CO2Under concentration conditions, cell suspending liquid is transferred to 15cm2More poly-1-lysine clad plates
Upper culture.A matrix was changed every 3 days until whole bottom surface is covered by one layer of cells.After the formation of spongiocyte layer, small glue
Cell plastid can form the suspension cell layer of one layer of (with bottom) adhesion.Applicant relatively easily can receive to these cells
Collection, transfer and further culture.After having collected suspension cell layer, microglia is placed under conditions of no GM-CSF and is incubated 3
My god, then carry out transduce operation or functional analysis.The purity of collected microglia is entered using immunocytochemistry
Row confirms.
Aβ1-42Phagocytosis and Degrading experiment
The isolated test method provided using Griciuc et al. is to monocyte or microglia to A β1-42Phagocytosis
It is measured with degradation capability[9].By person monocytic cell or mouse microglia and 5 μM of A β1-42It is common to be incubated 6h, utilize
EUSA (ELISA) measure internalization A β1-42Quantity, to assess cell to A β1-42Phagocytosis.First by people
The microglia of monocyte or mouse and 5 μM of A β1-42It is common to be incubated 6h, then cell is eluted down with fresh medium
To be placed in no A β1-42Serum in continue cultivate 6h, to determine cell to A β1-42Degradation capability.Afterwards, ELISA method is utilized
Determine intracellular A β1-42Residue it is horizontal.With A β1-42The ratio of surplus and internalization total amount represent cell to A β1-42Degraded
Ability.Ratio is higher to mean cell to A β1-42Degradation capability it is lower.
The preparation of lentiviral particle
In order that microglia is optionally overexpressed Trem1 albumen, applicant utilizes existing method[10], construct
Can be with encoding murine WT Trem1 cDNA (NM_021406.5) slow virus carrier under myeloid differentiation CD11b promoter regulations
And control group virus.In order to knock out Trem1, applicant utilizes existing method[11], synthesize in slow virus carrier and opened using U6
Mover has cloned following short hairpin (Trem1：5 '-AATTCAAAAAAGACCAAGGGTTCT-3 ' and control sequence：5’-
TTCTCCGAACGTGTCACGT-3’).Slow virus carrier after purification and suit carrier cotransfection is intracellular to 293FT.48
Supernatant is collected after hour, lentiviral particle concentration in supernatant is then concentrated into 1: 100 using supercentrifugation, uses phosphoric acid
Salt buffer salt solution carries out suspension recovery.Virus titer is carried out using business titration ELISA kit (Takara Bio companies)
Measure, does not find exist between the lentiviral particle containing Trem1 cDNA or Trem1 shRNA and its control group in titre
Significant difference.
Microglia is transduceed
On microglia transductive process, by microglia after purification with 4 × 105Cell/mL density plant in
On 24 orifice plates.Lentiviral particle and 8 μ g/mL polybrenes are added into culture medium, and centrifuge 90min.On being removed immediately after transfection
Clear liquid is simultaneously substituted for the basal medium containing 10%FBS and 50% colloid culture supernatant (being obtained before transfection from culture medium).
After 72h, quantitative polyase chain reaction (qRT-PCR) and ELISA method are measured to transfection efficiency during utilization.
Lentiviral particle intracerebral injection
Injection process follows " blind ", that is, is responsible for the technical staff of stereotaxis intracranial injection lentiviral particle to experimental group
Situation is ignorant.General procedure is：It is vertical by being fixed on after age in July APP/PSEN1 and the WT mouse anesthesias to match with its age
On body directional operation frame (David Kopf Instruments companies).Use the 30- being connected with Hamilton syringe
Slow virus preparation (2 μ l) is injected brain of the mouse per side cerebral hemisphere by gauge micropipettes (Sigma-Aldrich companies)
Cortex (injection point at two) and hippocampus (injection point at one).The stereotaxis coordinate of injection position is followed successively by since bregma：(1)
Cerebral cortex：It is front and rear：- 0.3, middle outside：2, carry on the back veutro：- 1.5mm and front and rear：- 2, middle outside：1.2, carry on the back veutro：-
1.2mm；And (2) hippocampus：It is front and rear：-2；Middle outside：1.2；Carry on the back veutro：-2mm.Complete injection 2 months after to knock out efficiency and
Trem1 is overexpressed situation and verified.
