CN107385029A - LBP-X suppresses the experimental method of glioma growth by adjusting blood-brain barrier - Google Patents
LBP-X suppresses the experimental method of glioma growth by adjusting blood-brain barrier Download PDFInfo
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Abstract
LBP-X suppresses the experimental method of glioma growth by adjusting blood-brain barrier, belongs to tcm field.Male SD rat is taken, LBP-X solution is given and raises 34 weeks；Then rat glioma model is built；Continue to give LBP-X solution after modeling and detect：1. the life span of every group of rat, survival rate and median survival interval；②Qu each groups rat cerebral tissue observes and measures tumor size；3. each group rat is taken equivalent brain tissue to detect Evans blue content, judge that blood-brain barrier permeability changes by vena femoralis injection Evans Blue solution；4. electron microscopic observation blood-brain barrier Change of Ultrastructure；5. carrying out apex of the heart perfusion with paraformaldehyde solution to each group rat, immunohistochemical assay is done after taking brain tissue slice；6. taking each group rat, directly broken end takes brain respectively, RNA and albumen are extracted, carries out RT PCR and Western blotting experiments.The present invention provides new therapy approach to treating a variety of blood-brain barrier diseases.
Description
Technical field
The invention belongs to traditional Chinese medicine application field, more particularly to a kind of LBP-X suppresses colloid by adjusting blood-brain barrier
The experimental method of knurl growth.
Background technology
Glioblastoma is the most common malignant brain tumor of adult, and its most typical feature is to surrounding brain tissues height
Infiltration, because the tumour cell of infiltration can not be removed completely, new tumor tissues are formed so as to recur, cause patient's prognosis pole
Difference, although giving positive operation, radiotherapy, chemotherapy, and it is aided with new molecular biology therapy, the median survival interval of patient
Only it is extended to 2 years.Diagnosis of molecular biology shows that glioblastoma pathomorphism is various and variable, and what this made controls
Treat further difficult.Studies have shown that antineoplastic Temozolomide has positive effect to treatment glioma at present, but due to blood
The effect of brain barrier, the concentration that medicine enters in knurl is little, causes total effect nor highly desirable.Effectively done it can be seen that finding
Method promotes blood-brain barrier permeability to increase, and passes through medicine, and valid density is reached in tumour, the treatment meeting to glioma
Play vital effect.
Lycium barbarum is the traditional rare traditional Chinese medicine in China, and is uniquely loaded into《Chinese Pharmacopoeia》Matrimony vine species.《This
Careless detailed outline》Record:" property sweet taste, Return liver, kidney channel is nourishing liver and kidney, benefiting shrewd head ", hard smart bone, goes fatigue, easy color, bleaches,
Improving eyesight is calmed the nerves, and makes us long-lived.”《Sheng Nong's herbal classic》In point out：Long term usage heavily fortified point muscles and bones；《Mingyi Bielu》Meaning matrimony vine is good at " help essence
Gas "；《Dietetic materia medica》Also matrimony vine " the beneficial people of energy, remove consumptive disease " is recorded.LBP-X is main active in matrimony vine, and content exists
5.42%~8.23%, modern pharmacology research confirms that LBP-X has a variety of pharmacotoxicological effects, including removes free radical, antioxygen
Change, anti-aging, antitumor, nerve modulation, immunological regulation, hypoglycemic, reducing blood lipid etc. act on, and have animal experiment study to confirm Chinese holly
Qi polysaccharide is without any side effects, can extend the glioma surviving rats phase, but its mechanism is unknown.
The content of the invention
The purpose of the present invention is for existing because blood-brain barrier causes present in the little situation of drug effectiveness not
A kind of foot part, there is provided the technical scheme that LBP-X suppresses the experimental method of glioma growth by adjusting blood-brain barrier.
The present invention explores emphatically LBP-X pair on the basis of research LBP-X influences on the glioma surviving rats phase
The influence of rat glioma growth and blood-brain barrier permeability, it is found that LBP-X can adjust blood-brain barrier, produced in encephalic
In the case of tumour, increase the permeability of blood-brain barrier, promote body peripheral system immunocyte to enter encephalic, it is thin to glioma
Intracellular growth plays suppression or killing action.
