CN104611294A - In-vitro culture method of visual cortex neuron - Google Patents
In-vitro culture method of visual cortex neuron Download PDFInfo
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Abstract
The invention relates to a neuron in-vitro culture method for synaptic plasticity research on a visual cortex neuron. The method is characterized by comprising the following steps: (1) carrying out pretreatment on cerebral tissues of fetal rats, separating visual cortex tissues and carrying out proteinase digestion and centrifugation; (2) adding an inoculation medium to terminate digestion; (3) blowing a single-cell suspension, sieving and counting; and (4) inoculating with the inoculation medium of which the density is 2.5*10<4>/cm<2> to 5*10<4>/cm<2>, carrying out adherent culture in a full liquid-exchange manner, and after cell attachment, carrying out subsequent culture in a half liquid-exchange manner. The invention further provides a visual cortex neuron cell line of rats, which is cultured by the neuron in-vitro culture method, and an application of the visual cortex neuron cell line in synaptic plasticity research on the visual cortex neuron. The culture method is efficient and simple; the neuron is high in purity, and good in state, and has a structural basis of neuronal synaptic plasticity; the functional state of the neuronal synaptic plasticity can be reflected; and the culture method can be applied to research on the synaptic plasticity of visual cortex of rats.
Description
Technical field
The present invention relates to cell engineering field, be specifically related to a kind of visual cortex neurone extracorporeal culturing method.
Background technology
Primary visual cortex (brodmman17 district), being positioned at hemicerebrum pillow rear portion near cerebellum region, is the important component part of vision high-level center.Post-natal humans's class and mammiferous visual system can change its original nerve connections and synaptic structure according to visual environment, the most tricky time that this change occurs is called as visual cortex critical period of plasticity, the mankind this period for birth after 2-10 year, rat is 2-6 week after birth.Period, the visual experience of exception will cause visual deterioration, but eye is without organic disease, the amblyopia that namely clinical ophthalmology is common.Its diseased region is mainly at visual cortex, in close relations with visual cortex plasticity-.In visual cortex critical period of plasticity, give effective visual stimulus, amblyopia can be cured; After critical period stops, visual cortex plasticity-is suppressed, and now abnormal visual environment no longer will cause amblyopia, but established amblyopia is also difficult to recover, and this is also the reason that more than 10 years old children and grownup's amblyopia are difficult to cure.For disclosing the pathogenesis of amblyopia and finding amblyopia treatment approach of growing up, the termination mechanism of visual cortex plasticity-and critical period thereof becomes the point of penetration of research at present.
Be based upon in living animal and brain sheet level to the plastic research of visual cortex at present more, often be confined on neural circuitry, be difficult to further investigate neurone itself, and in neurone aspect, show as neuronic synaptic plasticity in view of visual cortex plasticity-, therefore the plastic point of penetration of further investigation visual cortex is become to the research of visual cortex synaptic plasticity.Simultaneously because of projection branch that the neuron loss of acute isolation is complete, surface receptor is by partial destruction or loss, intercellular cannot form contact, is unfavorable for the research of synaptic plasticity, so the research of synaptic plasticity is mainly based upon in the cultivation level of neuronal cell at present.Based on this, set up a neurone culture system being applicable to the research of visual cortex neuronal synaptic plasticity to be very important to further investigation visual cortex plasticity-, this system also can play a role in ophthalmology pathology and drug reaction and the research field such as amblyopia, therapy mechanism simultaneously.And such culture system there is not been reported at home, external similar research system is based upon on hippocampus more, there is not been reported set up be used for visual cortex neuronal synaptic plasticity research neurone culture system in vitro.
Simultaneously, at present the research of visual cortex critical period of plasticity termination mechanism is thought, the maturation of GABA energy inhibitory interneuron and neurone ambient network (Perineuronal nets, PNs) increasing expression stops there is obvious promoter action to visual cortex critical period of plasticity: later stage critical period, neurone ambient network PNs increasing expression and change soluble mixture into by soluble complex, solidification synapse structure, and surround around GABA neurone and promote that it is ripe; Suppress synaptic plasticity further after GABA neuronal maturation, and PNs participates in the termination of critical period of plasticity jointly.
