CN105754943B - A kind of naked mole cultured hippocampal neuron method - Google Patents
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
The present invention relates to technical field of cell biology, in particular to a kind of naked mole hippocampal neuron isolate and purify and cultural method.The present invention is comprehensive to separate and purifies hippocampal neuron from newborn naked mole cerebral cortex using a variety of culture mediums, has found out the reasonable cultural method of the naked mole hippocampal neuron of rodent mammal suitable for alternating temperature.The method of the present invention being capable of the normal naked mole hippocampal neuron of a large amount of functional activities of acquisition easy, efficiently, economic, culture under hypoxia condition can guarantee that this cell is still able to maintain the biological characteristics under body state in ex vivo environment, consequently facilitating the special physiological function of naked mole hippocampal neuron is directly further studied in pure in-vitro cell culture model, to provide important theoretical foundation to explore biological mechanism therein and being applied to clinical relevant.
Description
Technical field:
The present invention relates to technical field of cell biology, and in particular to a kind of cell isolate and purify and cultural method, it is special
Be not related to a kind of naked mole hippocampal neuron isolate and purify and cultural method.
Background technique:
Spatial cognition, ability of learning and memory are one of significant capabilities necessary to the mankind and other animal survivals.Hippocampus is
One important component of limbic system, very important effect is played in body multiple functions, is especially remembered
(Memory systems of the during acquisition acquisition and integration, the acquisition of learning ability and space navigation etc.
brain:a brief history and current perspective.Squire LR.Neurobiol Learn
Mem.2004Nov；82(3):171-7.；Place cells,grid cells,and the brain's spatial
representation system.Moser EI,Kropff E,Moser MB Annu Rev Neurosci.2008；31
():69-89.).The study found that the disease occurred in a variety of neurodegenerative diseases and nervous system development process is all certain
Have in degree with the structure of hippocampus and dysfunction this it is a degree of contact, and the exception of hippocampal formation and function may be most
It can aggravate the occurrence and development of the nervous system disease eventually.Therefore, it deeply discloses hippocampal neuron physiologic function and feature will be advantageous
In the function of illustrating hippocampus and its effect in the nervous system disease occurrence and development.
Naked mole is a kind of alternating temperature mammal, is able to maintain in the stuffy cave that oxygen concentration only has 6% or so
Brain structure and function intact simultaneously carries out normal vital movement.And hippocampus is used as one kind based on hippocampal neural metamember
Structure, it is no matter awake or all the moment remains height vigor in sleep procedure in individual, participate in body space orientation with
The input and integration of various information, it is huge to oxygen and other nutrient requirement amounts, and itself does not store energy
Mechanism can only absorb oxygen and other nutrients from ambient enviroment in time to guarantee its normal activities (The
organization and neural substrates of human memory.Squire LR Int J
Neurol.1987-1988；21-22:218-22.；Cohen N.J.,Eichenbaum H.Memory,Amnesia,and the
Hippocampal System.Cambridge,Mass,USA:MIT Press；1993).Common is moved on ground
For animal, it is irreversible dead to remember to collective study that the anoxic or hypoxemia of several seconds can all cause hippocampal neuron to occur
Recall space orientation ability and brings huge threat.And naked mole can carry out in the cavern that oxygen concentration only has 6%
Normal cave is excavated, looks for food and mate the vital movements such as breeding, and guarantees body for by any injury.Therefore,
Disclose under naked mole hippocampal neuron hypoxia condition survival and its special hypoxemia tolerance mechanism, will for solve hypoxic-ischemic or
Low-oxygen environment provides certain strategy to the defence of mankind's bring pathological, it is therefore desirable to establish in vitro naked mole sea
Horse neuron models are to make up the defect in body observation.