Ventricles of the brain interior antibody conveys
Complete to convey the operation of antibody into the ventricles of the brain according to existing method[12].By excitability Trem1 antibody (100 μ g,
catalog#：MAB1187-100, R＆D Systems companies) or its IgG2AIsotype control Ab (100 μ g, catalog#：
MAB006, R＆D Systems companies) settling flux in 200 μ L synthetic cerebrospinal fluids is added to, and inject Osmotic minipumps (model
2006, ALZET companies) in.Operation on pump transplanting：By age in July APP/PSEN1 mouse and the WT to match with its age
It is fixed on after mouse anesthesia on stereoscopic localized crosshead (David Kopf Instruments companies).Will borrow vinyl tube with
The connected brain perfusion cannula (ALZET companies) of osmotic pumps is transplanted into diacele dorsal part (bregma rear 0.5mm, under skull surface
Square 3mm).The subcutaneous position that osmotic pumps are placed between the shoulder blade of mouse both sides simultaneously.Afterwards in 6 time-of-weeks, with 0.15 μ L/h
Pump degree, continue antibody being pumped into intracerebral.It is postoperative, operative incision is carefully sutured to closing.In whole surgical procedure, do not observe
There is any neurotoxicity performance to mouse.
Y- labyrinths are tested
Last day before animal is condemned to death, according to the method described before[10], Y- labyrinths have been carried out using " blind "
Test to assess the short-term spatial memory function of animal.In exposure period, mouse is placed on " starting " arm end, and allow its
The first arm section (being defined as " originating " arm) and the second arm section that Y- labyrinths are freely explored in 5 minutes (are defined as " other "
Arm).The stage, the entrance Basilar artery of the arm section of labyrinth the 3rd (being defined as " new " arm) will be led to.Afterwards, mouse is by from labyrinth
In be transferred to its inhabitation cage in treat 2min.In experimental stage, mouse is placed again into " starting " arm in labyrinth.Together
When, " new " arm entrance is opened, and allows mouse freely to explore 1rnin in labyrinth.UtilizeTracing system
(Noldus Information Technology companies) record each mouse in two stages in each arm area in labyrinth
The time of interior cost.For exposure period, we calculate mouse and stay in the total time of " other " arm with staying in the total of " starting " arm
The ratio of time.For experimental period, we calculate an index of discrimination=[new arm time/(during new arm time+other arms
Between)] × 100.
Morris water mazes are tested
In last 6 days before putting to death experimental animal, according to preceding method[7], water maze examination has been carried out using " blind "
Test.The experiment training of 4 times/day of mouse was given in continuous 5 days.And mouse is recorded from section start to finding hiding platform under water
Between route total length, while calculate four times experiment used in the time average value.In last day, one is carried out to mouse
The exploratory experiment after platform is removed, and is utilizedTracing system (Noldus Information Technology
Company) record its path of swimming.
QRT-PCR (real-time quantitative-polymerase chain reaction)
Extracted using Trizol reagents from person monocytic cell or mouse microglia and obtain total serum IgE.Utilize
PrimeScriptTMRT Master Mix (Takara Bio companies) make the total serum IgE of equivalent take a turn for the worse at the standard conditions
Record reaction.Then according to manufacturers instruction, SYBR Green Premix Ex Taq (Takara Bio companies) and specific are utilized
Mouse primer (GAPDH forward direction chains：GGTGGTCTCCTCTGACTTCA, GAPDH reverse strand：TCCTTGGAGGCCATGTGGGC；
Gapdh forward direction chains：CAACAGCAACTCCCACTCTTC, Gapdh reverse strand：GGTCCAGGGTTTCTTACTCCTT；TREM1 is just
To chain：AGTGGGGAGGATCATACTAGAAGAC, TREM1 reverse strand：AGGCTGGTAGATCACACACTGATAC；Trem1 is just
To chain：TGCTGTGCGTGTTCTTTGTCT, Trem1 reverse strand：CTTCTGGCTGTTGGCATACTT；TREM2 forward direction chains：
CTATGACTCCATGAAGCA, TREM2 reverse strand：GATTCCGCAGCGTAATGG；Trem2 forward direction chains：
GACCTCTCCACCAGTTTCTCC, Trem2 reverse strand：TACATGACACCCTCAAGGACTG；TYROBP forward direction chains：
AGCGATTGCAGTTGCTC, TYROBP reverse strand：GTGATACGCTGTTTCCGGGT；Tyrobp forward direction chains：
CGTACAGGCCCAGAGTGAC, Tyrobp reverse strand：CACCAAGTCACCCAGAACAA real-time quantitative-polymerase chain) has been carried out
Reaction.
