CN103940985A - Method for studying migration, proliferation and differentiation of brain endogenous neural stem cells - Google Patents
Method for studying migration, proliferation and differentiation of brain endogenous neural stem cells Download PDFInfo
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Abstract
The invention relates to a method for studying migration, proliferation and differentiation of brain endogenous neural stem cells. The method comprises the following steps: (1) performing double-labeled immunofluorescent staining on an animal brain section by 5-bromndeoxyuridine and doublecortin, 5-bromndeoxyuridine and neuronal nuclei antigen as well as 5-bromndeoxyuridine and glial cell line-derived neurotrophic factor, and single-labeled immunofluorescent staining by sialyl neural cell adhesion molecules; and (2) carrying out statistic analysis on data and drawing by GraphPad Prism6.0 software, and performing one-way variance analysis on single-factor multi-group data of the 5-bromndeoxyuridine positive cells, wherein the animal brain section is subjected to the following treatment: stereotactically injecting a 6-hydroxydopamine solution into a corpus striatum of the right side of the brain, injecting a PSA (prostate-specific antigen) specificity lyase stock solution into the lateral ventricle, and then injecting a 5-bromndeoxyuridine solution in the abdominal cavity. An effective method for studying migration influence of the brain endogenous neural stem cells is provided.
Description
Technical field
The invention belongs to preclinical medicine field, the present invention relates to particularly a kind of method of the impacts such as migration of studying neural stem cell in brain, increment, differentiation.
Background technology
Parkinson's (Parkinson ' s disease, PD) are a kind of common carrying out property of central nervous system degenerated diseases that nigrostriatum path pathology is principal character of take.The pathogenesis of PD is not yet clear and definite at present, is the synergistic result of number of mechanisms.Therapy clinically all can not fundamentally stop the carrying out property regression of patient's PD nigrostriatum path, and recently increasing people thinks that starting brain endogenous neural stem cell participates in the strategy that brain damage reparation probably develops into a brand-new treatment brain damage.This therapeutic strategy is particularly useful for repairing chronic brain injury disease-Parkinson's.
Current classical drug therapy can alleviate or relief of symptoms in the early stage as oral levodopa (L-dopa), but can not delay the progress of the course of disease, and long-term use has obvious spinoff, i.e. the dyskinesia of L-dopa induction.Operation also can only temporarily alleviate or relief of symptoms as DBS nucleus hypothalamicus.These therapies all can not fundamentally stop the carrying out property regression of patient's PD nigrostriatum path, and starting the reparation of brain endogenous neural stem cell participation PD brain damage is the strategy of a brand-new treatment chronic brain injury.
The neural whole process that neuronal development occurs just to refer to, since a precursor division, up to new neuronal maturation, integrate and have the process of function.Neural precursor energy self, but remain static more.When CNS is impaired or during sex change, cell micro-environment changes, NSCs is activated, and breeds, moves, breaks up, integrates, and promotes the recovery of neuron regeneration and damage location brain tissue 26S Proteasome Structure and Function.Adult Nao Youliangge neurogenesis district, nerve regneration is more active there: one is the subventricular zone (SVZ) of telocoele adjacency; One is the granulocyte inferior segment (SGZ) of hippocampus.The immature neurocyte (Neuroblast) that these two position neural stem cell produce has legacy migration path separately, the prematurity neurocyte that SVZ produces forms cell cluster, along mouth side migration stream (RMS), moves to olfactory bulb, is divided into intrerneuron; The prematurity neurocyte that SGZ produces moves to the GCL of hippocampal dentate, is divided into intrerneuron and joins in neural circuit.The molecular mechanism and the rule that disclose these cell migrations will provide important theoretical foundation to the migration of brain damage district to these cells of guiding.Large quantity research finds that the common feature of these two kinds of cells is sialylated neural cell adhesion factors that cell surface is all expressed high level.When these cells arrive destination, are divided into after ripe intrerneuron, the PSA of cell surface expresses and will disappear, and prompting PSA-NCAM plays an important role in prematurity neurocyte directional migration.Have experiment to show if Endo-N is expelled to telocoele, legacy migration path RMS will be destroyed, and the prematurity neurocyte that SVZ produces comprises corpus straitum dystopy migration showed increased to other brain tissues.
Summary of the invention
The object of the present invention is to provide a kind of method of studying brain endogenous neural stem cell migration, propagation, differentiation, for the impacts such as the cell migration of research brain endogenous neural stem, propagation, differentiation provide a kind of effective method.
The method of research brain endogenous neural stem provided by the invention cell migration for achieving the above object,, propagation, differentiation:
1) animal brain section is carried out to BrdU(5 bromodeoxyuridine) and DCX(both adrenal glands cortin), BrdU and NeuN(neuron core antigen), BrdU and GFAP(glial cell line-derived neurotrophic factor) two mark immunofluorescence dyeings, and the sialylated neural cell adhesion factor of PSA-NCAM() single immunofluorescence dyeing of marking;
2) use GraphPad Prism6.0 software statistics analyze data and draw, BrdU positive cell is that single factor multi-group data is used one-way ANOVA to analyze.
In described method, animal brain section is through following processing: at three-dimensional locating injection 6-OHDA(6-hydroxydopamine in the corpus straitum of brain right side) after solution, at intracerebroventricular injection Endo-N(PSA specificity lyases) stoste, then lumbar injection BrdU solution.
In described method, animal brain section refers to animal left and right brain section.
In described method, in described animal brain section, include control group brain section.
In described method, after described control group brain section is three-dimensional locating injection 6-OHDA solution, at intracerebroventricular injection PBS damping fluid, then lumbar injection BrdU solution.
