CN108148901A

CN108148901A - Applications of the TFEB as cerebral apoplexy biomarker and therapy target

Info

Publication number: CN108148901A
Application number: CN201711482143.0A
Authority: CN
Inventors: 杨静玉; 吴春福; 刘月阳
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2018-06-12
Anticipated expiration: 2037-12-29
Also published as: CN108148901B

Abstract

The present invention relates to applications of the transcription factor EB (Transcription factor EB, TFEB) as cerebral apoplexy biomarker and therapy target.It is a discovery of the invention that the expression quantity by detecting TFEB, can be used for the diagnosis of cerebral apoplexy, it has also been found that, the overexpression of TFEB can be used for treating cerebral apoplexy.It is above to find to be of great significance in the diagnose and treat of cerebral apoplexy.

Description

Applications of the TFEB as cerebral apoplexy biomarker and therapy target

Technical field

The present invention relates to a kind of biomarker and therapy target, more particularly to TFEB as cerebral apoplexy biomarker and The application of therapy target.

Background technology

Cerebral apoplexy is a kind of the nervous system disease caused by the various causes of disease make the angiogenesis lesion of supply brain blood. Worldwide, cerebral apoplexy is the main reason for adult permanently disables and is dead.Become three big lethal in the world One of property disease.Clinical treatment is mainly thrombolysis, the neuron being at death's door for saving ischemic area (Penumbra zone) and promotion The recovery of nervous function after damage.It is current mankind difficult medical problem in the urgent need to address to prevent ischemic cerebrovascular disease.Mesh Preceding U.S. FDA only has approved the tissue plasminogen activator factor (tPA) for the thromboembolism treatment after apoplexy, but its therapeutic time window It is very narrow, using just effective only in apoplexy 4.5 hours；But also there are the danger that bleeding and Ischemia Reperfusion aggravate cerebral injury Property.In recent years, some neuroprotective agents are in preclinical study, but the therapeutic effect having in them it is imprecise or it is specific not By force, some toxic side effects are larger, tolerance is small, thus most of are terminated at clinical trial.Some drugs are also in preclinical Or clinical investigation phase, it is difficult to play actively impact in prevention cerebral arterial thrombosis.Thus, develop fast and effective, safety surely It is extremely urgent that fixed and therapy target explicitly prevents brain ischemia medicament.

At present, TFEB is still not clear in the effect of cerebral apoplexy, therefore, illustrates TFEB in the changing rule of cerebral apoplexy and seeks A drug by selective regulation TFEB is looked for have great importance the treatment of cerebral apoplexy.

Invention content

Technical problem

In order to solve the problems in the existing technology, it is a discovery of the invention that transcription factor-EB (Transcription Factor-EB, TFEB) expression quantity and the cerebral apoplexy state of an illness progress there are correlation, and find TFEB can by regulation and control from - lysosome access is bitten, changes the disease process of cerebral apoplexy.Therefore, the purpose of the present invention is to provide a kind of new cerebral apoplexies Biomarker and therapy target.

Solution

In order to solve the above technical problem, the present invention provides a kind of detection transcription factor-EB (Transcription Factor-EB, TFEB) expression quantity reagent prepare diagnosis cerebral apoplexy reagent or kit in purposes.

The object of the detection is neuronal cell.

The present invention also provides a kind of objects for targeting transcription factor-EB (Transcription factor-EB, TFEB) Purposes of the matter in the drug for preparing treatment cerebral apoplexy.

TFEB of the drug targeting in neuronal cell.

The drug makes TFEB be overexpressed in neuronal cell.

The TFEB of overexpression is made by regulating and controlling the activation of autophagy-lysosome access after cerebral ischemia to play Cerebral ischemia protection With.

The TFEB of the overexpression can improve the function of lysosome, accelerate the degradation of autophagosome.

The treatment of the cerebral apoplexy includes reducing cerebral infarction volume, reduces brain water content or reduce in Neuroscore It is at least one.

The present invention also provides a kind of transcription factor-EB (Transcription factor-EB, TFEB) to prepare treatment Purposes in the drug of cerebral apoplexy.

The drug makes TFEB be overexpressed in neuronal cell.

The TFEB of the overexpression plays Cerebral ischemia protection by regulating and controlling the activation of autophagy-lysosome access after cerebral ischemia Effect.

The present invention also provides a kind of pharmaceutical compositions for treating cerebral apoplexy, it includes above-mentioned drug and clinically normal The drug of anti-headstroke.

The drug of the clinically common anti-headstroke includes 3-methyl-1-phenyl-2-pyrazolin-5-one, recombination group Knit fiber type pepsinogen activator (r-tPA).

Described pharmaceutical composition also includes pharmaceutically acceptable carrier.

Described pharmaceutical composition is tablet, capsule, granule, powder, pill, oral liquid or parenteral solution.

The present invention also provides a kind of purposes of above-mentioned pharmaceutical composition in the drug for preparing treatment cerebral apoplexy.

Advantageous effect

The present invention's research shows that, TFEB can be used as the biomarker of cerebral apoplexy diagnosis, and can be lacked as brain Therefore the target spot of blood treatment, detects the product of TFEB expression quantity and the drug of targeting TFEB, has to the diagnose and treat of cerebral apoplexy Play the role of positive.

