CN108728418A - A kind of preparation method and application of the single neuron dynamic studies model of chicken embryo central nervous system - Google Patents
A kind of preparation method and application of the single neuron dynamic studies model of chicken embryo central nervous system Download PDFInfo
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- CN108728418A CN108728418A CN201810630993.9A CN201810630993A CN108728418A CN 108728418 A CN108728418 A CN 108728418A CN 201810630993 A CN201810630993 A CN 201810630993A CN 108728418 A CN108728418 A CN 108728418A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
- G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
Abstract
The present invention relates to a kind of preparation method and applications of the single neuron dynamic studies model of chicken embryo central nervous system, belong to gene functional research technical field.Live body electricity transgenic technology and tissue culture are combined by the present invention, to carry out the gene functional research of the single neuron of chicken embryo central nervous system, specifically by chicken embryo spinal cord or optic tectum tissue through live body electricity transgenic technology, obtain genetically modified organism block, histotomy is cultivated after being sliced, foreign gene functional study is carried out after culture.It can be with the single complete structure of neuron visible in detail in the tissue obtained using the method culture of the present invention, and the projection of neuron axon is more regular, the tissue of culture of the present invention does not pass through the digestion of enzyme, it is also influenced simultaneously by other cells of tissue and related secretion factor, closer to live body, the single neuron in tissue can simulate the process of the in vivo dynamic change of neuron.
Description
Technical field
The present invention relates to a kind of preparation method and application of the single neuron dynamic studies model of chicken embryo central nervous system,
Belong to gene functional research technical field.
Background technology
Organotypic tissues' piece culture technique is a kind of effective vitro tissue cultural method, and the tissue of culture can be kept
The almost connection between normal cell, and almost therefore normal physiological function and morphosis feature avoid for holding
The shortcomings that cell culture of routine and in vivo experimental study.Organotypic tissues' piece culture is development of central nervous system process
Research provides a kind of good method.It is in 1981 that this technology, which really makes a breakthrough,(BH(1981)Organotypic monolayer cultures of nervous tissue.J Neurosci
Methods 4:329-342) the rolling tubular type culture developed.Using the cultural method, the brain pieces of different parts can be cultivated several
The time in week.In (Stoppini L, Buchs PA, Muller D (1991) the A simple method such as 1991, Stoppini
for organotypic cultures of nervous tissue.J Neurosci Methods 37:173-182) utilize
The culture that rat hippocampus is carried out with transparent and culture dish with a certain size aperture film, obtains well as a result, simultaneously
Establish a kind of simple organotypic brain piece culture technique.The brain piece of culture can successfully simulate in vivo neuronal development
Process, and it is capable of providing the experimental result as in vivo organ.Therefore, it is raw to have been applied to many nerves for this method
Object research field.
Currently, organotypic brain piece culture technique only sees the report of mouse, rat, rabbit and monkey isotype animal
(Mewes A,Franke H,Singer D(2012)Organotypic brain slice cultures of adult
transgenic P301S mice--a model for tauopathy studies.PLoS One 7:e45017；
Harwell CS,Coleman MP(2016)Synaptophysin depletion and intraneuronal Aβin
organotypic hippocampal slice cultures from huAPP transgenic mice.Mol
Neurodegener 11:44；Minami N,Maeda Y,Shibao S,Arima Y,Ohka F,Kondo Y,Maruyama
K,Kusuhara M,Sasayama T,Kohmura E,Saya H,Sampetrean O(2017)Organotypic brain
explant culture as a drug evaluation system for malignant brain tumors.Cancer
Med6:2635-2645；Lee NM,Chae SA,Lee HJ(2017)Effects of neural stem cell media
on hypoxic injury in rat hippocampal slice cultures.Brain Res 1677:20-25), do not have
It is reported about the research of chicken embryo organotypic brain piece culture.Chicken embryo is a kind of good experimental model animal, in Developmental Biology
It is widely used with neurobiological study field, especially chicken embryo development of central nervous system process.Chicken embryo is dynamic as pattern
The advantages of object has its own, such as abundance, easily operated and be easy to get material.With chicken embryo live body electricity transgenic technology
Occur, realizes that the unconventionality expression of chicken embryo central nervous system foreign gene is possibly realized in chick embryo development process, but external source base
Because of the phylogenetic influence of Central nervous after unconventionality expression, the research of live body and cellular level is only seen at present.