In described method, described animal brain section lumbar injection BrdU solution is to inject a couple of days continuously.
In described method, the mean+/-standard error for result (Mean ± SEM) of described statistical data analysis represents, usings p<0.05 as the boundary at significant difference.
Method of the present invention can be for the migration feature of research neural stem cell, the differentiation capability of the cell that the migration in the corpus straitum of analysis 6-OHDA damage comes, has established important working foundation for how further further investigation starts the impacts such as brain endogenous neural stem cell migration, propagation, differentiation.
Accompanying drawing explanation
Fig. 1 is the method study route of one embodiment of the invention.
Fig. 2 is that one embodiment of the invention is removed after PSA C57BL/6J mouse intracerebroventricular injection Endo-N, the immunofluorescence dyeing result of PSA-NCAM; Wherein: figure a is control group, figure b is experimental group, cuts into slices as sagittal plain section, and that in figure, dotted line represents is RMS.LV, telocoele; OB, olfactory bulb; RMS, mouth side migration stream.Engineer's scale, 250 μ M.
Fig. 3 is the immunofluorescence dyeing result of one embodiment of the invention 6-OHDA injection BrdU+ cell after 13 days; Wherein: a is the left brain striatum sagittal plain section of control group, b is that the right brain striatum sagittal plain section of control group, d are that the left brain striatum sagittal plain section of experimental group, e are the right brain striatum sagittal plain section of experimental group.Figure c is the sagittal plain section focused view altogether of amplification of the dotted line frame of figure b, figure f is the amplification of the dotted line frame of figure e, and figure g is the results of analysis of variance figure of single factor multi-group data, control mice n=5, experimental mice n=5, * p<0.05 has statistical significance.
Fig. 4 is the immunofluorescence dyeing result of the 6-OHDA injection two mark of BrdU/DCX+ cell after 13 days of one embodiment of the invention; Wherein: a is the left brain striatum sagittal plain section of control group, b is that the right brain striatum sagittal plain section of control group, d are that the left brain striatum sagittal plain section of experimental group, e are the right brain striatum sagittal plain section of experimental group.Mouse brain sagittal plain section corpus straitum altogether focused view figure f is the laser co-focusing orthogonal projection of the dotted line frame of enlarged drawing c, and two mark cells of demonstration are BrdU/DCX+ cell; Figure c engineer's scale 500 μ M, figure e engineer's scale 25 μ M.
Fig. 5 is that the immunofluorescence dyeing result sagittal plain section of 6-OHDA injection BrdU+ cell after 23 days of one embodiment of the present of invention is total to focused view.Wherein: a is the left brain striatum sagittal plain section of control group, b is that the right brain striatum sagittal plain section of control group, d are that the left brain striatum sagittal plain section of experimental group, e are the right brain striatum sagittal plain section of experimental group.Figure c is the amplification of the dotted line frame of figure b, and figure f is the amplification of the dotted line frame of figure e.Figure g is the results of analysis of variance figure of single factor multi-group data.(* p<0.05#p<0.05 has statistical significance for control mice n=5, experimental mice n=5)
Fig. 6 is the immunofluorescence dyeing result of the 6-OHDA injection two mark of BrdU/GFAP+ cell after 23 days of one embodiment of the present of invention.Wherein: a is the left brain striatum sagittal plain section of control group, b is that the right brain striatum sagittal plain section of control group, d are that the left brain striatum sagittal plain section of experimental group, e are the right brain striatum sagittal plain section of experimental group.Mouse brain sagittal plain section corpus straitum altogether focused view figure f is the laser co-focusing orthogonal projection of the dotted line frame of enlarged drawing c, and two mark cells of demonstration are BrdU/GFAP+ cell, figure c engineer's scale 100 μ M, figure e engineer's scale 25 μ M.
Fig. 7 is the immunofluorescence dyeing result of the 6-OHDA injection two mark of BrdU/NeuN+ cell after 23 days of one embodiment of the present of invention.Wherein: a is the left brain striatum sagittal plain section of control group, b is that the right brain striatum sagittal plain section of control group, d are that the left brain striatum sagittal plain section of experimental group, e are the right brain striatum sagittal plain section of experimental group.Mouse brain sagittal plain section corpus straitum altogether focused view figure f is the laser co-focusing orthogonal projection of the dotted line frame of enlarged drawing c, and two mark cells of demonstration are BrdU/NeuN+ cell, figure c engineer's scale 100 μ M, figure e engineer's scale 25 μ M.
Embodiment
The some English abbreviation relating in to instructions of the present invention at this is described, and the English abbreviation of mentioning hereinafter is all understood by this explanation:
PD(Parkinson ' s disease) Parkinson's;
AD(Alzheimer disease) alzheimer disease;
DA(dopamine) dopamine;
L-DOPA(levodopa) levodopa;
PB(phosphate buffer) phosphate buffer;
PBS(phosphate buffered saline) phosphate buffered saline(PBS);
SN(Substantia nigra) black substance;
TH(tyrosine hydroxylase) tyrosine hydroxylase;
6-OHDA(6-Hydroxydopamine) 6-hydroxyl DOPA;
Endo-N(endoneuraminidase) PSA specificity lyases;
EGF(epidermal growth factor) epidermal growth factor;
BDNF(brain derived neurotrophin factor) Brain Derived Neurotrophic Factor;
GFAP(glial fibrillary acidic protein) colloid source fibrillary acidic protein;
SGZ(subgranular zone) infragranular layer;
SVZ(subventricular zone) subependymal region;
RMS(rostral migratorystream) mouth side migration stream;
NSCs(neural stem cells) neural stem cell;
NPCs(neural precursor cells) neural precursor;
DW(distilled water) distilled water;
NeuN(neuron special protein) neuron core antigen;
DCX(doublecortin) both adrenal glands cortin;
BrdU(5-bromo-2 ,-deoxyuridine) 5 bromodeoxyuridines;
NGS(normal goat serum) normal goats serum;
ANOVA(analysis of Variance) variance analysis;
PFA(paraformaldehyde) paraformaldehyde;
PSA-NCAM(Polysialic acid-neural cell adhesion molecular) the sialylated neural cell adhesion factor;
GDNF(glial cell derived neurotrophic factor) glial cell line-derived neurotrophic factor.