Description of the drawings

Comprising in the description and the attached drawing of a part for constitution instruction and specification together illustrate the present invention's Exemplary embodiment, feature and aspect, and principle for explaining the present invention.

Figure 1A is expressions of the different time points TFEB in nucleus after pMCAO.

Figure 1B is the sxemiquantitative statistical chart of Figure 1A.

Fig. 1 C are to investigate positioning scenarios of the different time points TFEB in neuronal cell core after pMCAO.

Fig. 1 D are to investigate positioning scenarios of the different time points TFEB in microglia after pMCAO.

Fig. 1 E are to investigate positioning scenarios of the different time points TFEB in astroglia after pMCAO.

Fig. 2 is positioning scenarios of the different time points TFEB in nucleus after primary neuron OGD.

Fig. 3 A are the expression effects that virus is overexpressed with westernblot technical identifications TFEB.

Fig. 3 B are the sxemiquantitative statistical charts of Fig. 3 A.

Fig. 3 C are the expression effects that virus is overexpressed with immunofluorescence technique verification TFEB.

Fig. 3 D are the influences for being overexpressed TFEB to autophagy-lysosome access.

Fig. 3 E are the sxemiquantitative statistical charts of Fig. 3 D.

Fig. 3 F are the survival feelings that different Group Animals cortical neurons after TFEB are overexpressed with immunofluorescence technique verification Condition.

Fig. 3 G are the influences for being overexpressed TFEB to ischemic injuries after pMCAO.

Fig. 3 H are the sxemiquantitative statistical charts of Fig. 3 G

Fig. 3 I are to be overexpressed statistical charts of the TFEB to rat nerve function score after pMCAO

Fig. 3 J are to be overexpressed statistical charts of the TFEB to rat brain water content after pMCAO.

Fig. 4 A are the expression effects that virus is overexpressed with westernblot technical identifications TFEB.

Fig. 4 B are the sxemiquantitative statistical charts of Fig. 4 A.

Fig. 4 C are the expression effects that low TFEB viruses are struck with immunofluorescence technique verification.

Fig. 4 D are to strike influences of the low TFEB to ALP correlative protein expressions with immunofluorescence technique verification.

Fig. 4 E are to strike influences of the low TFEB to ALP correlative protein expressions with westernblot technical identifications.

Fig. 4 F are the sxemiquantitative statistical charts of Fig. 4 E.

Fig. 4 G are to strike influences of the low TFEB to neuronal survival with immunofluorescence technique verification.

Fig. 4 H are the influences struck after low TFEB to ischemic injuries after pMCAO.

Fig. 4 I are the sxemiquantitative statistical charts of Fig. 4 H

Fig. 4 J are to strike statistical charts of the low TFEB to rat nerve function score after pMCAO

Fig. 4 K are to strike statistical charts of the low TFEB to rat brain water content after pMCAO

Fig. 5 is the experiment flow figure of embodiment 2

Fig. 6 A are to start EGFP using cortical neuron specificity promoter CaMKIIa, are struck with targeting TFEB low ShRNA forms the plasmid map of fusion protein

Fig. 6 B are that specificity T FEB strikes low shRNA using the sequential element based on miR30 structures, based on miR30 structures ShRNA interference frame

Fig. 7 is the TFEB that specificity is overexpressed in neuron, while viral vectors is made to have enough capacity expression TFEB The CDS sequences of gene

Specific embodiment

Embodiment 1：Rat permanent middle cerebral artery occlusion (pMCAO) afterwards different time points TFEB in nucleus Expression and primary neuron oxygen sugar deprive (OGD) positioning scenarios of different time points TFEB in nucleus afterwards

1. material：

Experimental animal

SD rats, male, weight 260-280g are provided by Shenyang Pharmaceutical University's animal center, animal quality certification number： 211002300015664

Cell

Rat primary cortex neurons：The cortical neuron of neonate rat (within birth 24 hours)

2. method：

The foundation of 2-1. rats permanent middle cerebral artery occlusion (pMCAO) model

PMCAO models are prepared according to Longa methods

(1) pMCAO operation consents Rat Fast can't help water 12 hours, then with 3.5% chloraldurate (0.1mL/kg) abdomen Chamber is anaesthetized, fixation of lying on the back, and the notch of an about 0.5cm is cut off at cervical midline, and with haemostatic clamp blunt separation muscle layer, exposure is right Side arteria carotis communis (CCA, common carotid artery), external carotid artery (ECA, external carotid artery) and Internal carotid (ICA, internal carotid artery)；

(2) it detaches and ligatures ECA, ICA is closed with artery clamp folder, cut an osculum for line and in proximal part in CCA, thus mouth is inserted Enter line bolt, open artery clamp, stop being inserted into line bolt when feeling and have resistance, fixing line bolt, the depth that line bolt is inserted into ICA with ECA crotches start to calculate about 18mm；

(3) art finishes, and cuts off extra line bolt, skin suture, being carried out disinfection with Iodophor prevents postoperative infection；

The foundation of 2-2. sham-operation groups

Other than being not inserted into line bolt, remaining surgical procedure is same as above.