Invention content
The object of the present invention is to provide a kind of preparation sides of the single neuron dynamic studies model of chicken embryo central nervous system
Method, this method can cultivate animal tissue in vitro, simulate the growth and development process of animal in-vivo tissue, be developed for chicken embryo spinal cord
Foreign gene unconventionality expression research in the process provides a kind of new method.
The present invention also provides the applications of above-mentioned dynamic studies model preparation method.
To achieve the goals above, the technical solution adopted in the present invention is：
A kind of preparation method of the single neuron dynamic studies model of chicken embryo central nervous system, includes the following steps：
1) plasmid containing target gene is transferred to chicken embryo spinal cord or chicken embryo optic tectum group by the method for using live body electricity to turn
In knitting, genetically modified organism block is obtained；
2) genetically modified organism block is wrapped up with gel, obtains package block；The package block is sliced, tissue is obtained and cuts
Piece；Obtained histotomy is cultivated, is observed.
Specifically, histotomy is transferred on the microporous barrier of suspension type Tissue Culture Dish, then suspension type cell is trained
Foster ware is transferred in ordinary cells culture dish, and cell culture medium is added and is cultivated.
After electricity turns spinal cord, continue to cultivate embryonic development to materials slice is carried out at 5.5-6.5 days, with research purpose gene
Dynamic function in single neuron；After electricity turns optic tectum tissue, continue to be taken when cultivating embryonic development to 11-13 days
Material is sliced, with dynamic function of the research purpose gene in single neuron.
A kind of single neuron dynamic studies model preparation method of chicken embryo central nervous system, the party are established in the present invention
Method is established based on chicken embryo live body electric transgenic technology in situ, and this method is suitble to chick embryo development process hub nervous system single
The research of the dynamics variation of neuron, the also research for chicken embryo development of central nervous system process foreign gene unconventionality expression carry
A kind of new strategy is supplied.
When the genetically modified organism block is optic tectum tissue, the step of package, is：Gel piece is pasted into tissue
On the pallet of slicer；Then cut out slot in gel piece side, by chicken brain olfactory bulb tip cut-off portion of tissue, olfactory bulb end downward,
Optic tectum is put into the slot of gel, is also pasted on the pallet of histotomy.In the present invention when wrapping up optic tectum tissue, gel
Block pre-production, therefore injury of the coagulant liquid of heat for tissue cell can be excluded.
The package or embedding, specific step are：Genetically modified organism block is embedded with liquid agar, it is fast
Quickly cooling but after according to tissue size trimmed.The liquid agar is cooled to 39-41 DEG C in advance before embedding, is preferably pre-chilled to 40
℃。
The gel can be agar or agarose, the preferably agar of 3.5-4.5%, further preferably 4%
Agar.Concentration is too high to cause agar block brittleness to increase, and concentration too soft is inadequate, do not have the fixed effect of embedding.
The thickness of the histotomy is 250-350 μm, preferably 300 μm.The too thin operation difficulty of slice increases, and is easy broken
Broken, the too thick tissue of histotomy can only switch to seldom several slices.
Histotomy is placed in artificial cerebrospinal fluid after the slice.Ice cube is added in slicer box when the slice.
The drying and denaturation of tissue can be prevented during slice in this way.
The cell culture medium is grouped as by the group of following volume parts：48 parts of Neurobasal, Serum-Free
0.5 part of 1 part of Supplement, 0.5 part of GlutaMAX and penicillin-streptomycin.