LE rat, complete by name long evans rat, because of its eye structure and the mankind more seemingly, often by as rat ophthalmology experimental study object, therefore this experiment employing LE rat is research object.Cultivate and find, compared with 1-3d newborn rat after birth, the neurone purity adopting tire mouse to obtain is high, and heteroproteose cell is few, and spongiocyte splitting ability is low; Adopt the tire mouse of pregnant 19-21d, its spongiocyte splitting ability is comparatively strong, and be less than the tire mouse of pregnant 14d, its brain visual cortex region growing is not yet ripe, locate more difficult, and neurone is comparatively fragile, the easy apoptosis of late stage of culture.Experiment finds, the visual cortex neurone purity higher (80-90%) that pregnant 17-18d tire mouse is cultivated, and active better, survival rate is high and the survival time is long.
Summary of the invention
The present invention uses B27 collocation serum free medium to cultivate visual cortex neurone, and neuronic purity, growth conditions are identified, Immunofluorescence test is carried out to the GABA relevant to critical period termination mechanism energy inhibitory interneuron (GAD 65/67 marks) and PNs positive cell (Neurocan marks) simultaneously, and adopt patch clamp technique to detect neuronic functional development situation, take the lead in developing the neurone extracorporeal culturing method for the research of visual cortex neuronal synaptic plasticity.
Technical scheme of the present invention is as follows:
For the neurone extracorporeal culturing method of visual cortex neuronal synaptic plasticity research, it is characterized in that, comprise the steps:
1) get tire mouse cerebral tissue and carry out pre-treatment, be separated visual cortex tissue and carry out protease digestion, centrifugal;
2) add inoculation medium and stop digestion;
3) sieve after blowing and beating into single cell suspension and count;
4) inoculate with the density inoculation medium of 2.5 × 104-5 × 104/cm2, entirely change the adherent culture of liquid mode; After cell attachment, partly change the follow-up cultivation of liquid mode.
Described tire mouse is the tire mouse of the female rats of pregnant 17-18 days; Described pre-treatment refers to described rat brain tissue to be immersed in the high sugar of serum and to dissect in liquid 10 minutes; Described separation visual cortex is organized in common dissection liquid and carries out.
The high sugar of described serum is dissected liquid and is referred to that adding volume ratio in DMEM/F12 substratum is the horse serum of 10% and the mycillin of 2 × 105U/L; Described common dissection liquid is add the 4-hydroxyethyl piperazine ethanesulfonic acid damping fluid that the mycillin of 2 × 105U/L and volume ratio are 1% in Earle's balanced salt solution.
Described piping and druming refers to, the Pasteur's pipe being respectively 2 μm, 1.5 μm, 0.8 μm with bore is is softly blown and beaten tissue successively, and blows and beats 5-6 time with often kind of Pasteur's pipe.
Described protease digestion refers to be cut into 1mm × 1mm fritter under getting visual cortex is organized in dissecting microscope, then in 37 DEG C of water-baths, digests 15-20 minute with the papoid of 37 DEG C of preheatings 14U/ml of 0.5 hour; Describedly centrifugally to refer under the rotating speed of 1500r/min centrifugal 3 minutes.
Described adherent culture of entirely changing liquid mode refers to: observe after inoculation 2h, if cell debris is more, entirely should changes liquid, entirely change liquid after 18h with maintain base after inoculation 6h with inoculation medium; As fragment is less, then entirely change liquid with maintain base after 24h; Described follow-up cultivation of partly changing liquid mode refers to that the mode of once partly changing liquid with maintain base for 2-3 days is cultivated.
Described maintain base refers to
the L-glutaminase that volume ratio is the B27 serum substitute of 2% and the mycillin of 2 × 105U/L and 0.5mmol/L is added in Medium serum free medium.
Described inoculation medium is add the L-glutaminase that volume ratio is the horse serum of 10%, the mycillin of 2 × 105U/L and 0.5mmol/L in DMEM/F12 substratum.
The Rat Visual cortical neuronal cells system that described neurone extracorporeal culturing method obtains.
The application of described neurone extracorporeal culturing method in the research of visual cortex neuronal synaptic plasticity.