The isolation and culture of 2-3. Primary cortical neurons

(1) neonate rat (within birth 24 hours) isolates brain part through 75% alcohol disinfecting in broken end on ice；

(2) the full brain separated is put into the plate for filling DMEM/F12 culture solutions, plate is placed on ice, is isolated Cortex is removed with the tunica vasculose that microforceps cover cortical surface, discards the tissue of other brain areas；

(3) cortex isolated is cut into 1mm with eye scissors³Fritter, every newborn rat adds in 0.25% pancreas of 1mL Trypsase and tissue block are transferred in centrifuge tube by protease together with rubber head dropper, and 37 DEG C of water-baths digest 15 minutes, with less It measures the DMEM/F12 culture solutions containing 10%FBS and terminates digestion, discard supernatant liquid, add in the DMEM/F12 culture solutions containing 10%FBS And with pasteur pipet piping and druming for several times, cell suspension is made；

(4) cell suspension is filtered through 200 mesh screens, and filtrate is counted with blood counting chamber, and adjustment cell density is 1 × 10⁶ A/mL；

(5) the cell suspension plantation of density will be mixed up in advance with poly-D-lysine processing overnight Tissue Culture Dish or plate In, containing 5% CO₂, cultivate 4 hours in 37 DEG C of incubators, full dose is changed into containing 2%B27 serum after cell is adherent Neurobasal culture solutions, effect renew culture solution after 72 hours, culture can be used to test for 5 days.

2-4. Primary cortical neurons oxygen sugar deprives the foundation of (OGD) model

At the 5th day of primary neuronal culture, cell was rinsed 2 times with 37 DEG C of PBS, then replaces DMEM carbonless base papers Cell is placed in anoxic cell by liquid, and anoxic cell is filled with containing 5% CO₂With 95% N₂Mixed gas, flow velocity is It 20L/ minutes, inflates 4 minutes.Anoxic cell is placed in 37 DEG C of constant incubator later and is incubated corresponding time, blank group Cell is replaced with DMEM and contains sugared culture solution after being rinsed with PBS, the equal time is incubated in 37 DEG C of constant incubators.In correspondence Time point carries out the detection of immunoblotting and immunofluorescence.

2-5. immunohistofluorescences investigate the common location of albumen

(1) it draws materials

For the pMCAO rat models that above-mentioned 2-1 is established after 3.5% chloral hydrate anesthesia, chest is opened rapidly in fixation of lying on the back Chamber, isolating cardiac and aorta are inserted into aorta from left ventricle with passivity syringe, are fixed with artery clamp, cut off right auricle of heart, Then physiological saline (4000U/L) 200mL containing heparin is quickly pushed into go out the blood in animal body (50mL/ minutes), When right auricle of heart trickle is substantially free of blood, 4% paraformaldehyde (PFA) 120mL is pushed into, speed is 5mL/ minutes.It It is changed to the PFA180mL of instillation 4% afterwards, speed is 2 drops/sec, continues about 40 minutes.

(2) it fixes afterwards

Rat takes brain after 4% PFA perfusion fixations, and 24 hours are fixed after brain is put into 4% PFA；

(3) it is dehydrated

After brain tissue is fixed after PFA, it is respectively put into 20% and 30% sucrose and is dehydrated 24 hours, treat that brain tissue is sunk to the bottom After take out.

(4) frozen section

Brain tissue blots the liquid on brain tissue surface with filter paper after 30% sucrose dehydration.Then around brain tissue Embedding medium (SAKUPA 4583) is uniformly coated with, is placed in (temperature is -65 DEG C) quick-frozen on the quick-frozen platform of slicer about 10 minutes, later It balances 5 minutes, is sliced under the operating temperature for being placed in -30 DEG C on the table, thickness is 20 μm, and room temperature, which air-dries, is placed on -80 It is preserved in DEG C.

(5) the double dyes of immunofluorescence

1) brain tissue slice takes out from -80 DEG C of refrigerators, and after 30 minutes, the PBS through 0.01M is rinsed 3 times rewarming, every all over 5 Minute；

2) histotomy is placed in microwave antigen retrieval in the citrate buffer (pH=6.0) for be previously heated to 95 DEG C. Primary every 3 minutes antigen retrievals, each low fire heating of microwave 6 minutes, 20 minutes altogether, temperature maintained 85-95 DEG C always Between, it is subsequently cooled to room temperature.It is rinsed 3 times with PBS, it is every all over 5 minutes；

3) it is closed 1 hour with the lowlenthal serum confining liquid room temperature containing 0.3% TritonX-100 (V/V), abandons confining liquid；

4) (Anti-TFEB antibody, Anti-NeuN antibody, Anti-Iba1 resist the primary antibody of every brain piece dropwise addition 30-40 μ L Body, Anti-GFAP antibody, Anti-Flag antibody, Anti-LAMP1 antibody, Anti-Cahtepsin D antibody, primary antibody dilution Liquid, the PBS of 0.2% TritonX-100,0.01M), 4 DEG C are overnight.After 16 hours, brain tissue slice is taken out from 4 DEG C, room temperature Rewarming 30-60 minutes.It is cleaned 3 times with PBST (0.01M PBS, 1mL Tween-20), it is every all over 10 minutes；

5) fluorescein-labeled secondary antibody (Cy3 labels goat anti-rabbit igg, the Cy3 label donkeys of 30-40 μ L is added dropwise in every brain piece The goat anti-mouse IgG of anti goat igg, FITC labels, secondary antibody diluent, the PBS of 0.01M), it is protected from light, is incubated at room temperature 2 hours. Then it is cleaned 3 times with PBST, it is every all over 10 minutes；

6) 37 DEG C of DAPI dyeing liquors (0.01M PBS configurations) are incubated 0.5 hour, and PBST is cleaned 3 times, every all over 5 minutes；

7) anti-fluorescence quenching (green skies P0128) mounting of 5 μ L is added dropwise in every brain piece, is protected from light and dries；

8) it is observed under confocal fluorescent microscopic, as shown in Fig. 1 C-E.