Currently, researcher has built up out cultural method (the Peng Wang, Li of rat and mouse hippocampal neuron
Wang,Shilei Wang,Shuhong Li,Yu Li,Lin Zhang.Effects of calcium-sensing
receptors on apoptosis in rat hippocampus during hypoxia/reoxygenation
through the ERK1/2pathway.Int J Clin Exp Pathol.2015；8(9):10808–10815.；Konrad
A.Szychowski,Anna K.Wójtowicz.TBBPA causes neurotoxic and the apoptotic
responses in cultured mouse hippocampal neurons in vitro.Pharmacol
Rep.2016Feb；68(1):20-6.).Chinese patent CN201210023408.1, application publication number CN102533654A, it is public
Opened a kind of culture solution and its preparation method and application of new born rat hippocampal neurons originally culture, the culture solution be
1,6 diphosphofructoses, fructose and nutritional additive are added in basic culture solution Neurobasalmedium.
Chinese patent application CN201410066557.5, application publication number CN103789267A disclose a kind of improvement
Primary hippocampal neurons method, culture medium be DMEM/F12 culture medium be added fetal calf serum, active fermentation filtrate and
Dual anti-, the active fermentation filtrate is a kind of distinctive bifidobacterium adolescentis LJM-001 fermentation acquisition；Its cell maintenance medium is
B27 and glutamine is added in Neurobasal culture medium.
Chinese patent application CN201510257025.4, application publication number CN104818251A, discloses adult rat
Hippocampal neuron In-vitro separation culture method, culture medium used are hibernate-A/B27.
Chinese patent application CN201510420060.3, application publication number CN105087492A disclose a kind of rat
The cultural method of primary hippocampal neurons, the culture solution are divided into the first culture solution and the second culture solution, the first culture solution packet
Include: the DMEM culture medium of 78.5 volume %, the fetal calf serum of 10 volume %, the horse serum of 10 volume %, 1 volume % L- paddy ammonia
The mycillin stock solution of amide and 0.5 volume %；Second culture solution includes: the DMEM culture medium of 85.5 volume %, 10 bodies
Product the horse serum of %, the N-2 additive of 1 volume %, the B-27 additive of 2 volume %, 1 volume %L- glutamine and
The mycillin and streptomysin of 0.5 volume %.
But in the prior art no matter the hippocampal neuron of mouse or rat isolate and purify it is not applicable with cultural method
In naked mole, domestic and foreign literature there is no the relevant report isolated and purified in relation to naked mole hippocampal neuron with cultural method at present.
Summary of the invention:
The purpose of the present invention is to provide a kind of naked mole hippocampal neuron isolate and purify and cultural method, with it is convenient,
It is efficient to obtain the good naked mole hippocampal neuron of purity is high, state, technology is provided to establish naked mole hippocampal neuron in vitro
It supports, while activation and hypoxemia tolerance and naked mole hippocampal neural for the naked mole hippocampal neuron of in vitro study
The elaboration of first hypoxemia tolerance mechanism and the screening of corresponding treatment drug or target molecule provide powerful guarantee.
We have tried out current mouse, the hippocampal neuron of rat isolates and purifies and cultural method, as a result, it has been found that can not
Culture obtains the good naked mole hippocampal neuron of purity is high, state, and the quantity of naked mole proliferation of hippocampal neuron is also less.
Feasible experimental method is not established out in culture about naked mole hippocampal neuron still, we analyze its reason,
Mainly: 1) naked mole is a kind of poikilotherm, and the temperature of body cell in vitro culture needs to be groped, and its non-constant machine
Body metabolic rate causes its body cell in-vitro culture medium to be different from common rat or mouse；2) naked mole existence is more dark
In moist environment, the culture of body cell has higher requirement to humidity；3) naked mole has been already adapted to the ring of extraneous hypoxemia
Border so the oxygen concentration in its body cell in vitro culture environment is different from the animal to live in normal oxygen environment, and needs to touch
Rope.4) naked mole pregnancy cycle is up to two months or more, and rat and mouse only need 3 weeks, therefore is tested in selection tire mouse
Time need carefully grope.
It is therefore, most important in the culture of naked mole hippocampal neuron to grope to suitable temperature, humidity and oxygen concentration,
And grope to suitable pregnancy cycle and culture medium.
