CN104777295A - Method for detecting cortical neuron axonal growth guidance and application thereof - Google Patents
Method for detecting cortical neuron axonal growth guidance and application thereof Download PDFInfo
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- CN104777295A CN104777295A CN201510199039.5A CN201510199039A CN104777295A CN 104777295 A CN104777295 A CN 104777295A CN 201510199039 A CN201510199039 A CN 201510199039A CN 104777295 A CN104777295 A CN 104777295A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5058—Neurological cells
Abstract
The invention relates to a method for detecting cortical neuron axonal growth guidance. The method comprises the following steps: preparing a stamp with a microscopy channel on a mold through polydimentylsiloxane, buckling the stamp on a glass slide for coating poly-L-lysine, adding a solution containing guidance factors and fluorescent molecules from one side of the channel so as to form parallel stripes with equal intervals after entering ducts; and performing neuron cultivation on the glass slide coated with the fluorescent stripe, observing whether newborn axon enters the stripe containing the guidance factors, and computing proportion of the newborn axon and the stripe, statistically analyzing. The method disclosed by the invention is not only in favor of understanding the forming mechanism of the neural circuit in a brain, but also has potential treatment value to the axonal degeneration and axonal injury diseases. On the basis of the method for detecting the cortical neuron axonal growth guidance, a disease model is established, the specificity expression of the secretion factor in the brain in the time and the space is simulated through exogenously giving cell secretion factor, the functional reconstruction of the neutron is induced, and the experimental basis is established for clinical translational medicine for treating above diseases.
Description
Technical field
The present invention relates to Neuscience technical field, specifically, is a kind of method and application thereof of detecting cortical neuron axon growth guiding.
Background technology
In brain development process, neuron differentiation goes out the different region of aixs cylinder and dendron two functions, and forms synaptic contact with other neurons, carries out information transmission.In axon growth process, the path finding growing distance be carried out, set up with neuron at a distance and contact.This process need is subject to strict regulation and control, could form exquisite neural circuit, complete the Premium Features of brain.Corticocerebral newborn neuron, after ventricles of the brain wall (Ventricular zone, VZ) produces, is divided into the cell (Beale's ganglion cells) of two projections in front and back, along radial neuroglia fiber to external migration.In transition process, leading projection is divided into apical dendrite, and rear urogomphus rises and is divided into aixs cylinder, thus in brain neuronic axle/dendron differentiation and migration carry out together.It should be noted that, the direction of growth of aixs cylinder is not consistent with neuronic migratory direction, but along cortex mesozone (Intermediate zone, and Xia Shi district (Subventricular zone IZ), SVZ), the tangential direction being 90 degree with migratory direction grows, and forms axonal conduction bundle.
The growth of aixs cylinder has strict directivity, and axon ends is called growth cone, and growth cone is extremely responsive to surrounding environment, the guide molecule on its surface receptor identifiable design extracellular matrix or peripheral cell.Growth factor or cell factor can be secreted in some region in brain, and peripherad regional diffusion, form concentration gradient, have directive function to the direction of growth of neuronic migratory direction and aixs cylinder.Guidance cues is divided into the attractability Summing Factor repellency factor, after they are combined with corresponding membrane receptor, by affecting Ca according to the difference of effect
2+, the signaling molecule such as cAMP, RhoGTP enzyme level, finally cause the reconstruct of cytoskeletal protein and the change of the direction of growth.
SDF-1 (Stromal cell-derived factor-1, CXCL12) is the cell factor of induction of lymphocyte chemotactic response.The mouse of gene knockout SDF-1 or SDF-1 receptor CXCR 4, show similar neurodevelopment defect, cerebellum, hippocampus and neocortex lose layer structure clearly, and prompting neuron can not move to correct position, form orderly flaggy arrangement.Studies have found that, the granular cell in SDF-1 participation regulation and control brain and the directional migration of intrerneuron, can also promote growth and the prolongation of cerebellar granule cell and spinal cord DRG neuron axon.SDF-1 plays an important role in the growth guiding of aixs cylinder.BDNF (brain-derived neurotrophic factor, Brain Derived Neurotrophic Factor) is a kind of neurotrophic factor of wide expression in brain, has the effect promoting nerve growth, survival, regeneration, migration.There are some researches show, BDNF may be a kind of chemical attraction factor to neural axon, and it is worked by acceptor TrkB.