2-6. immunocytes fluorescence investigates the common location of albumen

(1) cell that above-mentioned 2-4 is established takes out from anoxic cell, discards culture solution, and cell 2 is rinsed with 37 DEG C of PBS Time, it is every all over 5 minutes；

(2) 15 minutes are fixed with 4% PFA, is rinsed 2 times with PBS later, it is every all over 5 minutes；

(3) cell carries out permeabilization through 0.1% saponin(e (Amresco 0163), 10 minutes, is rinsed 2 times with PBS later, Every time 5 minutes；

(4) it is closed 30 minutes with 3% BSA, abandons confining liquid, add in the TFEB primary antibodies (Anti-TFEB of PBS configurations later Antibody, primary antibody dilution, the PBS of 0.2% TritonX-100,0.01M) dilution incubation, 4 DEG C are overnight；

(5) primary antibody is recycled, cell is rinsed 3 times with PBST, it is every all over 5 minutes；

(6) secondary antibody (Cy3 labels goat anti-rabbit igg, Cy3 label donkey anti goat igg, the secondary antibody of corresponding Cy3 labels are added in Dilution, the PBS of 0.01M) dilution, it is incubated at room temperature 1 hour, is rinsed 5 times with PBS later, it is every all over 5 minutes；

(7) PBS solution of the dyeing liquor containing DAPI is added in for a long time in 4 DEG C of storages；

(8) it is observed under confocal fluorescent microscopic, as shown in Figure 2.

2-7. immunoblottings investigate the expression of albumen

(1) extraction of Nuclear extract

Experimental animal carries out broken end at detection corresponding time point and takes brain, with the blood on cold normal saline flushing brain tissue surface Mark, selective separating brain area, is transferred in the centrifuge tube of 1.5mL on ice；Extraction agent Nc-Buffer is taken out before Protein Extraction A and Nc-Buffer C (Nuclear extract extracts kit, Kang Wei century CW0199S) are pre-chilled；Claim tissue weight, often The Nc-Buffer A of 1mL are added in 100mg tissues (according to 1:99 ratios add in protease suppression before Protein Extraction in 2-3 minutes Preparation), it is fully homogenized on ice with homogenizer, is put on ice for being incubated 20 minutes；Add in the Nc-Buffer B (cells of 55 μ L Nucleoprotein extracts kit, Kang Wei century CW0199S), 5 seconds are vortexed with abundant mixing, are put on ice for being incubated 1 minute；4℃ 12000 × g is centrifuged 15 minutes, is collected in supernatant (collecting clean supernatant as possible) to new centrifuge tube, and (this is carried for -20 DEG C of preservations It is suppressor proteins to take liquid)；The Nc-Buffer C of 500 μ L are added in precipitation obtained by upper step (using preceding according to 1:99 ratios Add in protease inhibitors), 5 seconds are vortexed with abundant mixing, and precipitation is resuspended, is incubated 40 minutes on ice, was vortexed every 10 minutes mixed It is even primary, about 15-30 seconds every time；4 DEG C of 12000rpm are centrifuged 15 minutes, collect supernatant (collecting clean supernatant as possible) to new Centrifuge tube in, -20 DEG C preserve (this be Nuclear extract extracting solution).

(2) SDS- polyacrylamide gel electrophoresises (SDS-PAGE) and dyeing

12% separation gel and 5% concentration glue is prepared respectively, records SDS-PAGE (6 × 8 × 0.1cm).Install electrophoresis After slot, electrophoretic buffer, loading, 30 μ g/ holes of tissue total protein, 5 μ g/ holes of marker are added in.Constant pressure electrophoresis, sample are concentrating With 60V electrophoresis in glue, migrated after bromophenol blue to separation gel interface, sample separation gel with 120V electrophoresis to bromophenol blue migrate to Power supply is closed in gel bottom.Gel is removed, carries out electric transfer.

(3) immunoblotting

1) pvdf membrane activates 15 seconds in methyl alcohol, later by film, wet turn filter paper and sponge in transfering buffering liquid and fully soak Bubble 15 minutes；

2) it after SDS polypropylene amine gel is taken out, by gel by " sandwich " shape is built up, is inserted into transfer cell, gel one Face connects cathode, at 4 DEG C 260mA constant currents transfer the corresponding time；

3) pvdf membrane after shifting rinses in TBST, adds in 5% skimmed milk power closing, room temperature 1 hour；

4) by primary antibody, (Anti-TFEB antibody, Anti-Flag antibody, Anti-Lamin B antibody, Anti-SQSTM1 resist Body, Anti-Ubiquitin antibody, Anti-LAMP1 antibody, Anti-Cathepsin B antibody, Anti-Cahtepsin D resist Body, Anti-LAMP2 antibody, Anti-LC3 (B) antibody, Anti- β-actin antibody) be diluted to 5% skimmed milk power it is appropriate dense Degree, 4 DEG C of overnight incubations；

5) film is rinsed 4 times, every time 5 minutes with TBST；

6) by 5% skimmed milk power of secondary antibody (horseradish enzyme label goat anti-rabbit igg, horseradish enzyme mark goat anti-mouse IgG) Debita spissitudo is diluted to, is incubated at room temperature 1 hour；

7) film is rinsed 3 times, every time 10 minutes with TBST；

8) ECL reagents (Perkinelmer NEL 105) are added on memebrane protein face in darkroom, after reacting 1 minute, put In exposing in magazine, lid x-ray film exposes certain time；

9) it takes out film to be put into developer solution, after band to appear, with tap water rinse, is placed in fixing solution, finally uses Tap water rinses, and dries, and scanned picture preserves, as shown in Figure 1A.