The formula of artificial cerebrospinal fluid is：120mM NaCl, 3.5mM KCl, 1.3mM MgCl2, 2.5mM CaCl2, 1.25mM
NaH2PO4, 25.6mM NaHCO3With 10mM glucose.
The step of live body electricity turns be：
1) plasmid containing target gene is injected into the chicken of culture 2.5-3.5 days with microinjection capillary glass needle
In embryo spinal cord；Or the plasmid containing target gene is injected into the chicken of culture 4-5 days with microinjection capillary glass needle
Embryo optic tectum tissue；
2) electrode is placed on to spinal cord or the optic tectum tissue both sides of oneself injection plasmid, electricity is carried out and turns.
Specifically, electricity turn when by the plasmid 0.15-0.25 μ L of 0.25 μ g/ μ L with microinjection capillary glass needle stereoscopic
It is injected under microscope in the chicken embryo live body spinal cord of culture 2.5-3.5 days；Or by the plasmid 0.45-0.55 μ l of 0.25 μ g/ μ L
The chicken embryo optic tectum tissue of culture 4-5 days is injected under stereomicroscope with microinjection capillary glass needle.
When electricity turns spinal cord, the parameter used is total electric shock 5-7 times, and voltage 17-19V, shock by electricity 55-65ms every time,
Every 95-105ms；When electricity turns optic tectum tissue, the parameter used is total electric shock 5-7 times, voltage 14-16V, is shocked by electricity every time
55-65ms is spaced 95-105ms.
Above-mentioned preparation method can be used for the research of dynamic function of the target gene in single neuron.
Specifically, after electricity turns spinal cord, continue to cultivate embryonic development to materials slice is carried out at 5.5-6.5 days, with research
Dynamic function of the target gene in single neuron；After electricity turns optic tectum tissue, continue to cultivate embryonic development to 11-13 days
Shi Jinhang materials slices, with dynamic function of the research purpose gene in single neuron.
The beneficial effects of the invention are as follows：
The preparation method of the single neuron dynamic studies model of chicken embryo central nervous system in the present invention is a kind of will to live
Body electricity transgenic technology and tissue culture are combined the new method for carrying out gene functional research, can be to foreign gene in chicken embryo
Function during development of central nervous system is analyzed in individual cells level, can realize chicken embryo central nervous system
The research for the influence that growth course foreign gene unconventionality expression forms neuronal migration and aixs cylinder.The technology is that foreign gene exists
Influence of the chicken embryo central nervous system to neuron provides a kind of new research method, is one between live body and cell
Kind investigative technique.
Migration and the aixs cylinder path of single neuron can be observed in the experimentation of the present invention in the tissue of culture
The indexs such as selection；Although the environment and vivo environment of culture, there are difference, single neuron in the tissue of culture can be with
Simulate the process of the in vivo dynamic change of neuron.
It is difficult to observe complete single neuron in histotomy in vivo for common in-vivo tissue slice
Structure, but can be with the single complete structure of neuron visible in detail in the tissue cultivated of the present invention.With detach
The neuron cultivated afterwards is compared, and the neuron cultivated after separation can be longer compared with the neuron in tissue culture of the present invention,
But the projection of the neuron axon in tissue is more regular.The neuron of cultured tissue on piece of the present invention and the list detached
A neuron is compared, and does not pass through the digestion of enzyme, while also being influenced by other cells of tissue and related secretion factor, more
Close to live body.