There is the factor of guide effect can be divided into two large classes according to its signal characteristic to nerve fibre, I quasi-molecule comprises netrin, BDNF, MAG, Ach etc., their guide effect depends on extracellular Ca2＋, produce and attract or repel cAMP or the PKA activity level depending on neurocyte inside, when in born of the same parents during cAMP level height, these factors produce sucking action; Otherwise, when cAMP level is low, produce repulsive interaction.II quasi-molecule comprises slit, NT3, Semaphorin etc., their guide effect does not rely on extracellular Ca2＋, performance attraction or repulsive interaction are decided by the activity level (Yuan little Bing of cGMP or PKG in born of the same parents, Cdc42 and RhoA can mediated cell extrinsic factor to the attraction of aixs cylinder and repulsive interaction, Postgraduate School, Chinese Academy of Sciences, 2001).
At present, for detect neuron axon lead the most frequently used method be Mr. Pu Muming found growth cone turn to analytical approach, the ultimate principle of the method is in glass capillary, pour into the guidance cues solution needing research, constantly the injection of solution in kapillary is out formed stable concentration gradient with a certain size pneumatic pressure pulses with certain frequency, capillary tip is placed in the side in the Xenopus laevis spinal neuron Axonal growth cone front of in vitro culture, under the effect of concentration gradient, growth cone will grow towards or away from the direction of capillary tip, namely guidance cues has played attraction or repulsive interaction.But this kind of method needs growth selection to bore comparatively large neuron (as Xenopus laevis spinal neuron), can only the direction of simple observation guide molecule on growth cone and the impact of structure.
Summary of the invention
The object of the invention is for deficiency of the prior art, a kind of method for detecting neuron axon growth guiding is provided.
Another object of the present invention is, provides the purposes of said method.
For achieving the above object, the technical scheme that the present invention takes is:
For detecting a method for cortical neuron axon growth guiding, comprise the steps:
A. preparation table wears the PDMS seal of microchannel
First the performed polymer of PDMS and coupling agent are uniformly mixed, pour casting mold into after vacuumizing degasification, be heated to 80 DEG C and be incubated 1h solidification, the demoulding obtains PDMS seal.
Described PDMS seal lower surface is with the equal parallel microscopic channels in interval, and the channel width on PDMS seal surface is 60 μm, height is 100 μm, is spaced apart 90 μm between each parallel passage.
B. band bag quilt
Double dish put into by the slide cancelling poison, add poly-D-lysine and carry out bag quilt, the PDMS seal prepared is buckled on the slide of poly-D-lysine bag quilt, activity is diluted to by needing the guidance cues FITC-BSA of research, then join one end, microchannel of seal, aspirate with passage other end pump and make solution be full of whole passage, after solution dries, remove seal, slide is formed the parallel stripes containing guidance cues.
C. neuron plantation
Slide obtained in step b is placed on ice, adds Matrigel glue, Matrigel glue carries out neuron cultivation.
D. dyeing and analysis
The neuron of immunofluorescence technique to plantation is used to dye, whether the aixs cylinder of observation new life enters the band containing guidance cues, calculate in newborn aixs cylinder the ratio entered containing guidance cues band, and carry out statistical study, determine that this kind of guidance cues has the effect attracting or repel to aixs cylinder according to analysis result.
In described step b, poly-D-lysine concentration is 0.1mg/ml.
In described step c, Matrigel glue addition is 50 μ l/cm
2.
For realizing above-mentioned second object, the technical scheme that the present invention takes is:
Described method plant detecting cortex block neuron axon growth guiding, detecting guidance cues, cortex to be planted to block neuron axon be attract or the application in repulsive interaction.