3. experimental result：

As shown in fig. 1,3 hours to 6 hours after pMCAO, expression of the TFEB in nucleus gradually increases, 6 Peak value is reached when hour.With the extension of Ischemia Time, expression of the TFEB in nucleus was gradually reduced, at 24 hours When less than normal level and reached floor level at 48 hours.Figure 1B is the result of band sxemiquantitative.

As shown in Figure 1 C, the red TFEB albumen to be marked by TFEB antibody, green are neuron marker (Neuronal Nuclei, NeuN), in sham-operation group, TFEB with the formal distribution of disperse in cytoplasm, the 3 of pMCAO During hour to 6 hours, TFEB is activated, and shows as red dotted aggregation and fluorescence intensity gradually increases and gradually to cell It is shifted in core, 6 hours after pMCAO, the expression in nucleus has reached peak value.With the extension of Ischemia Time, TFEB is again Gradually go out core, the reduction of simultaneous expression quantity.Common location result is prompted, and the expression position of TFEB albumen and neuron occurs Most coincidence prompts TFEB mainly to express in neuron.

As shown in figure iD, red is TFEB albumen, and green is microglia marker Iba1, and common location result is shown Expression quantity of the TFEB in microglia is relatively low.

As referring to figure 1E, red is TFEB albumen, and green is astrocyte marker object GFAP, and common location result is prompted Expression quantity of the TFEB in astroglia is relatively low.

Exist as shown in Fig. 2, having investigated primary neuron different time points TFEB after OGD modelings by immunofluorescence method Expression in nucleus.TFEB albumen of the red for TFEB antibody label, blue are nucleus.In blank group, TFEB With the formal distribution of disperse in cytoplasm, after by the stimulation of OGD, TFEB is activated, and shows as dotted aggregation and red Fluorescence intensity increase, with the extension of OGD times, the degree of the dotted aggregations of TFEB increases and is gradually shifted into nucleus, Reach peak value within 6 hours after OGD, and then 12 hours after OGD, expression of the TFEB in nucleus decreases again.With Above the result shows that, 3 hours to 6 hours after neuron OGD, TFEB is gradually activated and is shifted into nucleus, and 12 is small after OGD When expression of the TFEB in nucleus continuously decrease again.

4. conclusion：

1) time dependence for first entering core there are TFEB during 3 hours to 48 hours after pMCAO and then going out core becomes Change, after pMCAO the expression in nucleus in 6 hours reach peak value.

2) after pMCAO during 3 hours to 48 hours, TFEB appearance first enters core then and goes out the time dependence variation of core And TFEB is mainly expressed in neuron.

3) TFEB Primary cortical neurons oxygen sugar deprive (OGD) occur first entering afterwards during 1 hour to 12 hours core with Go out the time dependence variation of core afterwards.

Embodiment 2：Specificity is overexpressed or strikes influences of the low TFEB to ALP functions and ischemic injuries after pMCAO

1. material：

Experimental animal

2. method：

Experimental design and grouping

First part：3 hours to 48 hours after pMCAO, the changing rule of TFEB and cellular localization SD male rats, body Weight 260-280g, is randomly divided into sham-operation group, model group (3,6,12,24,48 hours), every group of 10 animals.Through pMCAO hands Art modeling, the tissue progress Western blot detections of different time points selective separating brain area of 5 animals after modeling, 5 Immunofluorescence test is carried out at the same time, to investigate changing rules and cellular localization of the TFEB after pMCAO.

Second part：After striking to neuronal specificity low/overexpression TFEB, the influence to ischemia injury after pMCAO

SD male rats, weight 120-150g (4 week old) are randomly divided into sham-operation+GFP Empty virus groups, and sham-operation+ TFEB strikes low/overexpression group, pMCAO+GFP Empty virus groups, and pMCAO+TFEB strikes low/overexpression group, every group of 20 animals, Wherein 10 animals detect for behaviouristics, 5 animal immunofluorescence experiments, and 5 animals are used for immunoblot experiment.In disease The 28th day of poison injection carries out the verification that adeno-associated virus strikes low/overexpression efficiency.At the 29th day of virus injection, carry out PMCAO modelings are performed the operation, and strike low TFEB groups being detected for 6 hours after pMCAO, and it is 24 small after pMCAO to be overexpressed TFEB groups When be detected.It investigates and rat model cerebral infarction volume, brain water content, nervous function is commented respectively after striking low/overexpression TFEB Point and neuron survival rate influence.