The concrete technical scheme of the present invention is such：
Male SD rat is taken, first gives running water and different gradient LBP-X solution respectively（25mg/kg.d、50mg/kg.d、
100mg/kg.d、200mg/kg.d、400/kg.d）Raising 3-4 weeks；Then stereotaxic instrument is fixed, and builds rat glioma mould
Type, rat caudate nucleus inoculation C6 glioma cells（1×105It is individual）；Continue to give LBP-X and the inspection of various concentrations after modeling
Survey following index：1. observing and recording the life span of every group of rat, survival rate and median survival interval are calculated；With blank control group
Rat median survival interval is defined, and is further continued for detecting as follows：②Qu each groups rat cerebral tissue observes and measures tumor size；It is 3. right
Each group rat takes equivalent brain tissue to detect Evans blue content, judges blood-brain barrier by vena femoralis injection Evans Blue solution
Permeability changes；4. electron microscopic observation blood-brain barrier Change of Ultrastructure；5. each group rat is carried out using 4% paraformaldehyde solution
The apex of the heart is irrigated, and immunohistochemical assay is done after taking brain tissue slice；6. taking each group rat, directly broken end takes brain respectively, extract RNA and
Albumen, carry out RT-PCR and Western blotting experiments（3., 4., 5. and be 6. selected from above-mentioned tumour most group's brain tissue,
To detect encephalic immune indexes and the change of blood-brain barrier correlation molecule）.
The present invention technology path be：Experimental animal is grouped and raising --- Glioma Model preparation --- life cycle is seen
Examine --- scanning electron microscopic observation blood-brain barrier ultra microstructure that tumor size compares --- blood-brain barrier permeability detection --- change
Change --- immunohistochemical assay --- RT-PCR detections --- Western blotting experiments --- interpretation of result.
The present invention has the advantages that：
1st, LBP-X is the extract of Chinese medicine matrimony vine, belongs to natural botanical source, and is planted extensively, relative low price, is
The development of new type antineoplastic medicine provides high-quality raw material, and experimental method of the present invention demonstrates LBP-X and suppresses glioma life
Long effect, the achievement in research will greatly mitigate the family burden of middle and low income patient；
2nd, experimental method result of the present invention shows that LBP-X has the function that regulation Blood Brain Barrier (BBB) opening, can effective auxiliary
Treat, particularly the expectant treatment to postoperative patient, promote chemotherapeutics to enter encephalic, strengthen curative effect of medication, so as to reach treatment
The effect of tumour；
3rd, experimental method of the present invention is researched and developed to LBP-X related drugs, combines the more efficient regulation blood-brain barrier of several formulations
Opening, provide new therapy approach for a variety of blood-brain barrier relevant diseases such as Alzheimer disease.
Brief description of the drawings
Fig. 1 is each group group glioma surviving rats phase and the survival rate schematic diagram of LBP-X different feeding amount；
Fig. 2 is LBP-X different feeding amount brain tissue slice tumor size schematic diagram；
Fig. 3 is content schematic diagram of the Evans blue in different groups of rat cerebral tissues；
Fig. 4 is different groups of blood-brain barrier key molecule change schematic diagrams；
Fig. 5 is immunohistochemical experiment schematic diagram.
As shown in Figure 1：A, B, C, D, E be respectively 400/kg.d, 200mg/kg.d, 100mg/kg.d, 50mg/kg.d,
The rat that 25mg/kg.d LBP-Xs mixture solution is fed, F feed for running water.
As shown in Figure 2：A, B, C, D, E be respectively 400/kg.d, 200mg/kg.d, 100mg/kg.d, 50mg/kg.d,
The rat that 25mg/kg.d LBP-Xs mixture solution is fed, F feed for running water.
As shown in Figure 4：RT-PCR and Westernblotting experimental results are shown, adjust the key molecule of blood-brain barrier
Expression of the ANXA1 in tumour will be significantly lower than in normal cerebral tissue（P﹤ 0.05）；And respectively in control group（Normal rat）
And in tumor group, ANXA1 expression and blank group in the rat cerebral tissue that LBP-X is fed（Running water is fed）Compare, equally
It is substantially to reduce（P﹤ 0.05）；Immunohistochemical experiment is shown, in tumor tissues, ANXA1 protein expression positive cells are in matrimony vine
Significantly reduced in the rat cerebral tissue that the rat cerebral tissue that polysaccharide is fed feeds than running water.
Embodiment
The embodiment of the present invention is as follows.