One embodiment of the present of invention are to prepare chronic brain injury model by the one-sided injection of corpus straitum 6-OHDA.Because corpus straitum is the main target area of substantia nigra dopaminergic neuron, the 6-OHDA being injected in wherein can be absorbed by the former tip, by contrary axonal transport to the pericaryon in black substance, through monoamine oxidase, changing into free radical, optionally destroying these neurons and occur the symptom of similar PD.Because 6-OHDA must could arrive substantia nigra of midbrain by contrary axonal transport, thus the death of dopaminergic neuron be a kind of indirectly, progressive process.With directly damage method of black substance, compare, this damage process, closer to the clinical characters of mankind PD morbidity, is easier to dissect location at operation technique epistriatum compared with black substance.The present invention is more interested be corpus straitum at local damage near svz district, Endo-N stoste is expelled to telocoele and removes after PSA, intercellular same preferendum adhesion increases, therefore, the present invention has observed its effect situation to legacy migration path RMS.DCX is mainly expressed on the neural stem cell cell space and leading projection in migration, is usually used in neural stem cell in mark migration.Therefore, observe the situation of the two mark of BrdU and DCX cell simultaneously, analyzed the BrdU positive cell quantity in corpus straitum.The differentiation capability of the neural stem cell of also having observed migration and having come.This establishes the important working foundation of part by how starting the impacts such as brain endogenous neural stem cell migration, propagation, differentiation for further further investigation.
Object of the present invention:
(1) with Endo-N, remove PSA and can increase the corpus straitum that SVZ district neural stem cell dystopy moves to 6-OHDA damage;
(2) differentiation capability of the neural stem cell of research dystopy migration, and then provide part basis for studying Parkinsonian pathology.
The brain tissue frozen section that the present invention adopts, can be used following methods to make:
(1) choose C57BL/6J experiment and be divided into four groups with male mice, every group of five animals, all in the corpus straitum of brain right side, three-dimensional locating injection 2 μ l6-OHDA solution.
(2) the three-dimensional locating injection of 6-OHDA is after 3 days, experimental mice intracerebroventricular injection 5 μ lEndo-N stostes, the PBS damping fluid of control group injection same volume.
(3), after 6-OHDA injection, the 8th day starts, every day twice, respectively to 4 groups totally 20 animals press 50mg/kg lumbar injection BrdU solution, inject continuously 3 days.
(4) in 6-OHDA injection filling in latter the 13rd day, the 23rd day, kill and draw materials respectively, fixing dehydration embedding.With freezing microtome, the full brain of experiment mice is carried out to sagittal slices, slice thickness is 35 μ m.
(5) to four groups of all animals left and right brain sections, carry out the two mark of BrdU and DCX, BrdU and NeuN, BrdU and GFAP immunofluorescence dyeing, and the mono-mark of PSA-NCAM immunofluorescence dyeing.
(6) use GraphPad Prism6.0 software statistics analyze data and draw, BrdU positive cell is that single factor multi-group data is used one-way ANOVA to analyze.
In the brain tissue frozen section making:
1) in experimental group animal migration stream RMS, there is a small amount of PSA-NCAM positive cell, in control animals migration stream RMS, have a large amount of PSA-NCAM positive cells.
2) in experimental group animal corpus straitum, there are a large amount of BrdU positive cells, have the two mark of BrdU and DCX, BrdU and NeuN, BrdU and GFAP cell.Control animals corpus straitum has a small amount of BrdU positive cell, does not find the two mark of BrdU and DCX, BrdU and NeuN, BrdU and GFAP cell.
Can learn thus:
1) with Endo-N, remove after PSA, have a large amount of neural stem cell dystopys to move to the corpus straitum of 6-OHDA damage, these neural stem cell may come from SVZ district.
2) neural stem cell that migration comes has differentiation capability, can be divided into neuron and spongiocyte.
Below lifting a specific embodiment elaborates.
One, experiment material
1, animal used as test
The present embodiment animal used is C57BL/6J mouse, male, and in 8-10 week, body weight 22-26g, is provided by animal portion of the Capital University of Medical Sciences.Illumination 12 hours every days and 12 hours dark alternate environments, ingest drinking-water freely, and raising condition is SPF level.
Two, experimental technique
1, the lumbar injection mark of the three-dimensional locating injection of mouse brain and BrdU
1) the three-dimensional locating injection of 6-OHDA
Experiment mice is divided into four groups (A, B, C, D), five every group animals (seeing Fig. 1), all in the corpus straitum of brain right side, three-dimensional locating injection 6-OHDA solution (5mg/ml6-OHDA, 0.2% Vc, 0.9%NaCl).
2) step is as follows:
(1) mouse is weighed, and with Equitisin, presses 3ml/kg body weight, and intraperitoneal injection of anesthesia is downward by the mouse outside of belly of having anaesthetized, below pad a foam, and be fixed on stereotaxic apparatus.