Part III：After striking to neuronal specificity low/overexpression TFEB, autophagy after pMCAO-lysosome access is changed Influence

At the 29th day of virus injection, pMCAO modeling operations are carried out, low TFEB groups is struck and is carried out within 6 hours after pMCAO Detection is overexpressed TFEB groups being detected for 24 hours after pMCAO.Respectively investigate strike it is low/be overexpressed TFEB after it is big to model The influence of mouse ALP pathway associated proteins expression, including autophagosome GAP-associated protein GAP LC3, SQSTM1, Ubiquitin and lysosome The influence of GAP-associated protein GAP LAMP1, LAMP2, Cat.B and Cat.D expression.

Experiment flow is as shown in Figure 5.Wherein

(1) neuron TFEB strikes low adeno-associated virus (AAV) vector construction

The expression of TFEB in low cortical neuron is struck for specificity, this research uses cortical neuron specificity promoter CaMKIIa starts EGFP, and striking low shRNA with targeting TFEB forms fusion protein, and plasmid map is as shown in Fig. 6 A figures.To increase Strike inefficient and interference potential, the specificity T FEB of shRNA strikes low shRNA and uses the sequent based on miR30 structures Part, the shRNA based on miR30 structures interfere frame as shown in Fig. 6 B figures.This method expresses shRNA sequences in physiological conditions I.e. existing miR30 expression frame can dramatically increase the effect that shRNA sequences are identified and handled by endogenous Drosha enzymes Rate, miR30 sequences insertion position is between FLAG and WPRE expression cassettes.

(2) design of shRNA plasmids and sequencing result of low TFEB is struck

For the interference sequence of TFEB by being designed with first biology, interference sequence is as shown in table 1：

The interference sequence of table 1TFEB

ShRNA sequences carry out sequence verification after being connected into AAV interference plasmids, as shown in table 2, (the SEQ ID NO of sequence 1:1) and (the SEQ ID NO of sequence 2:2) it is to strike gene expression frame (the SEQ ID NO of low-quality grain:6) the shRNA hair clip sequences of targeting TFEB in It arranges (interference sequence of TFEB), (the SEQ ID NO of sequence 3:And (the SEQ ID NO of sequence 4 3):4) it is to strike the gene table of low-quality grain Up to frame (SEQ ID NO:6) flanking sequence of miR30 in, (the SEQ ID NO of sequence 5:5) it is to strike the gene expression frame of low-quality grain (SEQ ID NO:6) the Loop ring structures of miR30 in.Sequencing result shows do not occur being mutated and moving during plasmid construction Code phenomenon can be used for subsequent virus packaging.

Table 2shRNA sequences are connected into the result being sequenced after AAV interference plasmids

(3) neuron TFEB is overexpressed the structure of gland relevant viral vector

As shown in fig. 7, being overexpressed the TFEB in neuron for specificity, while make viral vectors that there is enough capacities charts Up to the CDS sequences of TFEB genes, this research is used compared with the SYN of CaMKIIa promoter fragments smaller and more strength with specifically TFEB is overexpressed in neuron to be overexpressed.

(4) TFEB plasmid gene sequencing results are overexpressed

After the CDS sequences of TFEB successfully are connected into virus particle, the full sequencing fragment of CDS sequences, sequencing result are carried out Such as (the SEQ ID NO of sequence 7:7) shown in, Flag protein gene sequences wherein included such as sequence 8 (SEQ ID NO:8) shown in, TFEB objective gene sequences such as sequence 9 (SEQ ID NO:9) shown in, sequencing result shows not dash forward during plasmid construction Change and frameshit phenomenon can be used for subsequent virus packaging.

(5) locating injection of adeno-associated virus

SD male rats, weight are 120-150g (4 week old), and for the locating injection of virus, rat is through 3.5% hydration Chloralization, prostrate are fixed on stereotaxic apparatus.Preserved skin after being carried out disinfection with 75% ethyl alcohol on head, the center line of two The mouth of 1cm is cut at place backward, cuts off fascia, and exposure skull is stopped blooding with dry cotton ball and wipes connective tissue, find bregma position, read Take coordinate.The coordinate of injection site is determined with reference to rat Naoliqing capsule Paxinos and Watson collection of illustrative plates and document.Cortex brain The coordinate in area is [AP:1.8 (site1),0.3(site2),1.2(site3)；ML:3.0；DV:2.5], syringe is moved on to note The top at position is penetrated, is then marked in the corresponding position of skull, bores game clock facial bones, is extracted with micro syringe corresponding Viral solution (2 μ L/site) inserting needle adjusts autosampler to site, and setting injection speed is 0.4 μ L/ minutes, and let the acupuncture needle remain at a certain point 10 Minute (ischemic side), pull out needle (2 minutes) skin suture afterwards.It is postoperative that penicillin is given to prevent from infecting.The expressing viral period is 4 weeks.

(6) foundation of the model of rat pMCAO

With embodiment 1

(7) Testing index

1) TTC is dyed：Quick broken end is carried out to postoperative rat for 24 hours and takes brain, complete -20 DEG C of brain tissue warp freezes 20min, from Pole starts to cut coronal brain piece, is cut into 6, is cut every 2mm a piece of.By brain piece as in the glass dish for filling 1%TTC solution, It is placed in 37 DEG C and is protected from light incubation 30min, brain piece is softly stirred when 15min, it is made uniformly to touch dyeing liquor.It, will after TTC dyeing Brain piece is placed in 4% paraformaldehyde solution to be protected from light after fixed 30min to take out and take pictures, and photo uses Image Pro Plus images point The analysis that software carries out cerebral infarct size is analysed, normal cerebral tissue region is red, and infraction and infraction affected area are white, according to Volume calculation formula calculates cerebral infarct volume ratio.