1. experimental animal is grouped and raising：1. take standard weight（180-200g）Male SD rat 200, is randomly divided into 6
Group, every group 25（Wherein 7 observation life cycles, 5 injection Evans blue detection blood-brain barrier changes, 7 4% paraformaldehydes fill
Note takes brain measurement tumor size and does immunohistochemical experiment, and it is real that 6 broken ends take brain to be used for RT-PCR and Western blotting
Test）, each group rat is randomly assigned again：4-5/cage；Each group has unnecessary rat, to prevent modeling failure or other unexpected factors
Experiment is impacted；2. blank control group rat drinking water whole process is running water, experimental group rat drinking water is various concentrations
LBP-X solution（Experiment start first 3 days be the rat laundering period, give running water nursing, record per cage rat daily averagely
Amount of drinking water, and calculate every rat and be averaged daily amount of drinking water, various concentrations LBP-X solution is configured with this, measured within every 5 days
Rat body weight, is adjusted to LBP-X solution concentration, ensures that every group of edible LBP-X of rat institute is respectively：
25mg/kg.d、50mg/kg.d、100mg/kg.d、200mg/kg.d、400mg/kg.d）.
2. prepared by Glioma Model：1. by C6 cells in blake bottle, with Trypsin Induced into single cell suspension, adjust
Cell density is 4 × 106Individual/mL, it is standby；2. anaesthetize：After rat weight, with 10% chloral hydrate anesthesia（4ml/kg,ip）；③
Rat prone position is fixed on stereotaxic apparatus, and adjustment makes bregma and posterior keep level, preserved skin, and scalp is being just after iodophor disinfection
Middle sagital incision, separate periosteum, exposure bregma；4. positioning left side caudate nucleus：3mm is opened on 0.5mm after bregma, left side；Mark backteeth
Section's drilling；5. drawing 25ul single cell suspensions with micro syringe, uniformly inject and finish in inserting needle 5.8mm, 6min, stop
5min, point 2 slow withdraws of the needle（Each 2-3 minutes, centre stop 5min）；6. the withdraw of the needle terminates rear bone wax sealing of hole, after iodophor disinfection
Skin suture；Operation terminates rear rat and fed ditto, pays attention to warming.
3. life cycle is observed：The start recording after modeling, until rat natural death, calculate rat the average survival time number of days.
4. tumor size compares：Cranium is opened after being irrigated with 4% paraformaldehyde solution and takes full brain, and full brain is put in rat brain slice
In mould, do the section of full brain Coronal, thickness 1mm, measurement tumour maximum major diameter a, maximum wide footpath b (coronal-plane, unit mm) and
Number of plies c, calculate gross tumor volume, gross tumor volume V=a × b × c × π/6.
5. blood-brain barrier permeability detects：10% chloraldurate solution is to rat anesthesia（Ditto）, rat, which lies on the back, is fixed on hand
On art platform, preserved skin, exposure femoral vein, puncture injection 2%（2g Evans blues add 100mlPBS buffer solutions）Evans Blue solution
（2ml/kg）, broken end takes mg of brain tissue 100 or so after 40min（Rat brain slice mould cuts same aspect, and right side is used for examining
Survey blood-brain barrier, left side detection blood flow barrier）, it is put into after weighing in the test tube for having 3 ml dimethylformamides, 60 DEG C of water-baths
In (lucifuge) extract 24 h.1 500 g centrifuge 10 min, take supernatant, using RF540 sepectrophotofluorometers (λ=
632 nm) measure EB fluorophotometrics value (opticaldensity, OD), distilled water is as blank control.According to optical density number
Value, calculates Evans blue content in brain tissue from standard curve, is represented with Evans blue content (the wet brain tissues of μ g/g)
The change of blood-brain barrier permeability.