Description of the drawings
Fig. 1 is chicken embryo central nervous system single neuron dynamic studies model preparation method each stage of embodiment 1
Operation diagram, A, B：Chick embryo culture；C：Remove 3-4ml egg white；D：Inject plasmid；E：Live body electricity turns；F：Chicken embryo sealing culture；G-I：
Materials；J-K：The collection of GFP positive expression spinal cords；L：In organization embedding to 4% agar；M：Histotomy；N-O：Tissue
It is transferred on suspension type culture dish film；Scale=5mm；
Fig. 2 is chicken embryo central nervous system single neuron dynamic studies model preparation method each stage of embodiment 2
Operation diagram；A：Plasmid is injected；B：Electric shock；C：Chicken brain when culture is to E12 after electricity turns；D-F：The collection of GFP positive expression chicken brains；
G：4% agar tissue block；H-I：Side cuts out the agar block of groove；J：The tissue block of 4% agar embedding；K：Histotomy；L：
Tissue is transferred to suspension type culture dish film and is transferred in 30mm culture dishes；Scale=5mm；
Fig. 3 is histotomy culture schematic diagram in embodiment 1 and embodiment 2；A-F：Myeloid tissue's piece culture；G-I：Depending on top
Lid tissue culture；J：Tissue training mode figure；Scale=5mm；
Fig. 4 is the myeloid tissue's piece cultivated in embodiment 1 and common biopsy institutional framework and neuron morphology
The contrast difference of structure schemes；A-C：The histotomy of control group pCAGGS-GFP positive expressions, A when E6：The cell of DAPI dyeing
Core, B：GFP expressions of results, C：The figure of superposition；D-F：When E6 after histotomy culture 48h pCAGGS-GFP positive expressions group
It knits；D：The nucleus of DAPI dyeing；E：GFP expressions of results, F：The figure of superposition；G-L：Cultured tissue piece is individually neural in spinal cord
Member, G：The nucleus of DAPI dyeing, H：The tissue of GFP positive expressions, I：Stacking chart；J is the partial enlarged view in G figures, K H
In partial enlarged view, L be I in partial enlarged view；Abbreviation：Sp is spinal cord, and drg is dorsal root ganglion；Arrow in B-C, E-F
Head expression commissural fibre, scale, 200 μm；
Fig. 5 is the optic tectum tissue cultivated in embodiment 2 and common biopsy institutional framework and neuron shape
The contrast difference of state structure schemes；A-C：Control group, the histotomy of pCAGGS-GFP positive expressions when E12；A：DAPI dyeing
Nucleus；B：GFP expressions of results, C are the amplified stacking charts of A and B；D-F：The tissue of pCAGGS-GFP positive expressions when E12
Cultivate the result after 48h；D：The nucleus of DAPI dyeing, E：GFP expressions of results, F are the result being superimposed after D and E amplifies；G-N：
The result of the series stratification scanning of optic tectum F figures；C：Arrow indicates single neuron；F-N：Arrow indicates that different layers are individually neural
Member；Scale, 200 μm.
Specific implementation mode
With reference to specific embodiment, the present invention is described in further detail.Fertilization kind egg purchase in following embodiment
It buys in kind of a chicken house, is cultivated in constant temperature and humidity incubator (HWS-150, essence is macro, China) incubator, cultivation temperature 37.8
DEG C, relative humidity 65%.It is tested when embryonic development is to E2.5, is drawn materials the latest in E12, every group of experiment is at least
Collect 5 embryos.Reporter gene pCAGGS-GFP plasmids (green fluorescent protein) are presented to so-and-so by this laboratory.With in plasmid in experiment
Green fluorescent protein (GFP) be used as reporter gene, all plasmids (pCAGGS-GFP) use the big extraction reagent kit of plasmid (health for, in
State) extraction, and concentrated using the NaCl and isopropanol of 5M, and use dissolved in purified water, final concentration of 2mol/L.
In addition to specified otherwise, equipment used in each embodiment and the equal conventional commercial of reagent can obtain.