Infarct volume：V=(A1+A2+ ...+An) t/2.

T is slice thickness, and A1 and A2 represent stripping and slicing mouth, caudal infarct size respectively, and An is represented between stripping and slicing mouth and caudal The infarct size of middle diencephalon piece.

Cerebral infarct volume is than %=infarct volume/full brain volume × 100.

2) measure of brain water content：Postoperative broken end for 24 hours records the weight (brain wet weight) of brain tissue, TTC dyeing when taking brain Afterwards, brain piece is put into the glass dish for being lined with masking foil, drying 72h in 37 DEG C to constant weight and records weight (brain stem weight), according to Formula calculates brain water content：Brain water content %=(brain wet weight-brain stem weight)/brain wet weight × 100.

3) Neuroscore：Selection improvement neurotrosis scoring (Modified Neurological Severity Score, mNss) nervous function detection is carried out to postoperative animal for 24 hours.

4) immunofluorescence investigates the common location of albumen

With embodiment 1

5) immunoblotting investigates the expression of albumen

With embodiment 1

Statistical analysis

Statistical analysis is carried out with 19.0 softwares of SPSS, cerebral infarction, brain water content and immunofluorescence and immunoblotting are real It tests result to be counted using one-way analysis of variance, and Tukey is selected to examine and carries out Multiple range test.Neurological deficits are adopted Mean value comparison is carried out with Kruskal-Wallis test, and Dunn ' s is selected to carry out Multiple range test.Numerical value is with mean value ± standard Accidentally represent.

P<0.05 thinks with statistical significant difference.

3. experimental result：

1) neuronal specificity is overexpressed influences of the TFEB to 48 hours ALP functions and ischemic injuries after pMCAO

We verify that TFEB is overexpressed the expression effect of virus first.As shown in Figure 3A, Western the result shows that, cross table The expression of Flag-TFEB fusion proteins and the expression of exogenous TFEB can significantly be increased after up to TFEB.Fig. 3 B are Fig. 3 A's Semi-quantitative results.

As shown in Figure 3 C, immunofluorescence results are shown, green is Flag albumen, and red is TFEB albumen, is being overexpressed The expression of exogenous TFEB after pMCAO can be dramatically increased after TFEB.

As shown in Figure 3D, neuronal specificity is overexpressed TFEB and significantly improves lyase body function within 48 hours after pMCAO, adds Fast autophagosome degradation.Fig. 3 E are the semi-quantitative results of Fig. 3 D.

As illustrated in Figure 3 F, immunofluorescence results are shown, green is TFEB albumen, and red is NeuN, and blue is DAPI, in mistake The survival condition of neuron after pMCAO can be dramatically increased after expression TFEB.

As shown in Figure 3 G, next it is overexpressed influences of the TFEB to ischemic injuries after pMCAO, it has been found that be overexpressed TFEB The cerebral infarction volume of 48 hours after pMCAO, brain water content and Neuroscore can significantly be reduced.Fig. 3 H are the half of Fig. 3 G Quantitative result.

2) neuronal specificity strikes influences of the low TFEB to ALP functions and ischemic injuries after pMCAO

We verify that TFEB strikes the expression effect of low virus first.As shown in Fig. 4 A to 4C, Western blot and exempt from Shown in epidemic disease fluorescence results, TFEB, which strikes low AAV and has under foundation level, higher strikes poor efficiency and can be during ischemic The TFEB albumen of core is called under significantly.

(1) influence that low TFEB closes pMCAO post-lysosomes albumen LAMP1 and Cat.D common location is struck

As shown in Figure 4 D, struck after low TFEB to lyase body associated protein LAMP1 by immunofluorescence method investigation and The influence of Cat.D common locations.Green is the green fluorescent protein GFP of exogenous expression, and blue is marked by LAMP1 antibody LAMP1 albumen, red are the Cat. D albumen marked by Cat.D antibody, give after TFEB strikes low AAV and reach in sham-operation It is substantially reduced the expression of LAMP1 and Cat.D and common location area.After pMCAO modelings, the expression of LAMP1 and Cat.D and fixed altogether Plane product, compared to dramatically increasing, is embodied in the increase of fluorescence intensity of the two and the increasing of overlapping area compared with sham-operation group Add.And it gives TFEB in rat model and strikes the LAMP1 that pMCAO can significantly be reversed after low AAV to mediate and Cat.D expression Increase, the above result shows that, cortical neuron, which specifically strikes low TFEB, can significantly reduce lysosome caused by pMCAO Activation.

(2) influence to ALP correlative protein expressions after pMCAO after low TFEB is struck

As shown in Figure 4 E, after the TFEB in successfully striking low neuron, had detected by Western blot methods strike it is low TFEB influences to ALP correlative protein expressions in 6 hours after pMCAO.As depicted in figs. 4 e and 4f, it is molten 6 hours after pMCAO The expression of enzyme body associated protein Cat.B and Cat.D, compared to dramatically increasing, after striking low TFEB, can significantly be dropped compared with sham-operation group The expression of target gene LAMP1, Cat.B and Cat.D of low TFEB regulation and control, and to the non-TFEB target gene LAMP2 albumen regulated and controled Expression is then without significant impact.In addition, can be dramatically increased after striking low TFEB after pMCAO 6 hours autophagosome LC3-II and The expression of autophagy substrate Ubiquitin.The above result shows that the activation of ALP can be significantly inhibited by striking low TFEB.