(2) head skin preservation, after conventional tincture of iodine alcohol disinfecting, gets center sagittal otch, cuts skin and hypodermis, and blunt separation, until expose skull, determines corpus straitum injection site coordinate according to mouse brain stereotaxic atlas.The injection site coordinate of selecting is, (AP)+0.5mm before bregma, and (ML)+1.9mm is opened on the right side of median line, (DV)-2.65mm under endocranium, front tooth rod is lower than biauricular line (TB)-1mm.
(3) under surgical operation microscope, the above-mentioned injection site in three-dimensional location, remaining at corresponding skull place by dental burr, careful boring opened an aperture, firmly not excessive, avoids damaging brain essence, with pincet, chooses out gently endocranium.
(4) use Hamilton micro syringe, slowly inject 2 μ l6-OHDA (5 μ g/ μ l) solution (Fig. 1), every injection 1 μ l let the acupuncture needle remain at a certain point 1min, after injection, let the acupuncture needle remain at a certain point ten minutes, prevents overflow, finally slowly exits Hamilton micro syringe.
(5) after using the tincture of iodine and alcohol wipe wound disinfection antibacterial, skin suture, is positioned over animal in warm environment, gives food and water after reviving.
2, the three-dimensional locating injection of Endo-N stoste
The three-dimensional locating injection of 6-OHDA is after 3 days, experimental group (B, D) mouse intracerebroventricular injection 5 μ l Endo-N stostes (1U/ μ l), the 0.01MPBS damping fluid (see figure 1) of control group (A, C) injection same volume.Step is as follows:
(1) mouse is weighed, and with Equitisin, presses 3ml/kg body weight, and intraperitoneal injection of anesthesia is downward by the mouse outside of belly of having anaesthetized, below pad a foam, and be fixed on stereotaxic apparatus.
(2) after mouse head alcohol disinfecting, get center sagittal otch, cut skin and hypodermis, blunt separation, until expose skull, determines intracerebroventricular injection site coordinate according to mouse brain stereotaxic atlas.The injection site coordinate of selecting is, bregma (AP)+0mm, and (ML)+0.9mm is opened on the right side of median line, (DV)-2.3mm under endocranium, front tooth rod is lower than biauricular line (TB)-1mm.
(3) under surgical operation microscope, the above-mentioned injection site in three-dimensional location, remaining at corresponding skull place by dental burr, careful boring opened an aperture, firmly not excessive, avoids damaging brain essence, with pincet, chooses out gently endocranium.
(4) use Hamilton micro syringe, slowly inject 5 μ l Endo-N stostes (1U/ μ l) or the PBS(of 0.01M and see Fig. 1), every injection 1 μ l let the acupuncture needle remain at a certain point 3min, after injection, let the acupuncture needle remain at a certain point ten minutes, prevents from overflowing, and finally slowly exits micro syringe.
(5) after using the tincture of iodine and alcohol wipe wound disinfection antibacterial, skin suture, is positioned over animal in warm environment, gives food and water after reviving.
3, the lumbar injection mark of BrdU
After 6-OHDA injection, the 8th day starts, every day twice, 4 hours, interval, respectively to 4 groups totally 20 animals press 50mg/kg lumbar injection BrdU solution, inject continuously 3 days (see figure 1)s.Concrete steps:
(1) with the syringe of 1ml, coordinate No. 4 syringe needles to carry out mouse peritoneal injection
(2) right hand syringes while injecting, the little finger of toe of left hand and the nameless tail of catching mouse, other three fingers are caught the neck of mouse, make the head of mouse downward.Organ in abdominal cavity will be swung to chest naturally like this, prevents that syringe from stinging the organs such as fashionable damage large intestine, small intestine.It is soft that the action of inserting needle is wanted, and prevents from stabbing abdomen organ.Especially for the less mouse of body weight, during lumbar injection, syringe needle can be walked a bit of distance in subcutaneous abdomen, preferably from belly one side inserting needle, through the opposite side at belly after ventrimeson, enter abdominal cavity, injected after medicine, slowly extract syringe needle, and slightly rotate syringe needle, prevent leakage.
4, draw materials with fixing
A, B group are filled with to kill and are drawn materials for the 13rd day after 6-OHDA injection, and C, D group are filled with and killed draw materials (Fig. 1) for the 23rd day after 6-OHDA injection.
(1) charge pump is cleaned in debugging: with 0.01M PBS flushing pipe, the ducted bubble of emptying, while being full of PBS in pipeline, shuts down standby.
(2) mouse is weighed, and with Equitisin, presses 3ml/kg body weight, intraperitoneal injection of anesthesia.
(3) the mouse outside of belly is upwards properly fixed on cake wax, with hemostatic forceps, mentions xiphoid-process place skin, cut off skin and hypodermis, then to both sides, cut off, until midaxillary line fully exposes splanchnocoel intersection and diaphram.From xiphoid-process, cut off diaphram again, separated to both sides, fully expose thoracic cavity and heart.Note upwards choosing and cutting gently, prevent from damaging internal organ and cause and bleed profusely;
(4) free heart, separated pericardium, inserts left ventricle by catheter needle, with hemostatic forceps, fixes.Cut off right auricle of heart, open charge pump.Perfusion 0.01M PBS.Expose as early as possible abdominal cavity, massage gently liver in abdominal cavity, to help the fast evacuation of blood, when animal's liver obviously turns white, close charge pump, intrusion pipe is moved in 4% paraformaldehyde solution, contain the beaker of paraformaldehyde solution and put in advance ice cube.Open charge pump, perfusion paraformaldehyde, front 1/3 amount quick filling, rear 2/3 amount is perfusion slowly, after animal foot twitch is stiff, continues perfusion paraformaldehyde 100ml left and right.