6. scanning electron microscopic observation blood-brain barrier Change of Ultrastructure：Cranium, which is opened, after being irrigated with 4% paraformaldehyde solution takes full brain, 4
DEG C normal saline flushing 3 times；Cut the same position rat cerebral tissue in right side（Size about 3mm × 3mm × 3mm）After being placed in fixer
Mark, 4 DEG C of fixed 30min；Tissue block is repaiied on slide small, be placed in the fixed 2-2.5h of 4 DEG C of fixer；Change buffer solution 2h
× 3 times；4 DEG C of 1% osmic acid soaks 2h；0.1M natrium cacodylicum wash buffers 15min × 2 time；Dehydration：1. 4 DEG C of 30% alcohol
It is dehydrated 10min；2. 4 DEG C of 50% dehydration of alcohol 10min；3. 4 DEG C of 70% dehydration of alcohol 10min；4. the dehydration of alcohol of room temperature 80%
10min；5. the dehydration of alcohol 10min of room temperature 90%；6. dehydration of alcohol 15min × 2 time of room temperature 100%；7. room temperature expoxy propane permeates
15min × 2 time；8. room temperature 1:1（Not exclusively embedding liquid:Expoxy propane）Permeate 1h；Room temperature 2:1（Not exclusively embedding liquid:Epoxy third
Alkane）Permeate 1h；Room temperature embeds liquid infiltration overnight completely；9. embedding liquid immersion completely, is placed in 35 DEG C of incubator 6h, is then transferred to bag
Buried plate（Embedding liquid completely）, 42 DEG C of incubators stay overnight；10. scanning electron microscopic observation blood-brain barrier Change of Ultrastructure.
7. immunohistochemical assay：Cranium is opened after being irrigated with 4% paraformaldehyde solution and takes full brain, and brain tissue is placed in 4% poly first
24h is soaked in aldehyde solution, the sucrose solution for changing 20% and 30% is dehydrated 24h (being completed in 4 DEG C of refrigerators), PBS bufferings respectively
After liquid rinses, it is placed on Lycra freezing microtome and cuts into slices after embedding medium embedding is fixed（Thickness 25um）, it is placed directly against and sticks load glass
On piece；It is placed in 37 DEG C of drying bakers and carries out antigen retrieval 1-2h；PBS moistens brain piece after taking-up；3%H2O2Deionized water is incubated
10min, to block endogenous peroxydase；PBS rinses 2min × 3 time；Lowlenthal serum closes 40min；Primary antibody is added dropwise（Rabbit-anti
Rat CD3 monoclonal antibodies, rabbit-anti rat CD8 monoclonal antibodies）, 37 DEG C of incubations 2h, PBS rinse 2min × 3 time；Add secondary antibody,
37 DEG C of incubations 20min, PBS rinse 2min × 3 time；Reagent 1,37 DEG C of incubations 20min, PBS are added dropwise and rinse 2min × 3 time；It is added dropwise
Reagent 2,37 DEG C of incubations 20min, PBS rinse 2min × 3 time；Haematoxylin is added dropwise and redyes 15min；Weathering liquid（75% alcohol 99ml+
Concentrated hydrochloric acid 1ml）2-5s is soaked, pbs rinses 2min × 3 time, dried；Graded ethanol（75%、80%、90%、100%）Dehydration（Each 2-
3min）；Dimethylbenzene is dehydrated transparent 2-3min；Neutral gum mounting；Micro- Microscopic observation is taken pictures.
8.RT-PCR is detected：After the direct broken end of rat takes brain, take brain tissue 100mg to be placed in tissue homogenizer and grind, add
Enter 1-1.5ml Trizol, be transferred in 2mlEP pipes, brain tissue is fully cracked at room temperature；Add chloroform（For Trizol volumes
1/5）, concussion is uniform, and room temperature places 15min；Centrifuge 15min（4 DEG C, 12000rpm）, three layers of liquid point in pipe；Draw most upper
Layer（RNA）It is placed in new no RNase EP pipes, adds isometric isopropanol, 10min is stored at room temperature after fully mixing；4 DEG C,
12000rpm centrifuges 10min, abandons supernatant, bottom precipitation is RNA；The alcohol of 1ml 75% is added to mix up and down to RNA progress clearly
Wash, 4 DEG C, 12000rpm centrifugation 10min, abandon supernatant；Appropriate DEPC water dissolving is added after naturally dry, determines RNA concentration；According to
ThermoRNA Reverse Transcriptase kits carry out reverse transcription to RNA, and RT-PCR detections are carried out further according to DBI fluorescence quantitative kits.