(3) influence to neuronal quantity after pMCAO after low TFEB is struck

As shown in Figure 4 G, the influence for striking low TFEB to the survival of neuron after pMCAO has been investigated by immunofluorescence method. Green is the green fluorescent protein GFP of exogenous expression, and red is neuron marker NeuN, and blue is nuclear marker object DAPI strikes low TFEB in sham-operation group and has no significant impact to the quantity of neuron.After pMCAO after modeling, neuron Quantity be significantly reduced, the cell quantity of the NeuN positives can be further reduced after giving TFEB and striking low AAV.

(4) influences of the low TFEB to ischemic injuries after pMCAO is struck

As shown in Fig. 4 H and Fig. 4 I, pMCAO modelings are carried out within 4 weeks after low TFEB is struck, 6 hours after modeling are detected.It gives The control AAV that GFP is marked is given to have no apparent influence to cerebral infarction, brain water content and the Neuroscore caused by pMCAO, and After pMCAO can be significantly increased after giving TFEB and striking low AAV the cerebral infarction volume of 6 hours and Neuroscore and The brain water content of 6 hours after pMCAO.

4. conclusion

1) neuronal specificity is overexpressed TFEB and significantly improves within 48 hours after pMCAO lyase body function, accelerates autophagy small Body is degraded.

2) cerebral infarction volume that TFEB can be reduced significantly 48 hours after pMCAO, brain water content and neural work(are overexpressed It can scoring.

3) cortex, which specifically strikes low TFEB, can aggravate the ischemia injury of 6 hours after pMCAO.

4) neure damage after pMCAO can be aggravated after striking low TFEB.

Although above TFEB is marked as the biology that cerebral apoplexy detects with a general description of the specific embodiments Remember that object and the possibility as treatment Treatment of Cerebral Stroke drug target have made detailed description, but on the basis of the present invention, it can To be made some modifications or improvements to it, this will be apparent to those skilled in the art.Therefore, without departing from the present invention These modifications or improvements on the basis of spirit, belong to the scope of protection of present invention.

Sequence table

<110>Shenyang Pharmaceutical University

<120>Applications of the TFEB as cerebral apoplexy biomarker and therapy target

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ggctcccctt tccccaacct gtccaagaag gatctggact taatgctcct agatgactcc 1500

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Claims

1. the reagent for detecting transcription factor-EB (Transcription factor-EB, TFEB) expression quantity is preparing diagnosis brain soldier In reagent or kit in purposes, the object preferably detected be neuronal cell.

2. the substance of transcription factor-EB (Transcription factor-EB, TFEB) is targeted in the medicine for preparing treatment cerebral apoplexy Purposes in object.

3. purposes according to claim 2, which is characterized in that TFEB of the drug targeting in neuronal cell, it is special Be not that the drug makes TFEB be overexpressed in neuronal cell, it is preferred that the TFEB of overexpression by regulate and control after cerebral ischemia from Bite-activation of lysosome access and play Cerebral ischemia protection effect, it is furthermore preferred that the TFEB being overexpressed can improve lysosome Function accelerates the degradation of autophagosome.

4. according to the purposes described in any one of claim 2-3, which is characterized in that the treatment of the cerebral apoplexy includes reducing brain Infarction volume reduces brain water content or reduces at least one of Neuroscore.

5. use of the transcription factor-EB (Transcription factor-EB, TFEB) in the drug for preparing treatment cerebral apoplexy On the way.

6. purposes according to claim 5, which is characterized in that the drug makes TFEB be overexpressed in neuronal cell, Preferably, the TFEB of overexpression is acted on by regulating and controlling the activation of autophagy-lysosome access after cerebral ischemia to play Cerebral ischemia protection, It is furthermore preferred that the TFEB being overexpressed can improve the function of lysosome, accelerate the degradation of autophagosome.

7. according to the purposes described in any one of claim 5-6, which is characterized in that the treatment of the cerebral apoplexy includes reducing brain Infarction volume reduces brain water content or reduces at least one of Neuroscore.

8. a kind of pharmaceutical composition for treating cerebral apoplexy, it includes the drug described in any one of claim 3-7 and clinically The drug of common anti-headstroke, it is preferred that the drug of the clinically common anti-headstroke includes 3- methyl-1-phenyl- 2- pyrazolin-5-ones, recombinant tissue plasminogen activator (r-tPA).

9. a kind of pharmaceutical preparation, which is characterized in that can it includes pharmaceutical composition according to any one of claims 8 and pharmaceutically connect The carrier received, it is preferred that the pharmaceutical preparation includes tablet, capsule, granule, powder, pill, oral liquid or parenteral solution.

10. the pharmaceutical preparation described in the pharmaceutical composition or claim 9 for the treatment of cerebral apoplexy according to any one of claims 8 is controlled in preparation Treat the purposes in the drug of cerebral apoplexy, it is preferred that the treatment of the cerebral apoplexy includes reducing cerebral infarction volume, reduces brain water content Or reduce at least one of Neuroscore.