Embodiment 1
The preparation method of the single neuron dynamic studies model of chicken embryo central nervous system in the present embodiment, including walk as follows
Suddenly：
1, spinal cord live body electricity turns to obtain genetically modified organism block：
Live body electricity shifting method specific operation process：Fertilized eggs lie in a horizontal plane in incubator culture to E2.5 when (Fig. 1, A,
B), 3-4mL egg white is removed in blunt end trepanning syringe, is open with scissors right over side, diameter 1-2cm (Fig. 1-
C).0.01% fast green dye liquor is added in the pCAGGS-GFP plasmids of 0.25 μ g/ μ L, capillary glass tube needle draws the matter mixed
Grain, is injected into spinal cavity (Fig. 1-D) by 0.2 μ L plasmids under stereomicroscope, the electrode of electroporation is placed in spinal cord both sides horse
Upper progress electricity turns (Fig. 1-E), and it is voltage 18V, pole time 60ms that electricity, which turns parameter, is spaced 100ms, is shocked by electricity 6 times.Electricity removes after turning
Opening medical proof fabric is sealed (Fig. 1-F) by electrode.By chicken embryo put back to incubator continue culture to E6 when, prepare draw materials simultaneously be sliced
Culture.
2, histotomy in vitro culture：
1) when embryonic development is to E6, select Stereo fluorescence microscope under visual report gene GFP embryos (Fig. 1, G-I) simultaneously
GFP positives position is cut for being sliced (Fig. 1-J, K).The tissue of selection is embedded with 4% agar, it is before embedding that agar is pre-
It is as cold as 40 DEG C (Fig. 1-L).
2) (VT1200S, Leica, Germany) is sliced in oscillating slice machine, and slice thickness is 300 μm, slice
After be quickly transferred to that on 30mm suspension type culture dish films (PICM03050, Millipore) (Fig. 1, M-O), ware is transferred to 30mm
In culture dish, 1ml culture mediums are added and are cultivated.
Culture medium prescription：48ml of Neurobasal (Thermo Fisher, USA), 1ml'sSerum-
The 1%GlutaMAX (Thermo Fisher, USA) of Free Supplement (50X) (Thermo Fisher), 0.5ml and
The 1%penicillin-streptomycin (Sigma) of 0.5ml is mixed.
Tissue is dry in order to prevent and is denaturalized for slicing processes, ice cube is added in slicer box, and be added in being sliced box
Artificial cerebrospinal fluid (Artificial cerebrospinal fluid, ACSF).
The culture formula of ACSF is：120mM NaCl, 3.5mM KCl, 1.3mM MgCl2, 2.5mM CaCl2, 1.25mM
NaH2PO4, 25.6mM NaHCO3With 10mM glucose.
3) culture dish is transferred in carbon dioxide incubator in 37 DEG C and 5%CO2Cultivated under condition of culture (Fig. 3,
B, H), culture solution is replaced in tissue culture afterwards for 24 hours, and culture 7d can be continued by replacing culture solution primary structure piece every 48h later
Time.Cultured tissue morphological structure and neuron 48h can laser confocal microscope (Olympus ix81,
Japan it is observed under).
In spinal cord slice incubation, live body electricity turn is carried out when embryonic development is to E2.5, continues culture when embryo sends out
It draws materials when educating E6, the embryo of GFP positive expressions is selected under fluorescence microscope, the region for cutting the GFP positives is cut
Piece (Fig. 3, A-F).6-8 piece tissues can be placed on each culture dish film and are cultivated (Fig. 3-A), in tissue incubation
Enough spaces, simultaneous selection and the tissue (Fig. 3, B, C) for retaining GFP positive expressions are reserved between tissue.In order to ensure
Fold cannot occur in tissue on accurate experimental result film, and GFP is selected only to express tissue (Fig. 3, D- to spinal cord side
F)。
Embodiment 2
The preparation method of the single neuron dynamic studies model of chicken embryo central nervous system in the present embodiment, including walk as follows
Suddenly：
1, optic tectum live body electricity turns to obtain genetically modified organism block：
Optic tectum live body electricity turns specific method：6mL egg white is removed when chick embryo development is to E2.5, continues culture to E3.5
When, with scissors from lateral opening, openings of sizes 1-2cm.By the fast green mixing of the pCAGGS-GFP and 0.01% of 0.25 μ g/ μ L,
The plasmid mixed liquor of 0.5 μ L is injected by side optic tectum (Fig. 2-A) using glass tube capillary needle under stereomicroscope.Electrode
Optic tectum both sides are respectively placed in be shocked by electricity (Fig. 2-B).Total electric shock 6 times, voltage 15V, shock by electricity 60ms every time, is spaced 100ms
(Fig. 2-B).After electricity turns (CUY-21 Electroporator, Nepa Gene, Japan), removes electrode and cure chicken embryo opening
It is sealed with adhesive plaster, when continuing culture to E12, carries out material collection (Fig. 2-C).