ELISA (enzyme linked immunosorbent assay (ELISA))
For level of the measure total length TREM family proteins on cell membrane, collect human monocyte and the small colloid of mouse is thin
Born of the same parents, and the method described according to Geumann and its colleague[13]It is extracted epicyte protein.The handbook provided according to manufacturer, I
Using special ELISA kit (for mankind TREM1：catalog#：EHTREM1, Thermo Fisher
Scientific companies；For mouse Trem1：catalog#：EMTREM1, Thermo Fisher Scientific companies；It is right
In mankind TREM2：catalog#：MBS2022102, MyBioSource, Inc.；For mouse Trem2：catalog#：
MBS2023874, MyBioSource, Inc.) total length TREMs albumen is detected.
In order to assess intracellular A β1-42Level, we with reference to before method[7]Collect and cracked human monocyte
With mouse microglia.For detection intracerebral solubility and insolubility A β1-42Level, we utilize 10 under the conditions of 4 DEG C
The trimethylolaminomethane buffered saline (TBS) of the ethylenediamine tetra-acetic acid containing 5mM (EDTA) of volume, inhibitors of phosphatases, no
Protease inhibitor cocktail and 2mM 1,10- phenanthrolene containing EDTA are independently carried out to cerebral cortex and hippocampal tissue
Homogenize process.Obtained homogenate centrifuges 1h under the conditions of 4 DEG C, 100,000g (rotating speeds).We are received to supernatant
Collect to determine soluble A β1-42Content.Obtained particulate material adds 70% formic acid, and (FA, the volume of formic acid used are equal to
For the volume of the TBS homogenate centrifuged) make homogenize process.Sample centrifuges 1h and received under the conditions of 4 DEG C, 100,000g (rotating speeds)
Collect supernatant.The supernatant containing FA is neutralized with 1Mtris alkali, gained sample is used for determining insolubility A β 1-42 content.Root
Handbook is provided according to manufacturer, A β 1-42 are determined using A β 1-42 ELISA kits (Thermo Fisher Scientific companies)
Level.
Immunohistochemical analysis
Our methods according to before have prepared brain tissue slice so that immunohistochemical analysis uses[7].By brain tissue and mouse
Anti- A β monoclonal antibody (Covance companies) is incubated together, then adds biotinylated goat anti-mouse igg antibody
(Zhongshan Golden Bridge companies).Immune response is detected using diaminobenzidine.Afterwards, by tissue
Dehydration, film-making are placed in optical microphotograph Microscopic observation.According to the method described before us[7], using " blind " to total starch
Sample protein level is measured.
Statistical analysis
Data analysis is carried out using GraphPad Prism 6 (GraphPad Software companies).Examined using single sample t
Test, independent samples t test or one-way analysis of variance (ANOVA) and Tukey ' s post hoc are examined to poor between average
It is different to be compared.For Morris water mazes, this hides platform test, with two factor duplicate measurements variance analyses and
Bonferroni multiple comparative tests are analyzed path length data.In this research, data representation form is：Average ±
Standard deviation.P ＜ 0.05 are considered as with statistical significance.
The Trem1 of embodiment 2 can adjust phagocytosis of the mouse microglia to A β
As central nervous system (CNS) unique mononuclear phagocytic cells, microglia participates in A β by phagocytosis activity
Removing.To understand fully effects of the TREM1 in microglia swallows A β, we are manipulated from WT children mouse using slow virus mechanism
Trem1 expression in the microglia isolated.After the 72h that transduces, qRT-PCR and elisa technique are utilized respectively to Trem1
The change of mRNA expression and Trem1 protein levels is measured.Afterwards, the in vitro examination provided using Griciuc et al.