The present invention relates to a kind of method and the application thereof that detect cortical neuron axon growth guiding, first get out mould, described mould can be made up of the material of resistance to more than 100 DEG C of silicon, metal, pottery, glass etc.; Be preferably silicon master mold.Die surface is carved with the equal parallel fine groove in interval, and the groove of die surface wide is 90 μm, and groove is high is 100 μm, is spaced apart 60 μm between each groove.By dimethyl silicone polymer (the PDMS glue of mixing and stirring, comprise performed polymer and coupling agent) vacuumize de-bubble, pour mould into, then dimethyl silicone polymer curing molding is made through vacuumizing, heating, obtain the PDMS seal that surface has parallel microchannels, each channel width is 60 μm, and height is 100 μm, is spaced apart 90 μm between the passage be parallel to each other.Lurching of microchannel is had by seal to be buckled in bag by the slide of poly-D-lysine, duct is formed between the microchannel of seal and slide, the guidance cues FITC-BSA that will study is diluted to activity, join the one end, duct between seal and slide, other end vacuum pump suction in duct makes the solution containing guidance cues be full of whole passage, seal is removed after solution dries, slide is formed the parallel stripes containing guidance cues, bar bandwidth is 60 μm, is spaced apart 90 μm between parallel stripes.Neuron cultivation is being carried out containing on the parallel stripes of guidance cues, immunofluorescence technique is utilized to dye, whether the aixs cylinder of observation new life enters the band containing guidance cues, calculate the ratio entering the band containing guidance cues in newborn aixs cylinder, carry out statistical study, determine that this kind of guidance cues has the effect attracting or repel to aixs cylinder according to analysis result.
The present invention utilizes dimethyl silicone polymer to prepare seal, and dimethyl silicone polymer has excellent optical clarity, nontoxicity and biocompatibility, can according to different stripe size needs, and mold the seal of different size, method is flexible.
Apply method of the present invention, can deeply disclose in brain environment, the mechanism of axon guidance is moulded in the interaction of different guidance cues sequential expression in time and multiple guidance cues jointly.Method of the present invention not only contributes to the Forming Mechanism understanding neural circuit in brain, and has potential therapeutic value to axonal degeneration and axonal injury class disease.On basis of the present invention, set up disease model, give cell-secretion factor by exogenous, specific expressed in the time and space of simulation brain endocrine factor, the reconstruction of Induction of neuronal, for the clinical translational medicine for the treatment of above disease establishes experiment basis.
The invention has the advantages that:
1, at present to the research that neuron axon is grown and guiding is machine-processed, major part utilizes experiment in vitro, studies the single molecular signaling mechanisms affecting growth cone guiding; Methods combining cortical neuron aixs cylinder of the present invention tangentially grows this physiological phenomenon in IZ/SVZ district, simulation brain environment, explores the impact that the interaction of multiple factor and order of representation in time lead on axon growth.
2, the model of existing research axon guidance mechanism is the Xenopus laevis spinal neuron that growth selection cone is larger, the direction of simple observation guide molecule on growth cone and the impact of structure; The present invention selects the Fetal Rat cerebral hippocampal district neuron cultivated, and utilizes unique microscopic imprint technology, sets up single neuron axon growth guided modes, observes guidance cues to the attraction of aixs cylinder or repulsive interaction, better simulate brain environment.
3, in the past to the research of cAMP signal, be utilize molecular biology method measured signal content, cannot the distribution and variation of realtime dynamic observation signal level; The present invention utilizes advanced FRET imaging technique, in Real Time Observation aixs cylinder cAMP activity change and at intracellular conductive process.
4, study SDF-1 in the effect of central nervous system in the past, all concentrate on the growth prolongation aspect of neuronic directional migration and aixs cylinder; The present invention pays close attention to the guide effect of SDF-1 in aixs cylinder path finding process first.
Accompanying drawing explanation
Accompanying drawing 1 prepares newborn neuron axon growth photo for utilizing method of the present invention.
The schematic diagram of accompanying drawing 2 for utilizing PDMS seal to prepare parallel stripes on slide; Wherein a part is slide seal being buckled in poly-D-lysine bag quilt; B part is the PDMS seal of lower surface with microchannel; The parallel stripes of c part for being formed after removing seal.
Embodiment
Below embodiment provided by the invention is elaborated.