Rat Visual cortical neuron culture method of the present invention has many improvements.First, the present invention finds through cultivating, and compared with 1-3d newborn rat after birth, the neurone purity adopting tire mouse to obtain is high, and heteroproteose cell is few, and spongiocyte splitting ability is low; Adopt the tire mouse of pregnant 19-21d, its spongiocyte splitting ability is comparatively strong, and be less than the tire mouse of pregnant 14d, its brain visual cortex region growing is not yet ripe, locate more difficult, and neurone is comparatively fragile, the easy apoptosis of late stage of culture.Experiment finds, the visual cortex neurone purity higher (80-90%) that pregnant 17-18d tire mouse is cultivated, and active better, survival rate is high and the survival time is long, and therefore the present invention innovates and adopts the tire mouse of pregnant 17-18d female rats as object of drawing materials.Secondly, be maintain neurone eubolism, reduce neuronal damage, the cerebral tissue of described tire mouse is immersed in the high sugar of serum and dissects in liquid by cultural method of the present invention, makes to draw materials to inoculation to complete shortening consuming time in 2h; In addition, for reducing the existence can dividing heteroproteose cell (as vascular endothelial cell etc.), pia mater is divested totally as far as possible; In digestive process, for ensureing that tissue digestion is complete, before digestion, institute's visual cortex tissue of getting first is cut into 1mm × 1mm fritter under dissecting microscope, and papoid also first carries out preactivate at 37 DEG C of preheating 0.5h; In piping and druming process, for avoiding neuronal cell excessive damage and stimulating spongiocyte division, piping and druming action is as far as possible soft, and piping and druming number of times is too much unsuitable; About inoculum density, when the present invention studies and finds that inoculum density is greater than 50*104/cm2, intercellular space resource is inadequate, it is abnormal that cultivation is greater than 10d cellular form, be unfavorable for electro physiology and Senile Mouse, and density is less than 1*104/cm2, be then interconnected minimizing between neurone, be difficult to supply nutrition each other, affect late growing stage; Repeatedly after experiment, suggestion inoculum density is 2.5-5*104/cm2 is comparatively suitable; On substratum, though have research by containing adding cytosine arabinoside to suppress spongiocyte merisis in blood serum medium, but cytosine arabinoside also has certain toxic action [14] to neurone, therefore present method first adopt containing blood serum medium help neurone adherent and growth, after 24h neurone to be seeded is substantially adherent, be changed to the serum free medium [16] containing B27, to suppress spongiocyte merisis, promote neure growth simultaneously; Follow-up cultivation is ensure that neurone can use the somatomedin of self secreting and avoid causing violent change to culture environment (osmotic pressure, pH etc.), takes within 2-3 days, partly to change liquid principle and changes liquid; When needing long-term cultivation (cultivation is more than 20d), late stage of culture (cultivate 15d after) is changed liquid number of times and can be gradually reduced to and within 7 days half, change liquid once, to ensure the stable of neurone culture environment.In order to the neurone that upper method is turned out, its survival rate is high, growth conditions good, and purity reaches 80%-90%.
The present invention adopts GAD65/67 and Neurocan to mark GABA neurone and PNs respectively, result shows that the expression of Neurocan and GAD65/67 on the visual cortex neurone that this law obtains all is positive, PNs positive cell ratio maintains 80%-85% always, and GABA positive neuron ratio maintains 40%-45% always.
The visual cortex neurone cultivated by cultural method of the present invention is under Constant Electric Current stimulates, neurone after Patch-clamp techniques obtains 1-3d, 7-14d, 14d is induced out single peak type respectively, the action potential of transition type and sustained, reflect the functional development state of institute's developing approach, and AP variation tendency and and body in the variation tendency basically identical [21] of PSCs in critical period of plasticity, show that visual cortex neurone that this law is cultivated possesses the function of synaptic plasticity research basic.
The described Rat Visual cortical neuronal cells system purity that the present invention obtains is high, in good condition, can reflect the structure and function state of visual cortex neuronal synaptic plasticity, can be used for the correlative study of visual cortex neuronal synaptic plasticity.