(5) after perfusion, with rongeur, peel off skull and endocranium, remove cerebellum curtain, brain curtain.Put into 4% paraformaldehyde solution and continue fixingly, preserve after 24 hours for 4 ℃ and take out brain tissue, put into 4 ℃ of 30% sucrose solutions and be saved to sample and sink to the bottom.
5, mouse brain tissue freezing section
(1) brain tissue after dehydration is completed takes out from 30% sucrose solution, with the PBS of a small amount of 0.01M, rinses, and with OCT embedding, carries out mark, puts into-40 ℃ of low temperature refrigerators and spends the night.
(2) freezing microtome precooling, installs fixed blade, until temperature stabilization, during at-20 ℃, freezes and ice platform, prepares one, little writing brush, standby.
(3) take out embedded block, be placed in freezing microtome, with reference to mouse brain stereotaxic atlas, by mark, from left brain, start, to right brain, full brain is carried out to sagittal slices, slice thickness is 35 μ m.
(4) full brain serial section, left and right brain is divided into respectively 5 holes to be placed, and every like this hole is a set of serial section.When the left brain of mouse switches under cortex 1ml, stay sheet, be put in order in the hole of placing left brain section.When the left brain olfactory bulb of mouse and diacele lose, start to stay right brain section.And be put in order in the hole of placing right brain section.24 orifice plates that fill anti freezing solution are put in all sections, 4 ℃ of preservations.
6, mouse brain sagittal slices immunofluorescence dyeing,
Brain sagittal sections position, four groups of all animals left and right brain section, has carried out the immunofluorescence dyeing of BrdU.A group and B group (within after 6-OHDA injection the 13rd day, fill with and kill) have also been carried out the two mark of PSA-NCAM immunofluorescence dyeing, BrdU and DCX immunofluorescence dyeing simultaneously.C group and D group (fill with for after 6-OHDA injection the 23rd day and kill) animal, the partially sliced two mark of BrdU and the NeuN immunofluorescence dyeing that simultaneously also carried out of right brain, the two mark of BrdU and GFAP immunofluorescence dyeing.
1) immunofluorescence dyeing of BrdU
(1) section rinsing 3 times in the PBS of 0.01M, 5min/ time;
(2) 2N-HCL is put in section, at 37 ℃, hatches 40min
(3) with 0.1M Borate buffer rinsing 3 times, 5min/ time;
(4) rinsing 3 times in the PBS of 0.01M, 10min/ time;
(5) 10% NGS solution (1%BSA+0.25%TritionX-100) sealing.Hatch 1h, RT;
(6) 5% NGS solution (1%BSA+0.25%TritionX-100) dilution antibody, hatches ambient temperature overnight.Ratio is large mouse-anti BrdU monoclonal antibody 1:300;
(7) rinsing 3 times in the PBS of 0.01M, 10min/ time
(8) two anti-5% NGS solution (1%BSA+0.25%TritionX-100) dilution, hatches room temperature 1h.Ratio is the mouse 1:200 of the CyTM3-conjugated-goat Chinese People's Anti-Japanese Military and Political College;
(9) rinsing 3 times in the PBS of 0.01M, 10min/ time
(10) section is mounted on microslide and dried, drip fluorescence mountant (Fluorescent Mounting Medium) on histotomy, covered, allows section contact mounting liquid, avoids bubble as far as possible.
(11) micro-Microscopic observation, copolymerization Jiao take pictures, and should notice that cancellation all easily occurs fluorescent material, and the sample after dyeing should keep in Dark Place.
2) the two mark of BrdU and DCX immunofluorescence dyeing
(1) section rinsing 3 times in the PBS of 0.01M, 5min/ time;
(2) 2N-HCL is put in section, at 37 ℃, hatches 40min;
(3) with 0.1M Borate buffer rinsing 3 times, 5min/ time;
(4) rinsing 3 times in the PBS of 0.01M, 10min/ time;
(5) 10% NGS solution (1%BSA+0.25%TritionX-100) sealing.Hatch 1h, RT;
(6) 5% NGS solution (1%BSA+0.25%TritionX-100) dilution antibody, hatches ambient temperature overnight.Ratio is large mouse-anti BrdU monoclonal antibody 1:300; The anti-Doublecortin(DCX of rabbit) 1:500;
(7) rinsing 3 times in the PBS of 0.01M, 10min/ time;
(8) two anti-5% NGS solution (1%BSA+0.25%TritionX-100) dilution, hatches room temperature 1h.Ratio is Alexa FluorR488-goat antirabbit 1:200; Cy
tMthe mouse 1:200 of the 3-conjugated-goat Chinese People's Anti-Japanese Military and Political College;
(9) rinsing 3 times in the PBS of 0.01M, 10min/ time;
(10) section is mounted on microslide and dried, drip fluorescence mountant (Fluorescent Mounting Medium) on histotomy, covered, allows section contact mounting liquid, avoids bubble as far as possible;
(11) micro-Microscopic observation, copolymerization Jiao take pictures; Should notice that cancellation all easily occurs fluorescent material, the sample after dyeing should keep in Dark Place.
3) PSA-NCAM immunofluorescence dyeing.