9.Western blotting are tested:After the direct broken end of rat takes brain, brain tissue 100mg is taken to be placed in tissue homogenizer
Middle grinding, operated according to triumphant base Protein Extraction Reagent kit and carry out protein extraction, it is dense to carry out albumen according to BCA kits operating method
Degree measure；Then plus sample-loading buffer, 100 DEG C of 5 min make albuminous degeneration；Take 50 μ g albumen（25 μL）Loading, carry out
SDS-PAGE（90 V, 90 mi n）With protein isolate, then electric transferring film（300 mA, 70 min）, PVDF film skim milks
Close 1 h；It is separately added into rabbit-anti rat GAPDH and ANXA1 monoclonal antibody（Volume dilution ratio is 1: 1 000）, 4 DEG C
Reaction is overnight；After TBS buffer solutions wash film, the goat antirabbit fluorescence secondary antibody of IRDye 800CW marks is added（Volume dilution ratio
For 1: 4 000）, 1 h of normal temperature hybridization；After washing film again, it is placed in Odyssey Infrared Imaging instrument and is exposed, applies
ImageJ Setup softwares carry out the gray value scanning of protein band, with the gray scale of destination protein and internal reference GAPDH protein bands
The ratio between value represents the relative expression levels of each albumen.
Claims (10)
1. LBP-X suppresses the experimental method of glioma growth by adjusting blood-brain barrier, it is characterised in that：Take male SD big
Mouse, running water and different gradient LBP-X solution are first given respectively（25mg/kg.d、50mg/kg.d、100mg/kg.d、
200mg/kg.d、400/kg.d）Raising 3-4 weeks；Then stereotaxic instrument is fixed, and builds rat glioma model, rat shape of tail
Core is inoculated with C6 glioma cells（1×105It is individual）；Continue to give the LBP-X of various concentrations after modeling and detect following index：
1. observing and recording the life span of every group of rat, survival rate and median survival interval are calculated；Given birth to blank control group rat middle position
The phase of depositing is defined, and is further continued for detecting as follows：②Qu each groups rat cerebral tissue observes and measures tumor size；3. each group rat is led to
Vena femoralis injection Evans Blue solution is crossed, takes equivalent brain tissue to detect Evans blue content, judges that blood-brain barrier permeability changes；
4. electron microscopic observation blood-brain barrier Change of Ultrastructure；5. carrying out apex of the heart perfusion using 4% paraformaldehyde solution to each group rat, take
Immunohistochemical assay is done after brain tissue slice；6. taking each group rat, directly broken end takes brain respectively, RNA and albumen are extracted, carries out RT-
PCR and Western blotting are tested（3., 4., 5. and be 6. selected from above-mentioned tumour most group's brain tissue, exempt to detect encephalic
Epidemic disease index and the change of blood-brain barrier correlation molecule）, the technology path of the experimental method is：Experimental animal is grouped and raising --- glue
--- life cycle observation --- tumor size compares --- blood-brain barrier permeability detection --- ESEM prepared by matter knurl model
Observing blood-brain barrier Change of Ultrastructure, --- immunohistochemical experiment --- RT-PCR experiments --- Westernblotting is real
Test --- interpretation of result.
2. experimental method as claimed in claim 1, it is characterised in that：Described experimental animal packet and the specific experiment of raising
Method is：1. take standard weight（180-200g）Male SD rat 200, it is randomly divided into 6 groups, every group 25（Wherein 7 observations
Life cycle, 5 injection Evans blue detection blood-brain barrier changes, 7 4% paraformaldehyde perfusions take brain measurement tumor size and done
Immunohistochemical experiment, 6 broken ends take brain to be used for RT-PCR and Westernblotting and tested）, each group rat is randomly assigned again：
4-5/cage；Each group has unnecessary rat, and experiment is impacted to prevent modeling failure or other unexpected factors；2. blank pair
It is running water according to group rat drinking water whole process, experimental group rat drinking water is various concentrations LBP-X solution（Experiment starts preceding 3
It is the rat laundering period, gives running water nursing, records the averagely amount of drinking water, and it is daily to calculate every rat daily per cage rat
Average amount of drinking water, various concentrations LBP-X solution is configured with this, the rat body weight of measurement in every 5 days is molten to LBP-X
Liquid concentration is adjusted, and ensures that every group of edible LBP-X of rat institute is respectively：25mg/kg.d、50mg/kg.d、100mg/
kg.d、200mg/kg.d、400mg/kg.d）.