2, histotomy in vitro culture：
1) when the embryonic development after electricity turns is to E12, the embryo of observation selection GFP positive expressions under Stereo fluorescence microscope
(Fig. 2, D-F).
Both of which may be used when carrying out histotomy, first, according to GFP positive tables after 4% agar is cooled down
Size up to chicken brain is trimmed to cuboid (Fig. 2-G), and the agar block trimmed is pasted histotome with seccotine
On pallet (Fig. 2-G).Then a slot (Fig. 2, H-I) is cut out in side, by chicken brain olfactory bulb tip cut-off portion of tissue, olfactory bulb end court
Under, optic tectum is put into the slot of agar, is sticked with glue in (Fig. 2-J) on the pallet of histotomy.This kind is used in the present embodiment
Mode.
Second, the chicken brain of GFP positive expressions is embedded with 4% agar, it is rapid it is cooling after according to tissue size into
Row trimming (Fig. 2-J).
2) tissue block pasted is sliced under oscillating slice machine (VT1200S, Leica, Germany), is sliced
Thickness is 300 μm, and tissue is transferred on suspension type tissue culture dishes film (PICM03050, Millipore) after slice,
Further ware is transferred in 30mm culture dishes, 1ml culture solutions is included in and is cultivated (Fig. 2, K, L).In slicing processes, in order to
The fresh artificial cerebrospinal fluid (Fig. 2, K, L) that precooling treatment is added in slicer buffer solution disk of protective tissue.
The formula of culture medium and artificial cerebrospinal fluid is the same as embodiment 1.
3) culture dish is transferred in carbon dioxide incubator in 37 DEG C and 5%CO2Cultivated under condition of culture (Fig. 3,
B, H), culture solution is replaced in tissue culture afterwards for 24 hours, and culture 7d can be continued by replacing culture solution primary structure piece every 48h later
Time.Cultured tissue morphological structure and neuron 48h can laser confocal microscope (Olympus ix81,
Japan it is observed under).
Optic tectum histotomy incubation places 2 tissues (Fig. 3-G) on each film.It is trained in optic tectum tissue
During supporting, mainly observation is transfected the migration of neuron and the projection of aixs cylinder, and therefore, the tissue GFP of selection should be accurate
True transfection is at endyma area (Fig. 3, H-I).In incubation, it is not immersed in culture solution above tissue, and lower section
It is adequately to be contacted with culture solution, ensure that tissue will not necrosis (Fig. 3-J).
By the culture of both examples above histotomy, we have obtained following result.
1, group can be lost in tissue incubation be woven in the original morphosis of central nervous system
The tissue of culture is compared with directly from embryonic tissue slice (Fig. 4, A-C), and most significant difference is culture
Tissue loses the morphosis (Fig. 4, D-F) of original central nervous system.The dorsal root ganglion after tissue culture 48h
Structural fuzzy, spinal cord and surrounding tissue boundary thicken (Fig. 4-D).
Same result (Fig. 5, A-F) is also observed in the tissue of optic tectum culture.Embryo development procedure optic tectum
Structure (Fig. 5, A-C) with typical layer.But boundary thickens in the tissue of culture and the structure of layer is unclear
Clear (Fig. 5, D-F).
