CN109266598A - A method of establishing anoxia-induced apoptosis cell model - Google Patents
A method of establishing anoxia-induced apoptosis cell model Download PDFInfo
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Abstract
The invention discloses a kind of method for establishing anoxia-induced apoptosis cell model of biomedicine technical field, the method and step for establishing anoxia-induced apoptosis cell model is as follows: S1: cell culture；S2: plating cells；S3: culture medium replacement；S4: cell hypoxia culture: one layer of ventilated membrane is covered on culture vessel, is placed in the modularization culture cell containing certain gas component and cultivates；S5: cell detection.The anoxia-induced apoptosis cell model that this method is established, experimental animal participation is not needed, modeling cost is low, reacts sensitiveer for oxygen deprivation stress, anoxia-induced apoptosis can be inquired into molecular biology level, condition can be provided for the progress of simulation anoxic experiment and the screening and verifying of anti-anoxic medicine.
Description
Technical field
The present invention relates to biomedicine technical field, specially a kind of method for establishing anoxia-induced apoptosis cell model.
Background technique
Anoxic refers to because the oxygen supply of tissue is insufficient or with oxygen obstacle, and leads to the metabolism of body, function and form knot
Structure is abnormal the pathologic process of variation.Brain, the life entities vitals anoxic such as heart are also to lead to the important original of body death
Cause, in addition, the tissue caused by arterial oxygen content is substantially reduced will lead to hypoxemia for hypoxgia.
The normal vital movement of body all be unable to do without oxygen, from cell metabolism to heartbeat all with oxygen it is close
It is related.Body oxygen intake, and tissue is reached by blood, finally experienced and utilized by cell, therefore, the essence of anoxic is
A kind of reaction and adaptive change of the cell to hypoxia, when acute severe depletion of oxygen or Poisoning anoxic, cellular change
Based on cell is adjusted with the compensatory of oxygen receptor based on mitochondrial obstacle, when chronic mild anoxic.
With going deep into anoxic understanding and research, pathologic damage mechanism and drug caused by discussion anoxic act on it
Target spot, have increasing need for the cellular damage mechanism that anoxic is illustrated in the level of molecular biology.Hypoxic cell damage master
Will be with cell membrane, mitochondria, based on the organelles such as lysosome.Cause ATP to reduce when intracellular severe depletion of oxygen, intercellular membrane to from
Sub- permeability increases, and causes ion along concentration difference through cell membrane, sodium ion inflow is to cause edema.Blood vessel endothelium is thin
Born of the same parents' oedema can block capilary, further result in microcirculation anoxic, while efflux of K+ ions, lead to cellular anabolic obstacle,
The generation of enzyme is reduced, by the function of the further generation for influencing ATP and ionic pump.When mitochondria position, partial pressure of oxygen drops to critical point
0.1kPa(< 1mmHg) when, the respiratory function of mitochondria reduces, and crucial dehydrogenase activity decline, ATP, which is generated, to be reduced.When serious
Mitochondria may occur in which the lesions such as swelling, ridge are disintegrated, outer membrane rupture and matrix are excessive.Because glycolysis enhances when anoxic, lactic acid is generated
Increase and fat oxidation increases entirely in-between metabolite ketoboidies.Lead to acid poisoning.PH reduction can cause phosphatide enzyme activity
Property increase, so that lysosome membrane phosphatide is decomposed, membrane permeability increases, as a result make lysosome swelling, rupture and a large amount of lysosomal enzymes
Release, and then lead to the dissolution of cell itself and its surrounding tissue, necrosis.
Plateau is a kind of special geographical environment, and main environment feature is hypobaric hypoxia.With the heat of highland tour
Tide, many reasons such as implementation of national development strategy, by Plain it is radical enter highlands crowd gradually increase, based on it is above-mentioned lack
There is serious acute mountain sickness symptom, including high protocerebrum archicerebrum after Acute Exposed Altitude in cellular damage mechanism caused by oxygen, part human body
Oedema, pulmonary edema etc., threat to life when serious.
Acute mountain sickness pathogenesis is complicated at present, constrains the plateau rational use of medicines and the research and development of related drugs.To scarce
The research of oxygen injury pathomechanism is inquired into greater need on the basis of anoxia model.It is relevant anti-for the prior art
The zoopery that is all based on of anoxic medicine is researched and developed, the Atropurpuran announced such as number of patent application 201610616740.7
Application of the derivative composition in anti-anoxic medicine, the anoxia model being wherein previously mentioned in specific embodiment use Kunming
It is mouse, this damages animal.In addition it is cultivated with also having that directly cell is placed under low-oxygen environment, simulates anaerobic condition
Model, but the shortcomings that this model, is that cell itself is more sensitive, and normal oxygen demand is 1% ~ 5%, due to Tissue Culture Flask
When being put into anaerobic environment, in bottle just containing in normal atmosphere 21% oxygen, and culture bottle be closed, gnotobasis, wherein
It is oxygenous be enough to maintain the normal existence environment of cell within a certain period of time, this cell model can not delicately reflect carefully
The side of the pharmaceutical agent for the treatment of anoxia-induced apoptosis is accurately screened or verify to born of the same parents' anoxia-induced apoptosis therefore, it is necessary to a kind of more convenient
Method, and must be set up inquiring into anoxia-induced apoptosis in molecular biology level, it could be to simulate the progress of anoxic experiment and resist
The screening and verifying offer condition of anoxic medicine.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for establishing anoxia-induced apoptosis cell model, to solve above-mentioned background technique
The needs of middle proposition are sensitiveer, simply, stable advantage, can Fast simulation cell hypoxia environment, reflect radical anoxic ring
Behind border the problem of the status method of the partial pressure of oxygen variation of cell peripheral and cellular damage.