Embodiment
One, the preparation of PDMS seal
Starting material and instrument: (die surface is carved with the equal parallel fine groove in interval to mould, and groove is wide is 60 μm, and groove is high is 100 μm, is spaced apart 90 μm between each groove.), dimethyl silicone polymer (PDMS) (Momentive RTV 615A liquid, B liquid, Suzhou Wen Hao chip Science and Technology Ltd.), thick matter aluminium foil, double dish (150mm); Vacuum tank, baking box, scalpel.
Flow process:
1, large double dish middle berth aluminium foil, weighs up A liquid, B liquid (being respectively performed polymer and coupling agent), fully stirs, and is rolled up at the edge of aluminium foil, and puts to vacuumize 10min in vacuum tank (note 1: when vacuumizing, can take out always; Also can take out while put);
2, new double dish middle berth aluminium foil, flattens around it, mould is being put on aluminium foil, the glue vacuumized in 1 is poured into (note 2: before being poured on mould, if also have bubble, available punctures) in the groove of mould, take out 5min vacuum again, be placed on 85 DEG C of 1h;
3, aluminium foil and mould are taken out from double dish, with edge careful separate of suitable scalpel along mould, remove aluminium foil, seal is taken out, cutting.Seal size: length and width 10mm × 5mm, its lower surface is with the equal parallel microscopic channels in interval, and channel width is 60 μm, height is 100 μm, is spaced apart 90 μm between each parallel passage.
Two, band bag quilt
Experiment reagent and apparatus: poly-D-lysine (PDL, Sigma company), band slide double dish (35mm+20mm slide), aqua sterilisa, absolute ethyl alcohol, soap, FITC-BSA (Life Technologies company), CXCL12 (R & D company), pipettor, rifle head; Vacuum pump, super-clean bench.
Flow process:
1, the PDMS seal prepared is placed in suds 70 DEG C to spend the night, cleans with distilled water, and spend the night with alcohol at normal temperature cleaning, ultraviolet disinfection; Band slide double dish PDL poly-D-lysine (0.1mg/ml) 4 DEG C spends the night, and aqua sterilisa dries after cleaning three times.
2, seal is placed on the slide in double dish, check whether bubble, the factor FITC-BSA of seal will be diluted to activity, then in addition in one end of seal, aspirate at other end vacuum pump, make solution be full of whole seal duct, form the parallel stripes (Fig. 2) that interval is identical.
3, be placed into super-clean bench, spend the night and dry.
4, seal is taken away, carry out subsequent experimental.
Three, cortex plants the plantation of block
Animal: pregnant 18d SD rat;
Experiment reagent and utensil: HBSS, Matrigel glue (BD company), the rifle head of precooling, eye scissors, anatomical lens, CO
2incubator.
Flow process:
1, cortex plants the preparation of block: the SD rat of the pregnant 18d of 10% chloral hydrate anesthesia, take out Embryonic Cerebral Cortex, be placed in ice-cold HBSS (Life Technologies company) solution, after peeling off meninx under anatomical lens, fritter is torn into ophthalmic tweezers, for subsequent use;
2, Matrigel Jiao Bao quilt: after thawed on ice Matrigel glue, with the rifle head mixing Matrigel glue of precooling.Be placed on ice, by 50 μ l/cm by wrapping before by the double dish of the band slide crossing band
2the concentration of growth area adds Matrigel glue.Place 30 minutes at 37 DEG C, can use;
3, cortex plants the plantation of block: planted on Matrigel glue by fritter, then adds the Matrigel glue mixed with the rifle head of precooling, is placed in 37 DEG C of incubators 30 minutes, then adds the Neurobasal nutrient culture media containing 1%B27, insert 5%CO
2, 37 DEG C of incubators are cultivated.
Four, dye
Experiment reagent and utensil: PBS, paraformaldehyde (PFA), TritonX-100, BSA, mountant, cover glass, primary antibodie polyclonal rabbit anti-Rat Tuj1 (Sigma), two anti-Alexa Fluor 568GoatAnti-Rabbit IgG (Life Technology).