Accompanying drawing explanation
Fig. 1 is the cellular form (A, the C:X100 that observe under phase microscope after the visual cortex culture of primary neurons of cultural method of the present invention acquisition supports 12h, 4d; B, D:X200)
Fig. 2 is cellular form (Neun dyes, and green, DAPI redyes, the blue) (A:X200 that visual cortex culture of primary neurons that cultural method of the present invention obtains supports at fluorescence microscopy Microscopic observation after Neun chemical staining after 6d; B:X400)
Fig. 3 to support after 4d after Neurocan chemical staining in the cellular form (A:X200, B:X400) of fluorescence microscopy Microscopic observation for visual cortex culture of primary neurons that cultural method of the present invention obtains
Fig. 4 is cellular form (GAD65/67 dyes, and DAPI redyes) A1, A2 at fluorescence microscopy Microscopic observation after GAD65/67 chemical staining after the visual cortex neurone of cultural method of the present invention acquisition cultivates different number of days: cultivate 5d (A1:X200; A2:X400), B1, B2: cultivate 13d (B1:X200; B2:X400)
Fig. 5 be the visual cortex culture of primary neurons that cultural method of the present invention obtains support 3,7, neuron action potential through Whole-cell recording after 14d brings out situation.Current clamp mode operation of recording current potential: pulse constant current stimulates (A, C:3d and 14d:40pA × 1000ms, B:7d:40pA × 300ms)
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but do not limit the scope of the invention.If no special instructions, the operation used in following embodiment is ordinary method, and the reagent adopted all can be commercially available.
The source of biomaterial and record source
The female mouse of other lang evans of cleaning grade is bought and moves probatio inspectionem pecuoarem word SCXK (Chongqing) 20020003 in three aseptic sursery institute of great Ping hospital of army medical university Experimental Animal Center
Main agents
DMEM/F12 substratum,
medium, B27, EBSS (Gibco, USA), mycillin (PS), horse serum (Hyclone, USA), laminin, L-glutamin, D-poly-lysine, Hepes (Sigma, USA), papoid (worthington, USA), little anti-rat NeuN monoclonal antibody, rabbit Chinese People's Anti-Japanese Military and Political College mouse GAD67 antibody (Millipore, USA), fluorescent mark rabbit anti-mouse Alexa Fluor 647 (Abcam, USA), rabbit Chinese People's Anti-Japanese Military and Political College mouse Neurocan (doctor's moral, China), fluorescent mark donkey anti-rabbit Alexa Fluor 647 (Gibco, USA), DAPI core dye liquor (the green skies, China)
Main solution and formula
DMEM/F12 substratum: Dulbecco`s improved culture medium and Ham`s F12 substratum mix with 1:1 (mass ratio)
Liquid dissected by the high sugar of serum: add 10% horse serum and 2*105U/L PS in DMEM/F12 substratum.
Common dissection liquid: add 2*105U/L PS and 1%Hepes in EBSS.
Inoculation medium: DMEM/F12 (Gibco) adds 10% horse serum and 2*105U/L PS and 0.5mmol/LL-glutamin.
Maintain base:
medium adds 2%B27 and 2*105U/LPS and 0.5mmol/L L-glutamin.
Liquid in electrode: Potassium Gluconate 230mmol/L, MgCl2 2.1mmol/L, Na2AT P 2mmol/L, Na2GTP 0.3mmol/L, EGTA 0.5mmol/L, HEPE 5mmol/L, adjustment pH to 7.2 ~ 7.5, filter bore filter with 0.22nm, divide and be filled in 0.5mlEP pipe, be placed in-20 DEG C and save backup.
Extracellular fluid: NaCl 120mmol/L, KCl2.7mmol/L, CaCl22.5mmol/L, MgCl21.3mmol/L, NaH2PO41.2mmol/L, NaHCO326.5mmol/L, glucose 17mmol/L, Sodium.alpha.-ketopropionate 1.7mmol/L, Sodium.alpha.-hydroxypropionate 3.8mmol/L, adjustment pH to 7.2 ~ 7.5, passing to volume fraction is 95%O2 and 50%CO2 10min, makes it reach capacity.