To reach this purpose, naked mole hippocampal neuron of the invention isolation and purification method the following steps are included:
A, naked mole cerebral hippocampus composite nerve cell is collected:
Hippocampal tissue in the naked mole brain of embryo of separation 60-65 days, hippocampal tissue is shredded, and tissue digestion liquid is added
After mixing, in 35-37 DEG C digestion 10-25 minutes；Then digestion, centrifugation removal supernatant are terminated with composite nerve cell culture medium
Later, precipitating is resuspended in composite nerve cell culture medium and blows and beats into single cell suspension；By single cell suspension kind in sterile
And it is coated in the culture plate of hypothallus in advance and (preferably selects 24 orifice plates)；
The tissue digestion liquid can be conventional enzyme of proteolysis digestive juice, such as trypsase, Papain
Enzyme etc. is aided with DNA enzymatic I to release cell aggregation caused by DNA fragmentation adhesion.Preferred tissue digestion liquid: contain the tissue
Digestive juice is the mixed liquor containing 0.25% pancreatin and 0.05mg/ml DNA enzymatic I.
The hypothallus can be conventional left-handed poly-D-lysine, dextrorotation poly-D-lysine etc..Preferred hypothallus
For laminin.
B, the culture of naked mole cerebral hippocampus composite nerve cell:
The composite nerve cell that step A is obtained is incubated in three gas incubators with composite nerve cell culture medium and is cultivated
10-15 hours, to its completely it is adherent after, be changed to neuronal panning culture medium continue culture 1-3 days, be then changed to mind again
It is maintained culture medium culture 1-3 days through member, is so a circulation, cultivates 2-4 circulation.
The composite nerve cell culture medium can be the conventional cell culture fluid of composite nerve containing serum, such as low sugar
DMEM etc..It is preferably the low sugar DMEM of 10% fetal calf serum containing percentage by volume.
The neuronal panning culture medium is the Nerobasal culture medium containing 10 μM of 5 FU 5 fluorouracils and adds body
The N2 that product percentage is 2%.
It is the Neurobasal culture medium for being 2%N2 containing volume fraction that the neuron, which maintains culture medium, is added simultaneously
Add 100ng/ml NGF.
The parameter setting of the three gas incubators are as follows: temperature is controlled at 35 ± 2 DEG C, and oxygen concentration 5%, carbon dioxide is dense
Degree is 5 ± 1%, and humidity is 96 ± 2%.
Further, in step A, the method for the hippocampal tissue in the naked mole brain of embryo of separation 60-65 days is as follows: taking bosom
The pregnant 60-65 days naked moles of female, by it in CO2It is middle make to be soaked in immediately after asphyxia processing disappear in 75% ethyl alcohol
Poison, then places it in sterile glass plate, carefully cuts open the belly and the uterus for being contained tire mouse is completely taken out, cut off uterine veil
And carefully take out tire mouse.Tire mouse skull is cut off, left and right brain is split into two halves from median line under Stereo microscope, it is then small
The heart removes hippocampus, and strips the meninx structure of Surface of Hippocampus.
Further, in step A, hippocampal tissue is shredded to 1mm with microscissors3, it is added after the mixing of tissue digestion liquid, in
37 DEG C of digestion 15min.Then digestion is terminated with 3ml composite nerve cell culture medium.
Further, in step B, with composite nerve cell culture medium culture 12 hours, to its it is completely adherent after, be changed to
Neuronal panning culture medium continues culture 2 days, then is changed to neuron and maintains culture medium culture 2 days, is so a circulation, training
Support 2 circulations.
Further, the parameter setting of the three gas incubator are as follows: temperature control is at 35 DEG C, oxygen concentration 5%, titanium dioxide
Concentration of carbon is 5%, humidity 96%.