2, neuron loses stringent regulation and control in the tissue cultivated
The migration of neuron and the projection of aixs cylinder are stringent (Fig. 4, A-C) by a variety of cytokine regulatories in body.
In spinal cord, we can observe that the spinal neuron fiber positioned at side accurately projects offside (Fig. 4, B, C).It is organizing
After piece culture 48h, the neuron of GFP positive expressions is still in the side of spinal cord, and still, the direction of neuronal migration and joint are fine
The projecting direction of dimension has occurred that change (Fig. 4, E, F).
3, tissue culture is more suitable for the research of single neuron dynamics
In single neural elemental study, the neuron in the tissue of culture have gem-pure raised structures (Fig. 4,
G-L；Fig. 5-F).In the case where copolymerization is burnt it is observed that the structure (Fig. 5, G-N) of complete single neuron.In general, from work
Body tissue direct slicing carries out being difficult the structure for observing complete neuron in neuron observation, because neuron has three-dimensional
Structure, would generally cut-out structure during histotomy so that can not observe structure (Fig. 5-of complete neuron
C).And in tissue incubation, neural precursor will be further formed neuron, and then form complete aixs cylinder and tree
Prominent structure (Fig. 5-F).It is difficult to have observed under low power lens although the tissue of culture of the present invention is thicker than biopsy
Still complete neuron knot may be implemented by stratification scanning under Laser Scanning Confocal Microscope in the structure of whole single neuron
Structure (Fig. 5, G-N).In addition, in tissue incubation, we can observe the variation of neuron dynamic under the microscope,
In conjunction with live body electricity transgenic technology, we can realize chicken embryo development of central nervous system process foreign gene unconventionality expression to god
The research of the influence formed through member migration and aixs cylinder.
4, it discusses
In development of central nervous system process, the unconventionality expression of gene can lead to the exception of structure and function.In maincenter god
It is a kind of common animal model through chicken embryo during system gene functional study.With going out for chicken embryo live body electricity transgenic technology
It is existing, realize that the unconventionality expression of gene is possibly realized in chicken embryo central nervous system.In central nervous system, the migration of neuron,
The projection of aixs cylinder, the selection in aixs cylinder path, the formation of neural circuit and the foundation of neural network are the hot spots of research.
Here, the present invention establishes a kind of chicken embryo central nervous system live body electricity transgenic technology and organotypic brain piece culture
Technology is combined the method for carrying out foreign gene functional study.Before carrying out tissue culture in chicken embryo spinal cord or optic tectum
It carries out live body electricity to turn, the unconventionality expression of foreign gene is realized, when chick embryo development is drawn materials to the specific stage and carries out tissue
Slice is cultivated.The observation of neuron morphology and structure can be carried out in the tissue of culture.We have found that in tissue
Tissue original physique structure in vivo can be destroyed in incubation, as the migration of spinal cord mediocommissure fiber nerve member changes
Become, the projecting direction of commissural fibre changes, and the structure of typical layer is lost in optic tectum.This result is also that we are pre-
It is that phase arrives as a result, because, during tissue culture, tissue is to be cultivated in the medium, can lose the various factors
Correct regulation and control to neuron.There are very big differences with intravital environment for the environment that neuron is survived in the medium, therefore,
Many functions cannot react intravital function completely, this is also to carry out the problem of tissue culture should pay attention to.Vitro tissue
The difference of piece culture and in vivo tissue is not only on general form, but also there is also differences in subtleer structure, such as set
Prominent length, the quantity etc. of clavula on dendron.