Embodiment 1 utilizes cultural method developing approach of the present invention
1. culture plate process:
Place the circular lid slide of diameter 14mm in 24 orifice plates in advance, add 0.05g/L poly-lysine and carry out bag quilt, spend the night, suck poly-lysine, aseptic ultrapure water rinsing 3 times, room temperature is dried, and adds 2mg/L laminin and wraps by 2h.
2. neurone is cultivated:
(1) draw materials: female mouse break neck put to death, open abdomen after 75% alcohol disinfecting belly and get tire.Take out tire mouse, get head.Ophthalmic tweezers fixes mouse head, and corneal scissors is cut to eyeball place from foramen magnum along both sides skull, removes skull, gets brain, in common dissection liquid, peel off pia mater.Visual cortex is positioned at dorsal surface region, brain occipital region, and size is about 2mm*3mm, and thickness is about 2mm*3mm, cuts visual cortex.Shred tissue, common dissection liquid washes twice.Add the papoid of 37 DEG C of preheatings 14U/ml of 0.5 hour, 37 DEG C of water-bath digestion 15 are to 20min, 1500r/min, centrifugal 3min, add appropriate inoculation medium and stop digestion, manage with 3 kinds of Pasteur that bore is descending and softly blow and beat tissue (Pasteur's pipe mouth of pipe should be mellow and full with spirit lamp polishing in advance), often kind of Pasteur's pipe is blown and beaten 5-6 time, to forming single cell suspension, basis of microscopic observation piping and druming effect.Cell suspension aperture is after the sieved filter of cell of 40nm, counts.
(2) inoculate and change liquid: with inoculation medium with the density inoculating cell of 2.5x104-5x104cell/cm2, observing after inoculation 2h, if cell debris is more, after inoculation 6h, entirely should changes liquid with inoculation medium, after 18h, entirely change liquid with maintain base.As fragment is less, then entirely change liquid with maintain base after 24h, the mode backward taking maintain base partly to change liquid changes liquid.Cultivate first two weeks, partly change liquid once every 2-3d maintain base, maintain base partly changes liquid once weekly backward.
Embodiment 2 Rat Visual cortical neuronal cells system of the present invention carries out the research of visual cortex neuronal synaptic plasticity
One, visual cortex neurone Purity
Method:
Get the Rat Visual cortical neuron creep plate cultivating 6d according to embodiment 1 step (1) and (2), 0.01M PBS rinsing 2 times, 3min/ time; 4% paraformaldehyde process 15min, 0.01M PBS rinsing 4 times, 5min/ time; 0.25%triton-100 breakthrough process 15min, 0.01M PBS rinsing 4 times, 5min/ time; Add the confining liquid containing 1%BSA and 10% rabbit anteserum, 37 DEG C of closed 1h; Add the Neun primary antibodie (1:500) of little anti-rat, place in wet box, 4 DEG C of overnight incubation.(5) 0.01M PBS rinsing 3 times, 5min/ time; Add two anti-(1:800) that rabbit anti-mouse Alexa Fluor 647 indicates, place in wet box, 37 DEG C of lucifuges hatch 45mins, 0.01M PBS lucifuge rinsing 2 times, 5min/ time; Lucifuge, 10%DAPI process 10min, 0.01M PBS rinsing 3 times, 5min/ time; The anti-quencher mounting of fluorescence, watches result under Laser Scanning Confocal Microscope.
Result:
Inoculation 2h observes, and visible cell major part is adherent, and has part to grow projection.24h is shown in when changing liquid, and cell attachment is good, and cell space is perfectly round bright, visible tiny enation.Observe after 4d and find, the full homogeneous of cell surface, diopter is good, and projection length rises appreciably, and sees Fig. 1.During 7-11d, the full increase of visible neuronal pyramidal cell cell space, smooth surface, the bright refractive power of cell space is good, and projection is crossed as net, and neurone can be survived about 30d.
Under fluorescent microscope, neurone (green) cell space full (Neun mark), karyon (blueness) is complete, neurite is obviously elongated, be interlaced with one another into net between cell process, see that (Neun is positioned at endochylema to Fig. 2, positive in green, DAPI contaminates core in blue), neurone purity is 80%-90%.