Chinese patent application 201510420060.3, application publication number are primary to disclose a kind of culture in 105087492A
The method of hippocampal neuron, wherein hippocampal neuron raising culture medium is used for the culture of hippocampal neuron.It is contemplated that from sea
The nerve cell that Malaysia and China obtain includes hippocampal neuron and a variety of spongiocytes, and wherein hippocampal neuron can not be proliferated in vitro, and
Then proliferative capacity is stronger for spongiocyte, so we have screened neuronal panning culture medium and neuron maintains the difference of culture medium
Combination, and the survival ratio of hippocampal neuron is counted, as a result as shown in the table, it has been found that will be adherent thin in composite nerve
Cell in born of the same parents' culture medium, first with neuron maintenance culture medium culture 2 days, then with neuronal panning culture medium culture 2 days, so
It is recycled for one, cultivates the hippocampal neuron of two available higher proportions of circulation.Neuronal panning culture medium and maintenance training
The basic ingredient for supporting base is to add the Neurobasal that volume fraction is 2%N2, and difference is added with final concentration of 10 μ at the former
M 5 FU 5 fluorouracil is used for the spongiocyte with proliferative capacity, and the latter adds final concentration of 100ng/ml NGF for maintaining mind
Through first vigor.
Chinese patent application 201510420060.3, application publication number are primary to disclose a kind of culture in 105087492A
The method of hippocampal neuron, wherein cell is derived from newborn one day Wistar suckling mouse.Compared with rat, naked mole pregnancy cycle is
70-80 days, the hippocampal neuron of higher proportion how is obtained, we are also groped, as shown in the table, it has been found that utilize
Above-mentioned culture medium and training method, the cell proportion for being derived from pregnant -65 days 60 days naked mole tire mouse are higher than the tire mouse of other age brackets.
The beneficial effects of the present invention are: it is 1. comprehensive that a variety of culture mediums is used to separate from naked mole tire mouse hippocampus and purify training
Hippocampal neuron is supported, the reasonable cultural method of the naked mole hippocampal neuron of rodent mammal suitable for alternating temperature has been found out.
We have found that cell state can preferably be kept under the conditions of 5% oxygen concentration, and it is able to maintain growth；2. operating method
Simply, repeatability with higher；3. it is thin effectively to control hippocampus composite nerve by using neuronal panning culture medium
The growth of spongiocyte in born of the same parents ensure that the purity of hippocampal neuron can reach 98% or more (for neuronal specificity mark
Remember the immunocytochemical stain result that object Tuj1 is carried out)；4. the growth factor type additionally added is few, it is only necessary in neuron
Maintain to add NGF in culture medium to guarantee the preferable functional status of neuron and vigor.
In conclusion the method for the present invention being capable of the normal naked mole sea of a large amount of functional activities of acquisition easy, efficiently, economic
Horse neuron, the culture under hypoxia condition can guarantee that this cell is still able to maintain under body state in ex vivo environment
Biological characteristics, consequently facilitating directly further studying naked mole hippocampal neuron in pure in-vitro cell culture model
Special physiological function, thus for explore biological mechanism therein and be applied to clinical relevant provide it is important it is theoretical according to
According to.
Detailed description of the invention:
Fig. 1 is the hippocampal neuron (ordinary optical microscope) of in vitro culture；
Fig. 2 is the hippocampal neuron (immunocytochemistry identification) of in vitro culture.
Specific embodiment:
Below in conjunction with the drawings and specific embodiments, the invention will be further described.It should be understood that following embodiment is only used for
It illustrates rather than for limiting the scope of the invention.
Embodiment 1: naked mole hippocampal neuron isolates and purifies and cultivates
1, experimental material
The naked mole of pregnant 60-65 days cleaning grades is provided by Second Military Medical University, PLA's Experimental Animal Center.
DNA enzymatic I is purchased from Solarbio Biotechnology Co., Ltd, and NGF, 5 FU 5 fluorouracil, trypsase, left-handed poly rely
Propylhomoserin, mycillin mixed liquor etc. are purchased from Sigma company, DMEM, low sugar DMEM, Australia source fetal calf serum, Neurobasal
Culture medium, N2 serum-free addition factor etc. are purchased from Thermo Fisher Scientific company, and 24 well culture plates are purchased from
Corning company.