Proved recipe method detects microglia to A β1-42Phagocytosis and degradation.Knocking out Trem1 causes WT children's mouse microglia internalization
Aβ1-42Level declines 52% (P ＜ 0.05), and Trem1 overexpressions cause microglia internalization A β1-42Level increase nearly 1.7
Again (P ＜ 0.05).It is worth noting that：Manipulate Trem1 expressions and do not significantly affect WT children mouse microglia to A
β1-42Degradation index.
Research past according to this is found：Significant AD Relevant phenotypes occur during APP/PSEN1 7 monthly ages of mouse[7].Therefore, it is
Found more than further being confirmed under the related backgrounds of AD, we pass through slow virus mechanism 7 monthly age APP/PSEN1 mouse of manipulation
Trem1 expression in microglia.After transduction 72h is completed, respectively using qRT-PCR and ELISA method to Trem1mRNA tables
It is measured up to changing with Trem1 protein levels.Knocking out Trem1 makes 7 monthly age APP/PSEN1 mouse microglia internalization A
β1-42Level be remarkably decreased 46% (P ＜ 0.05).On the contrary, Trem1, which is overexpressed, causes microglia internalization A β1-42Level increases
Add nearly 1.5 times (P ＜ 0.05).However, the change of Trem1 expressions is manipulated not to the 7 monthly age APP/PSEN1 small glue of mouse
Cell plastid is to A β1-42Degradation rate cause to significantly affect.
Embodiment 3 is relative in the progression of disease phase of APP/PSEN1 mouse, expressions of the Trem1 on microglia
It is stable.
Because TREM1 can promote the A β phagocytosis of microglia mediation under the conditions of AD, we reasonably make
Hypothesis below：TREM1 may play a crucial role in AD progress.In order to verify this it is assumed that we explore APP/ first
PSEN1 mouse are in Isosorbide-5-Nitrae, and 7 and during 10 monthly age, microglia Trem1 and its horizontal dynamics of adapter protein Tyrobp change.
During APP/PSEN1 mouse diseases are in progress, significant changes (Trem1 mRNA do not occur for Trem1 or Tyrobp expression
It is horizontal：P=0.31；Trem1 protein levels：P=0.4；Tyrobp mRNA level in-sites：P=0.77).Meanwhile we also do not have found
APP/PSEN1 mouse and the WT mouse to match with its age exist on microglia Trem1 or Tyrobp expression
Any significant difference.
Embodiment 4, which knocks out Trem1, can promote A β europathologies to occur
Further to explore effect of the TREM1 albumen in AD progress, we knocked out for 7 monthly ages using slow virus mechanism
In APP/PSEN1 mouse brains in microglia Trem1 expression, and observe its influence to A β pathology.Slow virus injection 2
After individual month, we confirm the table of Trem1 in microglia in APP/PSEN1 mouse brains by RT-PCR and ELISA method
Up to there occurs lower to change.Total amyioid levels rise 1.66 after Trem1 is knocked out in APP/PSEN1 mouse brains
Times.In addition, knocking out Trem1 makes insolubility and soluble A β in APP/PSEN1 Cerebral Cortexes1-42Level difference it is notable
Increase by 2.48 times (n=12, P ＜ 0.05) and 1.57 times (n=12, P ＜ 0.05).Make APP/PSEN1 small moreover, knocking out Trem1
Insolubility and soluble A β in mouse cerebral hippocampus1-42Level dramatically increase respectively 1.89 times (n=12, P ＜ 0.05) and
1.58 times (n=12, P ＜ 0.05).Result above shows：The A β pathology of APP/PSEN1 mouse brains can be aggravated by knocking out Trem1
It is horizontal, it means that TREM1 can suppress AD progress as protective factors.
Embodiment 5, which is overexpressed Trem1, can weaken A β pathology and save the related spatial cognition infringements of AD
For protective effects of the checking TREM1 in AD progress, we are to small colloid in 7 monthly age APP/PSEN1 mouse brains
The Trem1 of cell has carried out selective overexpression.After slow virus is injected 2 months, we are confirmed by RT-PCR and ELISA method
There occurs up-regulation change for Trem1 expression in APP/PSEN1 mouse brain microglias.