Two, visual cortex critical period of plasticity stops relevant immunocytochemical stain
Method:
(1) the Rat Visual cortical neuron creep plate cultivating 14d according to embodiment 1 step (1) and (2) is got, 0.01M PBS rinsing 2 times, 3min/ time; 4% paraformaldehyde process 15min on ice, 0.01M PBS rinsing 4 times, 5min/ time; 0.25%triton-100 breakthrough process 15min, 0.01M PBS rinsing 4 times, 5min/ time; Add containing 1%BSA and 10% donkey serum block, 37 DEG C of incubator process 1h; Add the GAD65/67 primary antibodie (1:2000) of rabbit Chinese People's Anti-Japanese Military and Political College mouse, place in wet box, 4 degree of refrigerators, overnight incubation; 0.01M PBS shaking table rinsing 3 times, 10min/ time; Add two anti-(1:1000) that donkey anti-rabbit FITC indicates, place in wet box, room temperature lucifuge hatches 60mins, 0.01M PBS lucifuge shaking table rinsing 3 times, 10min/ time; Add 10%DAPI lucifuge process 10min, 0.01M PBS lucifuge rinsing 3 times, 10min/ time; The anti-quencher mounting of fluorescence, watches result under Laser Scanning Confocal Microscope.
(2) the Rat Visual cortical neuron creep plate cultivating different number of days according to embodiment 1 step (1) and (2) is got, primary antibodie is rabbit Chinese People's Anti-Japanese Military and Political College mouse Neurocan (1:500), two resist for donkey anti-rabbit Alexa Fluor 488 mark two anti-(1:400), and other operations are identical with above-mentioned steps (1).
Result:
(1) original cuiture cultivates the expression of Neurocan on visual cortex neurone:
Neurocan (green) mainly expresses on cell space and projection, visible obviously expression (ratio about 85%) on 4d, along with cultivated days increases, it expresses stable, and display cell space increases, and projection extends, reflection neure growth developmental condition is ripe gradually, its positive ratio maintains 80%-85% always, sees Fig. 3 (in green, DAPI contaminates core in blue to the Neurocan positive).
(2) expression of GAD65/67 on original cuiture visual cortex neurone:
GAD65/67 mainly expresses on cell body and projection, 5d just can detect obvious expression (ratio about 45%), (the GAD65/67 positive is in green to see Fig. 4, DAPI contaminates core in blue), and increase with incubation time number of days, it expresses stable, and positive ratio maintains 40%-45% always.
Three, original cuiture Rat Visual cortical neuron patch clamp detects
Method:
Get and cultivate 1-15d visual cortex neurone creep plate, have in the diaphragm clamp basin of extracellular fluid in being positioned over, infusion pump continues input 95%O2+5%CO2 mixed gas in bath.The glass recording electrodes (tip diameter: 1-1.6um) made with horizontal electrode suppressor, in it, after perfusion electrode solution, glass electrode resistance is 5-10M Ω.With light transmission, good and ganoid visual cortex neurone is record object, after glass electrode and after birth sealing-in, after giving negative pressure-pumping rupture of membranes gently, forms Whole-cell recording pattern.In such a mode, pass through current clamp, operation of recording current potential (AP), subsequent data acquisition and analysis are completed by the pClmp10.1 software of Axon company, Detailed operating procedures is shown in " Shultz SR, MacFabe DF, Foley KA, et al.A single mild fluid percussion injury induces short-term behavioral and neuropathological changes in the Long-Evans rat:support for an animal model of concussion [J] .Behav Brain Res.2011, 224 (2): 326-335 " and " Yao Jun Ping Houwensheng Liu Hui etc., newborn LongEvans primary rat visual cortex neuronic culture & identification [J] Recent Advances in Ophthalmology, 2012, 32:204-207 ".