Flow process:
1, use vacuum pump Aspirate medium, add rapidly the PFA solution of 1ml 4 DEG C, leave standstill 15 minutes, cell is fixed;
2, blot liquid, add 1ml PBS, leave standstill 5 minutes, clean.Repeated washing three times;
3, blot PBS, add 0.5-1ml, the Triton X-100 of 0.4% leaves standstill 10-15 minute, carries out rupture of membranes.Three times are cleaned afterwards with PBS;
4, blot liquid, add the BSA of 0.5-1ml 1%, leave standstill 1 hour, close;
5, blot liquid, add primary antibodie polyclonal rabbit anti-Rat Tuj1 (1:200) 200ul with BSA preparation, 4 DEG C of overnight incubation;
6, primary antibodie is hatched complete, cleans three times with PBS.Add two anti-Goat Anti-RabbitIgG (Alexa Fluor 568, the 1:400) 200ul with PBS preparation, normal temperature hatches 1-2 hour;
7, PBS cleans three times;
8, add 1ml PBS 4 DEG C to keep in Dark Place, or with anti-cancellation mountant mounting, normal temperature keeps in Dark Place.
Five, result
Accompanying drawing 1 is when containing BSA, SDF-1, BNDF in band respectively, newborn neuron axon growth photo.As can be seen from the figure, time in band containing BSA, neuron axon self-sow, band grows without guide effect it; When in band containing SDF-1 time, newborn neuron axon along between band containing the region growing of SDF-1; When containing BDNF in band, the region growing of newborn neuron axon containing BDNF in band.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.
Claims (5)
1. detect a method for cortical neuron axon growth guiding, it is characterized in that, comprise the steps:
A. preparation table wears the PDMS seal of microchannel
The performed polymer of PDMS and coupling agent are uniformly mixed, pour casting mold into after vacuumizing degasification, be heated to 80 DEG C and be incubated 1h solidification, the demoulding obtains PDMS seal; Described PDMS seal lower surface is with the equal parallel microchannels in interval;
B. band bag quilt
Double dish put into by the slide cancelling poison, add poly-D-lysine and carry out bag quilt, the PDMS seal prepared is buckled on the slide of poly-D-lysine bag quilt, activity is diluted to by needing the guidance cues FITC-BSA of research, then join one end, microchannel of seal, make solution be full of whole passage, after solution is dry in the suction of the passage other end, remove seal, slide is formed the parallel stripes containing guidance cues;
C. neuron plantation
Slide obtained in step b is placed on ice, slide adds BD Matrigel, Matrigel carries out neuron cultivation;
D. dyeing and analysis
Immunofluorescence technique is used to dye, whether the aixs cylinder of observation new life enters the band containing guidance cues, calculate the ratio entering the band containing guidance cues in newborn aixs cylinder, and carry out statistical study this kind of guidance cues has attraction or repulsion effect to aixs cylinder.
2. method according to claim 1, is characterized in that, the channel width of described PDMS seal lower surface is 60 μm, height is 100 μm, is spaced apart 90 μm between each parallel passage.
3. method according to claim 1, is characterized in that, in described step b, poly-D-lysine concentration is 0.1mg/ml.
4. method according to claim 1, is characterized in that, in described step c, Matrigel glue addition is 50 μ l/cm
2.
5. cortex plants block neuron axon growth guiding to the arbitrary described method of claim 1-4, detecting guidance cues, to plant block neuron axon to cortex be attract or the application in repulsive interaction detecting.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105067810A (en) * | 2015-07-16 | 2015-11-18 | 上海市第十人民医院 | Method for detecting migration polarization of primary NSCs (neural stem cells) and application of method |
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| WO2002042779A2 (en) * | 2000-08-30 | 2002-05-30 | Georgetown University | Axonal response to chemotropic gradients |
| CN1861779A (en) * | 2005-05-09 | 2006-11-15 | 财团法人工业技术研究院 | Neural process stretching guiding apparatus and mfg. method thereof |
| CN104130943A (en) * | 2014-07-28 | 2014-11-05 | 民航总医院 | Neuronal and neuroglial cell ordered co-culture device and preparation method thereof, and neuronal and neuroglial cell ordered co-culture method |
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Cited By (1)
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| CN105067810A (en) * | 2015-07-16 | 2015-11-18 | 上海市第十人民医院 | Method for detecting migration polarization of primary NSCs (neural stem cells) and application of method |
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