First, we observe influence of the Trem1 overexpressions to brain A β Pathological levels.Trem1, which is overexpressed, makes big intracerebral
Total starch sample protein level have dropped 65.4%.Meanwhile Trem1 overexpressions make in APP/PSEN1 Cerebral Cortexes not respectively
Soluble and soluble A β1-42Level have dropped 61.7% (n=12, P ＜ 0.05) and 32.5% (n=12, P ＜ 0.05).This
Outside, Trem1 is overexpressed makes insolubility and soluble A β in APP/PSEN1 hippocampus of mice respectively1-42Level have dropped 58% (n
=12, P ＜ 0.05) and 35.9% (n=12, P ＜ 0.05).
In next step, we have detected influence of the Trem1 overexpressions to short-term spatial memory using Y- maze tests.In exposure
Phase, each group mouse are similar in the time that two arm regions are spent, it is believed that exploration degree of each group mouse to labyrinth is identical.Trying
Test the phase, it has been found that (reflection is relative " other arm regions ", and animal explores the preference of " new arm region " for " index of discrimination " of WT mouse
Tendency) it is more than 50% (n=24, P ＜ 0.05).When compared with WT mouse, exploration of the APP/PSEN1 mouse to " new arm region "
There is impaired (n=24 in tendency；Compared with 50%：P ＜ 0.05；Compared with WT mouse：P ＜ 0.05).And be overexpressed Trem1 can be with
This infringement (n=24 is reversed completely；Compared with 50% probability：P ＜ 0.05；APP/ with receiving the injection of control group slow virus
PSEN1 mouse are compared：P ＜ 0.05).It is worth noting that, injecting lentivirus operation does not influence two kinds of mouse in experimental period
Performance.Meanwhile Trem1 overexpression does not influence on experiment performance in WT mouse.
Afterwards, in last 5 days before putting to death animal, we are tested using water maze test to animal.First,
We compare the swimming rate of two kinds of mouse, do not observe significant difference.Slow virus is injected or Trem1 is overexpressed operation not
Swimming rate generation to two kinds of mouse (WT and APP/PSEN1 mouse) significantly affects.These results ensure that we exclude motivation
With the interference of sensorimotor factors on test performance.Next we test the space of animal using a hiding platform test
Learning functionality.As fig 5d：In from the 2nd to the 5th day time, WT mouse find the distance swum across position of platform and are intended to compare
APP/PSEN1 mouse are short (the 2nd day：8.45±1.03vs.9.14±1.04m；3rd day：7.52±0.98vs.8.96±
0.84m；4th day：6.05±0.42vs.8.11±0.57m；5th day：5.38±0.79vs.7.56±0.72m；N=24/ is every
Group, P ＜ 0.05).Two factor duplicate measurements variance analyses show：During whole task, the performance preferably (F of WT mousegenotype
(1,230)=148.8, P ＜ 0.05；Fdays(4,230)=173.9, P ＜ 0.05；Fgenotype×days(4,230)=15.59, P
＜ 0.05), the result confirms that September age APP/PSEN1 mouse show the infringement of spatial memory learning functionality really.Important
It is that Trem1 is overexpressed this functional lesion (F that can save APP/PSEN1 mousetreatment(1,230)=59.72, P ＜
0.05；Fdays(4,230)=85.07, P ＜ 0.05；Ftreatment×days(4,230)=7.49, P ＜ 0.05).It is noticeable
It is：Slow virus injection operation does not influence performance of two kinds of mouse in water maze test.Meanwhile Trem1 overexpressions are not also right
The test performance of WT mouse has an impact (Ftreatment(1,230)=2.27, n.s.；Fdays(4,230)=168.4, P ＜
0.05；Ftreatment×days(4,230)=0.95, n.s.).In order to detect influence of the Trem1 overexpressions to spatial memory, we
Platform is removed from underwater, and it is upper once train off-test 24h after allow mouse to carry out space exploration experiment again.It is small with WT
Mouse shows on the contrary, APP/PSEN1 mouse are nearly identical in the exploration time that target quadrant and other any quadrants are spent, meaning
Taste animal and obvious spatial memory infringement occurs.It is important that, it has been found that receive the APP/ of Trem1 slow virus injection
PSEN1 mouse will be long than other any quadrants in the time that target quadrant is spent, and difference has statistical significance (n=24；
37.57 ± 1.38%vs.20.81 ± 0.46%；P ＜ 0.05), this shows that the spatial memory function of animal is restored.It is worth
It is noted that slow virus injection operation does not influence performance of two kinds of mouse in exploratory experiment.Trem1 is overexpressed also not simultaneously
Influence the performance of WT mouse in test.