Patch clamp successfully records 170 neurones altogether, (1-7d:54,7-14d:66,14-18d:50), resting membrane electric potential is-50--70mV, under current clamp, give cell Constant Electric Current to stimulate, entice cell produces action potential (AP), and result is as follows:
Cultivate 1-3d neurone and belong to juvenile form neurone, single peak type AP can be induced out; Cultivate 7-14d neurone and belong to osculant neurone, transition type AP can be induced out; After cultivating 14d, neurone belongs to adult form neurone, can be induced out sustained AP, see that Fig. 5: 3d is single peak type AP, show as neurone in the burst length and can only be induced out an AP; 7d is transition type AP, show as neurone in the burst length be induced out quantity not wait AP, but AP pulse stop before end; 14d is sustained AP, show as after neurone in the burst length is stimulated by Constant Electric Current and can continue to produce AP, and the frequency of AP increases with stimulating current intensity and increases, AP judging criterion see reference document " Chen Zhongshan, cloudy positive diligent, Wang Shijun; etc.; LongE ~ rat retinal ganglion cell electrophysiology developmental characteristic research. [J] Third Military Medical University journal 2003,21-1927-03,1000-5404 ".
Claims (10)
1., for the neurone extracorporeal culturing method of visual cortex neuronal synaptic plasticity research, it is characterized in that, comprise the steps:
1) get tire mouse cerebral tissue and carry out pre-treatment, be separated visual cortex tissue and carry out protease digestion, centrifugal;
2) add inoculation medium and stop digestion;
3) sieve after blowing and beating into single cell suspension and count;
4) with 2.5 × 10
4-5 × 10
4individual/cm
2density inoculation medium inoculate, entirely change the adherent culture of liquid mode; After cell attachment, partly change the follow-up cultivation of liquid mode.
2. neurone extracorporeal culturing method according to claim 1, is characterized in that, described tire mouse is the tire mouse of the female rats of pregnant 17-18 days; Described pre-treatment refers to described rat brain tissue to be immersed in the high sugar of serum and to dissect in liquid 10 minutes; Described separation visual cortex is organized in common dissection liquid and carries out.
3. neurone extracorporeal culturing method according to claim 2, is characterized in that, the high sugar of described serum is dissected liquid and referred to add the horse serum and 2 × 10 that volume ratio is 10% in DMEM/F12 substratum
5the mycillin of U/L; Described common dissection liquid is
earle's balanced salt solutionin add 2 × 10
5the mycillin of U/L and volume ratio are the 4-hydroxyethyl piperazine ethanesulfonic acid damping fluid of 1%.
4. neurone extracorporeal culturing method according to claim 1, is characterized in that, described piping and druming refers to, the Pasteur's pipe being respectively 2 μm, 1.5 μm, 0.8 μm with bore is is softly blown and beaten tissue successively, and blows and beats 5-6 time with often kind of Pasteur's pipe.
5. neurone extracorporeal culturing method according to claim 1, it is characterized in that, described protease digestion refers to be cut into 1mm × 1mm fritter under getting visual cortex is organized in dissecting microscope, then in 37 DEG C of water-baths, digests 15-20 minute with the papoid of 37 DEG C of preheatings 14U/ml of 0.5 hour; Describedly centrifugally to refer under the rotating speed of 1500r/min centrifugal 3 minutes.
6. neurone extracorporeal culturing method according to claim 1, it is characterized in that, described adherent culture of entirely changing liquid mode refers to: observe, if cell debris is more after inoculation 2h, after inoculation 6h, entirely should change liquid with inoculation medium, after 18h, entirely change liquid with maintain base; As fragment is less, then entirely change liquid with maintain base after 24h; Described follow-up cultivation of partly changing liquid mode refers to that the mode of once partly changing liquid with maintain base for 2-3 days is cultivated.
7. neurone extracorporeal culturing method according to claim 6, is characterized in that, described maintain base refers to
the B27 serum substitute and 2 × 10 that volume ratio is 2% is added in Medium serum free medium
5the mycillin of U/L and the L-glutaminase of 0.5mmol/L.
8., according to the arbitrary described neurone extracorporeal culturing method of claim 1 or 6, it is characterized in that, described inoculation medium is add the horse serum, 2 × 10 that volume ratio is 10% in DMEM/F12 substratum
5the mycillin of U/L and the L-glutaminase of 0.5mmol/L.
9. the Rat Visual cortical neuronal cells system of the arbitrary described neurone extracorporeal culturing method acquisition of claim 1 ~ 8.
10. the arbitrary described application of neurone extracorporeal culturing method in the research of visual cortex neuronal synaptic plasticity of claim 1 ~ 8.
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