Composite nerve cell culture media component is that low sugar DMEM culture medium contains the fetal calf serum that volume fraction is 10%, sea
Horse neuronal panning culture medium is 10 μM of 5 FU 5 fluorouracils of Neurobasal+ volume fraction 2%N2+ final concentration.Hippocampal neuron
Maintaining culture medium is the Neurobasal culture medium for being 2%N2 containing volume fraction, while adding final concentration of 100ng/ml's
NGF。
2, naked mole hippocampal neuron isolates and purifies and cultural method
1. collecting naked mole hippocampus composite nerve cell: by it in CO2In make asphyxia processing after be soaked in immediately
It carries out disinfection, is then placed it in sterile glass plate in 75% ethyl alcohol, and with penicillin 100U/ml and 0.1mg/ml chain
Mycin mixed liquor wipes naked mole skin of abdomen, carefully cuts open the belly and the uterus for being contained tire mouse is completely taken out, cut off uterine veil
And carefully take out tire mouse.Number of units skull is cut off, left and right brain is split into two halves from median line under Stereo microscope, it is then small
The heart removes hippocampus, and strips the meninx structure of Surface of Hippocampus.Hippocampal tissue is shredded to 1mm with microscissors3, tissue digestion is added
After liquid mixes, in 37 DEG C of digestion 15min.Then digestion is terminated with composite nerve cell culture medium, after centrifugation removes supernatant,
Precipitating is resuspended in nerve cell mixed culture medium and blows and beats into single cell suspension.Then by single cell suspension kind in sterile
And it is coated in 24 orifice plates of hypothallus in advance.
2. the culture of naked mole cerebral hippocampus composite nerve cell: will 1. obtained in cell, with composite nerve cell train
Support and be incubated in three gas incubators and cultivate 12 hours in base, to its it is completely adherent after, be changed to neuronal panning culture medium and continue
Then culture medium is changed to neuron and maintained culture medium 2 days, be so a cycle, cultivated one week by culture 2 days.
The parameter setting of three gas incubators are as follows: temperature control is at 35 DEG C, oxygen concentration 5%, gas concentration lwevel 5%,
Humidity is 96%.
Embodiment 2: the identification of naked mole hippocampal neuron
Using the identification of the resulting naked mole hippocampal neuron of embodiment 1: being trained using 4% paraformaldehyde in serum-free
The hippocampal neuron supported in base is fixed, and the methods of combining form identification, immune refinement chemistry are identified.
1, cytomorphology is identified:
(Lei Xianga, Yanping Ren, Xun Li, Wen Zhao, Yijun described in identification method bibliography
Song.MicroRNA-204suppresses epileptiform discharges through regulating TrkB-
ERK1/2-CREB signaling in cultured hippocampal neurons.Brain Res.2016,pii:
S0006-8993(16)30104-4.)。
As a result as shown in Figure 1, under light microscopic, naked mole hippocampal neuron cell space is larger, has 1 or several shorter trees
Prominent, the other end has an elongated aixs cylinder.
2, immunocytochemistry is identified:
(Lei Xianga, Yanping Ren, Xun Li, Wen Zhao, Yijun described in identification method bibliography
Song.MicroRNA-204suppresses epileptiform discharges through regulating TrkB-
ERK1/2-CREB signaling in cultured hippocampal neurons.Brain Res.2016,pii:
S0006-8993(16)30104-4.).As a result as shown in Fig. 2, hippocampal neuron is carried out creep plate experiment, in proliferated culture medium
After middle culture 24 hours, using 4% paraformaldehyde, the cells are fixed, then utilizes the antibody of neuron surface specific antigen Tuj1
Carry out immunocytochemical stain.The results show that it is positive (red glimmering that cell is able to maintain Tuj1 in serum free medium
Light).
According to above-mentioned experimental result, the naked mole hippocampal neuron that the present invention isolates and purifies has typical hippocampal neuron
Form, cell space is larger, has 1 or several shorter dendrons, and the other end has an elongated aixs cylinder, immunocytochemistry dye
It is positive in Tuj1 that color shows it, and good vegetative state is able to maintain in serum free medium.