Embodiment 6 activates Trem1 signal paths by agonistic antibody can relax A β pathology and the cognition of AD correlation spaces
Infringement
In order to find to be transformed into clinically relevant background by more than, we continue 6 circumferential 7 monthly age APP/ using Osmotic minipumps
PSEN1 mouse brain diacele dorsal parts pour into a kind of excitability Trem1 antibody to activate Trem1 signal pathways.
First, we determine influence of the activation of Trem1 signal pathways to A β pathology.The note of excitability Trem1 antibody
Enter to make non-solubility and soluble A β in APP/PSEN1 Cerebral Cortexes1-42Level significantly reduce 31.4% (n respectively
=12, P ＜ 0.05) and 24.6% (n=12, P ＜ 0.05).Meanwhile the injection of excitability Trem1 antibody makes APP/PSEN1 small
Non-solubility and soluble A β in mouse hippocampus1-42Level significantly reduce 6.1% and 22.6% (n=12, P ＜ respectively
0.05)。
In next step, we have evaluated shadow of the activation to short-term spatial memory of Trem1 signal pathways using Y- maze tests
Ring.In exposure period, it is similar that each group mouse explores the time that two arm regions are spent.In experimental period, APP/PSEN1 mouse show
" the new arm " come explores the impaired (n=24 of tendency；Compared with 50% probability：n.s.；Compared with WT mouse：P ＜ 0.05) it can be swashed
Dynamic property Trem1 antibody saves (n=24；Compared with 50% probability：P ＜ 0.05；APP/ with receiving the injection of control group antibody
PSEN1 mouse are compared：P ＜ 0.05).It is pointed out that Osmotic minipumps were transplanted not to two kinds of mouse in experimental period
Performance has an impact.Meanwhile performance of the WT mouse in experimental period is not also influenceed by excitability Trem1 antibody.
Hereafter last 5 days before animal is put to death, we are tested using Morris water maze tests to animal.WT
Osmotic minipumps graft procedure or excitability Trem1 antibody implant operations are not affected by with the swimming rate of APP/PSEN1 mouse
Significantly affect.We determine the space learning function of animal followed by hiding platform test.Excitability Trem1 antibody
Inject and successfully saved the impaired Spatial learning ability (F of APP/PSEN1 mousetreatment(1,230)=62.79, P ＜ 0.05；
Fdays(4,230)=103.4, P ＜ 0.05；Ftreatment×days(4,230)=4.02, P ＜ 0.05).It is it is worth noting that, miniature
The implant procedure of osmotic pumps does not influence performance of two kinds of mouse in platform test is hidden.Meanwhile excitability Trem1 antibody
Inject the performance not on the WT mouse test phases and produce any influence (Ftreatment(1,230)=0.18, n.s.；Fdays(4,
230)=203, P ＜ 0.05；Ftreatment×days(4,230)=2.31, n.s.).In order to detect the activation of Trem1 signal paths
Influence to spatial memory, we remove platform and 24h allowed mouse to carry out space spy again after last training off-test
Rope is tested.Receive time that the APP/PSEN1 mouse of the injection of excitability Trem1 antibody are spent in target quadrant than other
What quadrant length, difference have statistical significance (Ftreatment(1,230)=0.18, n.s.；Fdays(4,230)=203, P ＜
0.05；Ftreatment×days(4,230)=2.31, n.s.), show that mouse spatial memory function is improved.It is noticeable
It is that the implantation of Osmotic minipumps does not influence performance of two kinds of mouse in exploratory experiment.Meanwhile excitability Trem1 antibody
Injecting the performance on the WT mouse test phases does not influence.