(1) section rinsing 3 times in the PBS of 0.01M, 5min/ time;
(2) 10% NGS solution (1%BSA+0.25%TritionX-100) sealing.Hatch 1h, RT;
(3) 5% NGS solution (1%BSA+0.25%TritionX-100) dilution antibody, hatches ambient temperature overnight.Ratio is the anti-PSA-NCAM monoclonal antibody of mouse 1:350;
(4) rinsing 3 times in the PBS of 0.01M, 10min/ time;
(5) two anti-5% NGS solution (1%BSA+0.25%TritionX-100) dilution, hatches room temperature 1h.Ratio is DyLightTM488-goat anti-mouse 1:200;
(6) rinsing 3 times in the PBS of 0.01M, 10min/ time;
(7) section is mounted on microslide and dried, drip fluorescence mountant (Fluorescent Mounting Medium) on histotomy, covered, allows section contact mounting liquid, avoids bubble as far as possible;
(8) micro-Microscopic observation laser, copolymerization Jiao take pictures, and should note: cancellation all easily occurs fluorescent material, and the sample after dyeing should keep in Dark Place.
4) the two mark of BrdU and NeuN immunofluorescence dyeing
(1) section rinsing 3 times in the PBS of 0.01M, 5min/ time;
(2) 2N-HCL is put in section, at 37 ℃, hatches 40min;
(3) with 0.1M Borate buffer rinsing 3 times, 5min/ time;
(4) rinsing 3 times in the PBS of 0.01M, 10min/ time;
(5) 10% NGS solution (1%BSA+0.25%TritionX-100) sealing.Hatch 1h, RT;
(6) 5% NGS solution (1%BSA+0.25%TritionX-100) dilution antibody, hatches ambient temperature overnight.Ratio is the anti-NeuN monoclonal antibody of mouse 1:300; Large mouse-anti BrdU monoclonal antibody 1:300;
(7) rinsing 3 times in the PBS of 0.01M, 10min/ time;
(8) two anti-5% NGS solution (1%BSA+0.25%TritionX-100) dilution, hatches room temperature 1h.Ratio is DyLightTM488-goat anti-mouse 1:200; The mouse 1:200 of the CyTM3-conjugated-goat Chinese People's Anti-Japanese Military and Political College;
(9) rinsing 3 times in the PBS of 0.01M, 10min/ time;
(10) section is mounted on microslide and dried, drip fluorescence mountant (Fluorescent Mounting Medium) on histotomy, covered, allows section contact mounting liquid, avoids bubble as far as possible.
(11) micro-Microscopic observation, laser co-focusing is taken pictures, and should note: cancellation all easily occurs fluorescent material, and the sample after dyeing should keep in Dark Place.
5) the two mark of BrdU and GFAP immunofluorescence dyeing
(1) section rinsing 3 times in the PBS of 0.01M, 5min/ time;
(2) 2N-HCL is put in section, at 37 ℃, hatches 40min;
(3) with 0.1M Borate buffer rinsing 3 times, 5min/ time;
(4) rinsing 3 times in the PBS of 0.01M, 10min/ time
(5) 10% NGS solution (1%BSA+0.25%TritionX-100) sealing.Hatch 1h, RT;
(6) 5% NGS solution (1%BSA+0.25%TritionX-100) dilution antibody, hatches ambient temperature overnight.Ratio is the anti-GFAP polyclonal antibody of rabbit 1:1000; Large mouse-anti BrdU monoclonal antibody 1:300;
(7) rinsing 3 times in the PBS of 0.01M, 10min/ time;
(8) two anti-5% NGS solution (1%BSA+0.25%TritionX-100) dilution, hatches room temperature 1h.Ratio is DyLightTM549-goat antirabbit 1:200; The mouse 1:200 of the CyTM3-conjugated-goat Chinese People's Anti-Japanese Military and Political College;
(9) rinsing 3 times in the PBS of 0.01M, 10min/ time;
(10) section is mounted on microslide and dried, drip fluorescence mountant (Fluorescent Mounting Medium) on histotomy, covered, allows section contact mounting liquid, avoids bubble as far as possible.
(11) micro-Microscopic observation laser, copolymerization Jiao take pictures, and should note: cancellation all easily occurs fluorescent material, and the sample after dyeing should keep in Dark Place.
7, statistical procedures
Data are used GraphPad Prism6.0 software statistics analyze and draw, and single factor multi-group data is used one-way ANOVA to analyze, and it is mean value ± standard error (Mean ± SEM) expression for result.Using p<0.05 as the boundary at significant difference.
The right brain of mouse that the present embodiment has been chosen A group and B group has carried out the dyeing of the distinctive albumen PSA-NCAM of neuroblast (Neuroblasts) containing the brain sagittal plain section of RMS, result is processed by Endo-N stoste (1U/ μ l), in the RMS of 13 days experimental mice (B group) that filling is killed afterwards of 6-OHDA injection, does not substantially express PSA-NCAM.And high expressed PSA-NCAM(is shown in Fig. 2 in the RMS of the control group mice (A group) of processing with the PBS of equivalent).
In the corpus straitum of the experimental group B group mouse of processing in Endo-N stoste (1U/ μ l) (fill with and kill afterwards for 13 days in 6-OHDA injection), in containing, there is a large amount of BrdU positive cell (see figure 3)s
Cut into slices and carried out the immunofluorescence dyeing of BrdU in a whole set of sagittal sections position of mouse left and right brain that the present embodiment has been chosen A group and B group, with inverted fluorescence microscope, observe whole brain sections, choose contain corpus straitum texture and optic tract simultaneously pure sagittal slices, carry out the statistics of BrdU positive cell and carry out statistics comparison.Be divided into following group: (raw data is in Table 1) Endo-N-left group and the Endo-N-right group (raw data is in Table 2) of Control-left group and Contorl-right group.Use two-photon laser Laser Scanning Confocal Microscope to take pictures, (a) BrdU positive cell is seldom for Fig. 3 to can be observed Control-left group, Contorl-right group (Fig. 3 b) and Endo-N-left group (Fig. 3 d) BrdU positive cell are few, and Endo-N-right group (Fig. 3 e) has a large amount of BrdU positive cells.Four groups of data are used one-way ANOVA to analyze, and it is mean value ± standard error (Mean ± SEM) expression for result.Using p<0.05 as the boundary at significant difference (Fig. 3 g).Result Endo-N-right group all has significant difference with other three groups.