3. experimental method as claimed in claim 1, it is characterised in that：Described Glioma Model prepares concrete operation method
For：1. by C6 cells in blake bottle, with Trypsin Induced into single cell suspension, adjustment cell density is 4 × 106Individual/mL, it is standby
With；2. anaesthetize：After rat weight, with 10% chloral hydrate anesthesia（4ml/kg,ip）；It is three-dimensional fixed that 3. brain is fixed in rat prone position
On the instrument of position, adjusting makes bregma and posterior keep level, preserved skin, scalp median sagittal otch after iodophor disinfection, separates periosteum, exposure
Bregma；4. positioning left side caudate nucleus：3mm is opened on 0.5mm after bregma, left side；Dental burr drills after mark；5. inhaled with micro syringe
25ul single cell suspensions are taken, uniformly injects and finishes in inserting needle 5.8mm, 6min, stop 5min, point 2 slow withdraws of the needle（Each 2-3
Minute, centre stops 5min）；6. the withdraw of the needle terminates rear bone wax sealing of hole, skin suture after iodophor disinfection；Operation terminates rear rat and fed
Ditto, pay attention to warming.
4. experimental method as claimed in claim 1, it is characterised in that：Described life cycle observation be after the modeling remember
Record, until rat natural death, calculate rat the average survival time number of days.
5. experimental method as claimed in claim 1, it is characterised in that：Described tumor size is relatively molten with 4% paraformaldehyde
Cranium is opened after perfusion and takes full brain, full brain is put in rat brain slice mould, does full brain Coronal section, thickness 1mm, measurement is swollen
Knurl maximum major diameter a, maximum wide footpath b (coronal-plane, unit mm) and number of plies c, calculating gross tumor volume, gross tumor volume V=a × b × c ×
π/6。
6. experimental method as claimed in claim 1, it is characterised in that：Described blood-brain barrier permeability detection is by 10% water
Chloral solution is closed to rat anesthesia, rat, which lies on the back, is fixed on operating table, preserved skin, exposure femoral vein, puncture injection 2%（2g Yi Wen
Think blue addition 100mlPBS buffer solutions）Evans Blue solution（2ml/kg）, broken end takes mg of brain tissue 100 or so after 40min（Greatly
Mouse brain section mould cuts same aspect, and right side is used for detecting blood-brain barrier, left side detection blood tumor barrier）, it is put into after weighing
In the test tube for having 3 ml dimethylformamides, (lucifuge) extracts 24 h in 60 DEG C of water-baths, and 1500g centrifuges 10 min,
Supernatant is taken, using RF540 sepectrophotofluorometers (λ=632 nm) measure Evans blue fluorophotometric
(opticaldensity, OD), distilled water, according to optical density numerical value, calculate brain tissue as blank control from standard curve
Middle Evans blue content, the change of blood-brain barrier permeability is represented with Evans blue content (the wet brain tissues of μ g/g).
7. experimental method as claimed in claim 1, it is characterised in that：Described scanning electron microscopic observation blood-brain barrier ultra microstructure
Change：Cranium, which is opened, after being irrigated with 4% paraformaldehyde solution takes full brain, 4 DEG C of normal saline flushings 3 times；Cut the same position rat in right side
Brain tissue（Size about 3mm × 3mm × 3mm）Marked after being placed in fixer, 4 DEG C of fixed 30min；Tissue block is repaiied on slide
It is small, it is placed in the fixed 2-2.5h of 4 DEG C of fixer；Change buffer solution 2h × 3 time；4 DEG C of 1% osmic acid soaks 2h；0.1M natrium cacodylicums
Wash buffer 15min × 2 time；Dehydration：1. 4 DEG C of 30% dehydration of alcohol 10min；2. 4 DEG C of 50% dehydration of alcohol 10min；③4
DEG C 70% dehydration of alcohol 10min；4. the dehydration of alcohol 10min of room temperature 80%；5. the dehydration of alcohol 10min of room temperature 90%；6. room temperature 100%
Dehydration of alcohol 15min × 2 time；7. room temperature expoxy propane permeates 15min × 2 time；8. room temperature 1:1（Not exclusively embedding liquid:Epoxy third
Alkane）Permeate 1h；Room temperature 2:1（Not exclusively embedding liquid:Expoxy propane）Permeate 1h；Room temperature embeds liquid infiltration overnight completely；It is 9. complete
Liquid immersion is embedded, 35 DEG C of incubator 6h is placed in, is then transferred to embedding plate（Embedding liquid completely）, 42 DEG C of incubators stay overnight；10. scanning electricity
Sem observation blood-brain barrier Change of Ultrastructure.