Bibliography：
1.Scheltens P, Blennow K, Breteler MM, et al.Alzheimer ' s disease.Lancet
2016.
2.Jiang T, Yu JT, Tian Y, et al.Epidemiology and Etiology of Alzheimer ' s
disease：From Genetic to Non-Genetic Factors.Curr Alzheimer Res 2013；10：852-
67.
3.Hardy J, Selkoe DJ.The amyloid hypothesis of Alzheimer ' s disease：
progress and problems on the road to therapeutics.Science 2002；297：353-6.
4.Mawuenyega KG, Sigurdson W, Ovod V, et al.Decreased clearance of CNS
beta-amyloid in Alzheimer′s disease.Science 2010；330：1774.
5.Jiang T, Yu JT, Zhu XC, et al.Temsirolimus promotes autophagic
clearance of amyloid-beta and provides protective effects in cellular and
animal models of Alzheimer′s disease.Pharmacol Res 2014；81：54-63.
6.Zondler L, Muller K, Khalaji S, et al.Peripheral monocytes are
functionally altered and invade the CNS in ALS patients.Acta Neuropathol
2016.
7.Jiang T, Tan L, Zhu XC, et al.Upregulation of TREM2 ameliorates
neuropathology and rescues spatial cognitive impairment in a transgenic mouse
model of Alzheimer′s disease.Neuropsychopharmacology 2014；39：2949-62.
8.Bliederhaeuser C, Grozdanov V, Speidel A, et al.Age-dependent defects
of alpha-synuclein oligomer uptake in microglia and monocytes.Acta
Neuropathol 2016；131：379-91.
9.Griciuc A, Serrano-Pozo A, Parrado AR, et al.Alzheimer ' s disease risk
gene CD33 inhibits microglial uptake of amyloid beta.Neuron 2013；78：631-43.
10.Jiang T, Zhang YD, Chen Q, et al.TREM2 modifies microglial phenotype
and provides neuroprotection in P301S tau transgenic mice.Neuropharmacology
2016；105：196-206.
11.Jiang T, Tan L, Zhu XC, et al.Silencing of TREM2 exacerbates tau
Pathology, neurodegenerative changes, and spatial learning deficits in P301S
tau transgenic mice.Neurobiol Aging 2015；36：3176-86.
12.Tan MS, Yu JT, Jiang T, et al.IL12/23 p40 inhibition ameliorates
Alzheimer′s disease-associated neuropathology and spatial memory in SAMP8
mice.J Alzheimers Dis 2014；38：633-46.
13.Geumann C, Gronborg M, Hellwig M, et al.A sandwich enzyme-linked
immunosorbent assay for the quantification of insoluble membrane and scaffold
proteins.Anal Biochem 2010；402：161-9.
Claims (3)
- The application of 1.TREM1 or its oligonucleotides coding in the medicine for preparing treatment Alzheimer disease, the few core of coding Thuja acid is present in the form of slow virus carrier.
- Application of the 2.TREM1 activity part or activity antibody in the medicine for preparing treatment Alzheimer disease.
- 3. the application described in claim any one of 1-2, it is characterised in that the medicine is formed sediment by strengthening microglia to β The phagocytosis of powder sample albumen and treat Alzheimer disease.
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| Publication number | Priority date | Publication date | Assignee | Title |
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Non-Patent Citations (3)
| Title |
|---|
| 《A TREM1 variant alters the accumulation of Alzheimer-related amyloid pathology》;Replogle JM等;《Ann Neurol》;20150301;第77卷(第3期);摘要第8-11行、14-15行、第68页第16-19行、第9页第3-6行 * |
| 《髓样细胞表达的触发受体-1分子的功能与信号转导机制研究进展》;朱江 等;《中国药业》;20090615;第18卷(第11期);第2页左栏第3点第5-9、13-16行 * |
| 《髓样细胞触发受体-1 在疾病中的作用》;刘娜 等;《中国医药导报》;20100131;第7卷(第1期);摘要 * |
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