In the corpus straitum of the experimental group B group mouse of processing in Endo-N stoste (1U/ μ l) (fill with and kill afterwards for 13 days in 6-OHDA injection), contain the two mark of a small amount of BrdU/DCX+ cell (see figure 4).The present embodiment has been chosen a set of sagittal sections of the right brain of the mouse position section of A group and B group and has been carried out the two mark of BrdU/DCX+ immunofluorescence dyeing, with two-photon laser Laser Scanning Confocal Microscope, take pictures, the right brain striatum sagittal slices of Endo-N-right(test group in B group) group, find the two mark of a small amount of BrdU/DCX+ cell (Fig. 4 c), in the right brain striatum sagittal slices of Contorl-right(control group of A group) organize and do not find visible marking's thing.This enforcement life example has been chosen the two mark of BrdU/DCX+ cell and has been carried out the burnt rectangular projection (Fig. 4 f) of copolymerization.
In the corpus straitum of the experimental group D group mouse of processing in Endo-N stoste (1U/ μ l) (fill with and kill afterwards for 23 days in 6-OHDA injection), contain a large amount of BrdU positive cell (see figure 5)s.Cut into slices and carried out the immunofluorescence dyeing of BrdU in a whole set of sagittal sections position of mouse left and right brain that the present embodiment has been chosen C group and D group, with inverted fluorescence microscope, observe whole brain sections, choose contain corpus straitum texture and optic tract simultaneously pure sagittal slices, carry out the statistics of BrdU positive cell and carry out statistics comparison.Be divided into following group: (raw data is in Table 3) Endo-N-left group and the Endo-N-right group (raw data is in Table 4) of Control-left group and Contorl-right group.Use two-photon laser Laser Scanning Confocal Microscope to take pictures, (a) BrdU positive cell is seldom for Fig. 5 to can be observed Control-left group, Contorl-right group (Fig. 5 b) and Endo-N-left group (Fig. 5 d) BrdU positive cell are few, and Endo-N-right group (Fig. 5 e) has a large amount of BrdU positive cells.Four groups of data are used one-wayANOVA to analyze, and it is mean value ± standard error (Mean ± SEM) expression for result.Using p<0.05 as the boundary at significant difference (Fig. 5 g).Result Endo-N-right group all has significant difference with other three groups.Endo-N-left group also has significant difference with Control-left group.
In the corpus straitum of the experimental group D group mouse of processing in Endo-N stoste (1U/ μ l) (fill with and kill afterwards for 23 days in 6-OHDA injection), contain the two mark of a small amount of BrdU/GFAP+ cell (see figure 6).The present embodiment has been chosen a set of sagittal sections of the right brain of the mouse position section of C group and D group and has been carried out the two mark of BrdU/GFAP+ immunofluorescence dyeing, with two-photon laser Laser Scanning Confocal Microscope, take pictures, the right brain striatum sagittal slices of Endo-N-right(test group in D group) group, find the two mark of a small amount of BrdU/GFAP+ cell (Fig. 6 c), in the right brain striatum sagittal slices of Contorl-right(control group of C group) organize and do not find visible marking's thing.The present embodiment has been chosen the two mark of BrdU/GFAP+ cell and has been carried out the burnt rectangular projection (Fig. 6 f) of copolymerization.
In the corpus straitum of the experimental group D group mouse of processing in Endo-N stoste (1U/ μ l) (fill with and kill afterwards for 23 days in 6-OHDA injection), contain the two mark of a small amount of BrdU/NeuN+ cell (see figure 7).The present embodiment has been chosen a set of sagittal sections of the right brain of the mouse position section of C group and D group and has been carried out the two mark of BrdU/NeuN+ immunofluorescence dyeing, with two-photon laser Laser Scanning Confocal Microscope, take pictures, the right brain striatum sagittal slices of Endo-N-right(test group in D group) group, find the two mark of a small amount of BrdU/NeuN+ cell (Fig. 7 c), in the right brain striatum sagittal slices of Contorl-right(control group of C group) organize and do not find visible marking's thing.The present embodiment has been chosen the two mark of BrdU/NeuN+ cell and has been carried out the burnt rectangular projection (Fig. 7 f) of copolymerization.
In method of the present invention, 6-OHDA is that a kind of neurotoxin can selective injury dopaminergic neuron.When 6-OHDA is directly injected after black substance, the DA serotonergic neuron that is rich in monoamine oxidase in mouse substantia nigra of midbrain can absorb it, and changes into neurotoxin under monoamine oxidase effect, as free radical and quinones cause damage to neuron.For Nervous System Anatomy, the fiber that black substance spreads out of has mainly formed Nigrostriatal projection, and namely projection fibre, after black substance sends, arrives striatal caudate nucleus head and shell core head along the hypothalamus outside of passing through on black substance back of the body inner side, capsula interna inner side.When 6-OHDA is injected to after mouse striaturn, by dopaminergic neuron tip, by aixs cylinder counter transport, transported in black substance portion cell body, by destroying the antioxidant system of dopaminergic neuron, make stability and the DNA integrality of mitochondrial function, film be subject to destruction, finally cause the hypofunction of nigrostriatum dopamine system and produce chronic brain injury symptom.Therefore, the target spot that uses 6-OHDA to prepare PD animal model mainly contains black substance compact part, corpus straitum and medial forebrain bundle, the most frequently used method is that one-sided injection 6-OHDA is to black substance or medial forebrain bundle, impel dopaminergic neuron dead fast within a short period of time, prepare like this PD acute injury model, be difficult for the early stage PD pathological manifestations of simulation.And chronic injury model can directly be injected 6-OHDA greatly to corpus straitum and cause the retrogression pathological changes of substantia nigra dopaminergic neuron, this method can slowly produce neuronal degeneration in one week to the time in several weeks, met the course of disease of carrying out property of PD regression.In the present invention, be exactly to adopt injection 6-OHDA in the one-sided corpus straitum of brain three-dimensional location mouse to manufacture chronic brain injury.