8. experimental method as claimed in claim 1, it is characterised in that：Described immunohistochemical assay is molten with 4% paraformaldehyde
Cranium is opened after perfusion and takes full brain, brain tissue is placed in 4% paraformaldehyde solution and soaks 24h, changes 20% and 30% sucrose solution
24h (being completed in 4 DEG C of refrigerators) is dehydrated respectively, after PBS rinses, Lycra frost is placed in after embedding medium embedding is fixed and is cut
Cut into slices on piece machine（25 μm of thickness）, it is placed directly against and sticks on slide；It is placed in 37 DEG C of drying bakers and carries out antigen retrieval 1-2h；Take
Go out rear PBS to moisten brain piece；3%H2O2Deionized water is incubated 10min, to block endogenous peroxydase；PBS flushings 2min ×
3 times；Lowlenthal serum closes 40min；Primary antibody is added dropwise（Rabbit-anti rat CD3 monoclonal antibodies, rabbit-anti rat CD8 monoclonal antibodies）,
37 DEG C of incubations 2h, PBS rinse 2min × 3 time；Add secondary antibody, 37 DEG C of incubations 20min, PBS rinse 2min × 3 time；Reagent 1 is added dropwise,
37 DEG C of incubations 20min, PBS rinse 2min × 3 time；Reagent 2,37 DEG C of incubations 20min, PBS are added dropwise and rinse 2min × 3 time；It is added dropwise
Haematoxylin redyes 15min；Weathering liquid（75% alcohol 99ml+ concentrated hydrochloric acids 1ml）2-5s is soaked, pbs rinses 2min × 3 time, dried；
Graded ethanol（75%、80%、90%、100%）Dehydration（Each 2-3min）；Dimethylbenzene is dehydrated transparent 2-3min；Neutral gum mounting；It is aobvious
Micro- Microscopic observation is taken pictures.
9. experimental method as claimed in claim 1, it is characterised in that：Described RT-PCR detections are that rat directly breaks end to take
After brain, take brain tissue 100mg to be placed in tissue homogenizer and grind, add 1-1.5ml Trizol reagents, be transferred to 2ml EP pipes
In, brain tissue is fully cracked at room temperature；Add chloroform（For the 1/5 of Trizol volumes）, concussion is uniform, and room temperature places 15min；
Centrifuge 15min（4 DEG C, 12000rpm）, three layers of liquid point in pipe；Draw the superiors（RNA）It is placed in new no RNase EP pipes,
Isometric isopropanol is added, 10min is stored at room temperature after fully mixing；4 DEG C, 12000rpm centrifugation 10min, abandon supernatant, bottom
Precipitation is RNA；Adding the alcohol of 1ml 75%, mixing is cleaned to RNA up and down, 4 DEG C, 12000rpm centrifugation 10min, is abandoned
Clearly；Appropriate DEPC water dissolving is added after naturally dry, determines RNA concentration；According to Thermo companies of U.S. RNA Reverse Transcriptase kits
Reverse transcription is carried out to RNA, RT-PCR detections are carried out further according to German DBI fluorescence quantitative kits.
10. experimental method as claimed in claim 1, it is characterised in that：The Westernblotting experiments:Rat is direct
After broken end takes brain, take brain tissue 100mg to be placed in tissue homogenizer and grind, operated according to triumphant base Protein Extraction Reagent kit and carry out egg
White extraction, determination of protein concentration is carried out according to BCA kits operating method；Then plus sample-loading buffer, 100 DEG C of 5 min make
Albuminous degeneration；Take 50 μ g albumen（25 μL）Loading, carry out SDS-PAGE（90 V, 90 mi n）It is then electric to turn with protein isolate
Film（300 mA, 70 min）, PVDF films 1 h of skim milk closing；It is separately added into rabbit-anti rat GAPDH and ANXA1 monoclonal
Antibody（Volume dilution ratio is 1: 1 000）, 4 DEG C of reactions are overnight；After TBS buffer solutions wash film, IRDye 800CW marks are added
The goat antirabbit fluorescence secondary antibody of note（Volume dilution ratio is 1: 4 000）, 1 h of normal temperature hybridization；After washing film again, it is placed in
Odyssey Infrared Imaging instrument is exposed, and the gray value that protein band is carried out using ImageJ Setup softwares is swept
Retouch, the relative expression levels of each albumen are represented with the ratio between gray value of destination protein and internal reference GAPDH protein bands.
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