It can be seen that, in central nervous tissue piece incubation, it is most suitable for single nerve from experimental result of the present invention
The research of member.The complete structure of single neuron is able to observe that in incubation.Although in tissue incubation
The morphosis of original in vivo tissue is lost, but neuron is still in the tissue, therefore, to be trained in experimentation
The indexs such as migration and the aixs cylinder Path selection of single neuron can be observed in foster tissue.Although culture environment and in vivo
There are difference for environment, but the single neuron in the tissue cultivated can simulate the mistake of the in vivo dynamic change of neuron
Journey.We compared the tissue and in-vivo tissue slice of culture in an experiment, it has been found that be difficult in histotomy in vivo
Observe the structure of complete single neuron, but can be complete with single neuron visible in detail in the tissue of culture
Whole structure.In addition to this, live body electricity is carried out before carrying out tissue culture to turn, realize foreign gene in spinal cord and optic tectum
Unconventionality expression, on this basis carry out tissue culture, we can be to foreign gene in chicken embryo development of central nervous system
Function in the process is analyzed in individual cells level.In the experimentation of early period, we are also to the nerve after transfection
Member is separately cultured.The neuron cultivated after separation can be longer compared with the neuron in tissue culture, but in tissue
Neuron axon projection it is more regular.The neuron of cultured tissue on piece does not have compared with the single neuron detached
There is the digestion by enzyme, while also being influenced by other cells of tissue and related secretion factor, closer to live body.In short,
This method provides the strategies that a kind of external effective single neuronal migration and aixs cylinder form research.
Claims (9)
1. a kind of preparation method of the single neuron dynamic studies model of chicken embryo central nervous system, it is characterised in that：Including such as
Lower step：
1) plasmid containing target gene is transferred in chicken embryo spinal cord or chicken embryo optic tectum tissue by the method for using live body electricity to turn,
Obtain genetically modified organism block；
2) genetically modified organism block is wrapped up with gel, obtains package block；The package block is sliced, histotomy is obtained；It will
Obtained histotomy is cultivated, is observed.
2. preparation method according to claim 1, it is characterised in that：When the genetically modified organism block is chicken embryo optic tectum group
When knitting, the step of package, is：Gel piece is pasted on the pallet of histotome；Then it is cut out in gel piece side a
Slot, by chicken brain olfactory bulb tip cut-off portion of tissue, downward, optic tectum is put into the slot of gel at olfactory bulb end, is also pasted on histotomy
Pallet on.
3. preparation method according to claim 1, it is characterised in that：The gel is the agar of 3.5-4.5%.
4. preparation method according to claim 1, it is characterised in that：The thickness of the histotomy is 250-350 μm.
5. preparation method according to claim 1 or 4, it is characterised in that：Histotomy is placed in manually after the slice
In cerebrospinal fluid.
6. preparation method according to claim 1, it is characterised in that：The step of live body electricity turns be：
1) plasmid containing target gene is injected into the chicken embryo ridge of culture 2.5-3.5 days with microinjection capillary glass needle
In marrow；Or the plasmid containing target gene is injected into the chicken embryo cultivated 4-5 days with microinjection capillary glass needle and is regarded
Head cover tissue；
2) electrode is placed on to spinal cord or the optic tectum tissue both sides of oneself injection plasmid, electricity is carried out and turns.
7. preparation method according to claim 6, it is characterised in that：When electricity turns spinal cord, the parameter used is total electric shock
5-7 times, voltage 17-19V, shock by electricity 55-65ms every time, is spaced 95-105ms；When electricity turns optic tectum tissue, the parameter that uses
Always to shock by electricity 5-7 times, voltage 14-16V, shock by electricity 55-65ms every time, is spaced 95-105ms.
8. preparation method as described in claim 1 answering in dynamic function of the research purpose gene in single neuron
With.
9. application according to claim 8, it is characterised in that：After electricity turns spinal cord, continue to cultivate embryonic development to 5.5-
Materials slice is carried out at 6.5 days, with dynamic function of the research purpose gene in single neuron；Turn optic tectum tissue in electricity
Afterwards, continue to cultivate embryonic development to materials slice is carried out at 11-13 days, with dynamic of the research purpose gene in single neuron
Function.
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