Daniela Battista in 2010 and Urs Rutishauser are used the specificity lyases of Endo-N(PSA) remove after PAS, they find that legacy migration path RMS is destroyed, and the prematurity neurocyte that SVZ produces comprises corpus straitum dystopy migration showed increased to other brain tissues.The present invention has also observed identical result in the corpus straitum of animal used as test, also find has a large amount of BrdU positive cells in the corpus straitum of right side of mice 6-OHDA damage simultaneously, with the unmarred left side comparison with an experiment mice of corpus straitum, this phenomenon is particularly evident.Therefore by method of the present invention, can consider, the local inflammation that 6-OHDA damage causes is obvious to the attractability migration of neural stem cell.Use Endo-N effectively to remove after PAS, lost a large amount of gatherings in HeRMSQi Shi district, neural stem cell svz district of adhesion for providing prerequisite to corpus straitum migration.This results suggest people use Endo-N to remove PSA will become an approach that effectively starts Endogenous neural stem cells treatment chronic brain injury.
Large quantity research is found neural stem cell surperficial PSA-NCAM that all expresses high level in transition process of subventricular zone.When these cells arrive destination, are divided into after ripe intrerneuron, the PSA of cell surface expresses and will disappear, prompting PSA-NCAM plays an important role in prematurity neurocyte directional migration, and also someone proves that PSA-NCAM has the effect that suppresses neural stem cell differentiation simultaneously.Therefore, the present invention can study moving to the differentiation capability of striatal neural stem cell.And having obtained gratifying result after having done dyeing, the migration of this class and the neural stem cell come have been broken up becomes ripe neuron and astroglia.Deiter's cells is the class neurocyte in mankind's central nervous system, and they and conduct electricity impulsion unlike neuron are considered to only rise supporting function for a long time.Until in the last few years, people just started to recognize the regulating action of Deiter's cells (especially astroglia) in brain relevant with brain cognitive ability.Result of study of the present invention is further pointed out the differentiation capability of the neural stem cell of migration, for further experiment provides cell base.
Table 1: the mouse brain corpus straitum BrdU of control group (A group)
+quantity
L1-L5, R1-R5 are animal number of cases; N can be used for the number of slices of statistics
Table 2: the mouse brain corpus straitum BrdU of experimental group (B group)
+quantity
L1-L5, R1-R5 are animal number of cases; N can be used for the number of slices of statistics
Table 3: the mouse brain corpus straitum BrdU of control group (C group)
+quantity
L1-L5, R1-R5 are animal number of cases; N can be used for the number of slices of statistics
Table 4: the mouse brain corpus straitum BrdU of experimental group (D group)
+quantity
L1-L5, R1-R5 are animal number of cases; N can be used for the number of slices of statistics.
Claims (7)
1. a method of studying brain endogenous neural stem cell migration, propagation, differentiation:
1) to animal brain, 5 bromodeoxyuridines and the two mark of both adrenal glands cortin, 5 bromodeoxyuridines and neuron core antigen, 5 bromodeoxyuridines and glial cell line-derived neurotrophic factor immunofluorescence dyeing are carried out in section, and sialylated neural cell adhesion factor list mark immunofluorescence dyeing;
2) use GraphPad Prism6.0 software statistics analyze data and draw, 5 bromodeoxyuridine positive cells are that single factor multi-group data is used one-way variance analysis.
2. method according to claim 1, wherein, described animal brain section is processed through following:
In the corpus straitum of brain right side after three-dimensional locating injection 6-OHDA solution, in intracerebroventricular injection PSA specificity lyases stoste, then lumbar injection 5 bromodeoxyuridine solution.
3. method according to claim 1, wherein, described animal brain section is animal left and right brain section.
4. method according to claim 1, wherein, includes control group brain section in described animal brain section.
5. according to the method described in claim 1 or 4, wherein, after described control group brain section is three-dimensional locating injection 6-OHDA solution, at intracerebroventricular injection phosphate buffered saline(PBS) damping fluid, then lumbar injection 5 bromodeoxyuridine solution.
6. method according to claim 1, wherein, described animal brain section lumbar injection 5 bromodeoxyuridine solution are to inject a couple of days continuously.
7. method according to claim 1, wherein, the result of described statistical data analysis represents with mean+/-standard error, usings p<0.05 as the boundary at significant difference.
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| CN110806485A (en) * | 2019-11-12 | 2020-02-18 | 广州医科大学附属第二医院 | Colloidal fiber acidic protein antibody detection kit and application thereof |
| CN112057634A (en) * | 2020-09-17 | 2020-12-11 | 深圳先进技术研究院 | Method for detecting new growth date of neurons of newborn animals and method for marking embryos by 5-ethynyl-2' deoxyuridine |
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