The preferred embodiment of the present invention has been described in detail above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make various equivalent on the premise of not violating the inventive spirit of the present invention
Variation or replacement, these equivalent variation or replacement are all included in the scope defined by the claims of the present application.
Claims (7)
1. a kind of naked mole cultured hippocampal neuron method, comprising the following steps:
A, naked mole cerebral hippocampus composite nerve cell is collected:
Hippocampal tissue in the naked mole brain of embryo of separation 60-65 days, hippocampal tissue is shredded, and tissue digestion liquid is added and mixes
Later, in 35-37 DEG C digestion 10-25 minutes；Then with composite nerve cell culture medium terminate digestion, centrifugation removal supernatant it
Afterwards, precipitating is resuspended in composite nerve cell culture medium and blows and beats into single cell suspension；By single cell suspension kind in sterile and
It is coated in the culture plate of hypothallus in advance；
B, the culture of naked mole cerebral hippocampus composite nerve cell:
The composite nerve cell that step A is obtained is incubated in three gas incubators with composite nerve cell culture medium and cultivates 10-15
Hour, to its completely it is adherent after, be changed to neuronal panning culture medium continue culture 1-3 days, be then changed to neuron again
It maintains culture medium culture 1-3 days, is so a circulation, cultivates 2-4 circulation；
The composite nerve cell culture medium is to contain the low sugar DMEM that percentage by volume is 10% fetal calf serum；
The neuronal panning culture medium is the Nerobasal culture medium containing 10 μM of 5 FU 5 fluorouracils and adds volume hundred
The N2 that score is 2%；
It is the Neurobasal culture medium for being 2%N2 containing volume fraction that the neuron, which maintains culture medium, is added simultaneously
100ng/ml NGF；
The parameter setting of the three gas incubators are as follows: temperature control is at 35 ± 2 DEG C, oxygen concentration 5%, and gas concentration lwevel is
5 ± 1%, humidity is 96 ± 2%.
2. a kind of naked mole cultured hippocampal neuron method according to claim 1, which is characterized in that described in step A
Tissue digestion liquid be mixed liquor that the pancreatin containing 0.25% mass concentration and concentration are 0.05mg/ml DNA enzymatic I.
3. a kind of naked mole cultured hippocampal neuron method according to claim 1, which is characterized in that described in step A
Hypothallus be laminin.
4. a kind of naked mole cultured hippocampal neuron method according to claim 1, which is characterized in that in step A, with aobvious
Micro- cut shreds hippocampal tissue to 1mm3, it is added after the mixing of tissue digestion liquid, in 37 DEG C of digestion 15min.
5. a kind of naked mole cultured hippocampal neuron method according to claim 1, which is characterized in that in step A, use
3ml composite nerve cell culture medium terminates digestion.
6. a kind of naked mole cultured hippocampal neuron method according to claim 1, which is characterized in that in step B, with mixed
Close the culture of neuronal cell cultures base 12 hours, to its completely it is adherent after, be changed to neuronal panning culture medium and continue culture 2
It, then be changed to neuron and maintain culture medium culture 2 days, it is so a circulation, cultivates 2 circulations.
7. a kind of naked mole cultured hippocampal neuron method according to claim 1, which is characterized in that three air culture is supported
The parameter setting of case are as follows: temperature control is at 35 DEG C, oxygen concentration 5%, gas concentration lwevel 5%, humidity 96%.
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| CN106520697B (en) * | 2016-10-21 | 2021-04-20 | 中国人民解放军第二军医大学 | Naked mole rat DRG neuron culture method |
| CN112410299B (en) * | 2020-11-26 | 2022-08-19 | 四川大学华西医院 | Method for acute separation of mammalian hippocampal cells |
| CN113652403A (en) * | 2021-09-15 | 2021-11-16 | 复旦大学附属中山医院 | Primary neural cell plating method for high content drug screening |
| CN116254228A (en) * | 2022-11-25 | 2023-06-13 | 深圳先进技术研究院 | Method for culturing primary nerve cells of transgenic animals |
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