To achieve the above object, the invention provides the following technical scheme: a kind of method for establishing anoxia-induced apoptosis cell model,
The method and step for establishing anoxia-induced apoptosis cell model is as follows:
S1: cell culture: cell is cultivated in 5% CO2 incubator, 80% is fused to when cell is grown in culture bottle ~
When 90% density, preparing becomes cell suspension；
S2: plating cells: cell suspension is then added in culture vessel, is carried out bed board, is regrowed to cell to 80% ~ 90%
Density；
S3: culture medium replacement: preparation culture medium equilibrated in anaerobic environment, and by the culture in S2 step in culture vessel
Base is changed to above-mentioned culture medium equilibrated in anaerobic environment, adds various concentration when carrying out anti-anoxic medicine screening simultaneously
Diluted drug；
S4: cell hypoxia culture: one layer of ventilated membrane is covered on culture vessel, then culture vessel is placed in containing certain gas
In the modularization culture cell of component, then by culture cell be put into 37 DEG C of room temperature, trained in the incubator of 5% CO2 content
It supports；
S5: cell detection: the hypoxic exposure of the cell in step S4 as needed is cultivated, and cell training is taken out after the completion of culture
Container is supported, oxidative damage parameters are detected.
Preferably, the cell in the step S1 can be attached cell, be also possible to suspension growth cell.
Preferably, the culture medium in the step S3 is the culture that partial pressure of oxygen reaches balance in dissolved oxygen and anaerobic environment
Base.
Preferably, the anaerobic environment in the step S3 is the anaerobic environment that oxygen content is lower than 21%.
Preferably, the culture vessel in the step S2 includes the culture dish that can be used for cultivating cell, culture plate, culture
Bottle.
Preferably, the modularization culture cell in the step S4 is to be able to maintain fixed gas componant, and airtightness is good
Good modularization culture cell.
Preferably, the ventilated membrane in the step S4 is that a kind of hydrone cannot pass through, and gas molecule can be with free exchange
Semi-permeable membrane.
Preferably, the semi-permeable membrane can be microporous polyethylene film, fluorocarbon film or polyethylene terephthalate film.
Preferably, the oxidative damage parameters in the step S5 are gene, the albumen, generation that cell generates in anaerobic environment
Thank to the change level of product.
Compared with prior art, the beneficial effects of the present invention are: the anoxia-induced apoptosis cell model that this method is established, is not required to
Experimental animal is wanted to participate in, modeling cost is low, reacts sensitiveer for oxygen deprivation stress, can inquire into molecular biology level
Anoxia-induced apoptosis can provide condition for the progress of simulation anoxic experiment and the screening and verifying of anti-anoxic medicine.
Detailed description of the invention
Fig. 1 is method for building up flow chart of the present invention；
Fig. 2 is CCK-8 experimental result picture after the processing of one BRL cell hypoxia of the embodiment of the present invention；
Fig. 3 is the relatively normal oxygen group survival results figure of anoxic group after the processing of one BRL cell hypoxia of the embodiment of the present invention；
Fig. 4 is that the HIF-1 α that two protein immunoblotting of the embodiment of the present invention detects in cell schemes；
Fig. 5 is two HIF-1 α albumen semi-quantitative analysis result figure of the embodiment of the present invention；
Fig. 6 is that the embodiment of the present invention three screens anti-anoxic medicine effective concentration according to HIF-1 α albumen semi-quantitative analysis result
Figure.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Referring to Fig. 1, the present invention provides a kind of technical solution: a method of anoxia-induced apoptosis cell model being established, this is built
The method and step of vertical anoxia-induced apoptosis cell model is as follows:
S1: cell culture: attached cell or suspension growth cell are cultivated in 5% CO2 incubator, when cell is being trained
When growth is fused to 80% ~ 90% density in feeding bottle, preparing becomes cell suspension；
S2: plating cells: being then added to the culture dish including can be used for cultivating cell for cell suspension, culture plate, culture bottle
In culture vessel, bed board is carried out, is regrowed to cell to 80% ~ 90% density；
S3: culture medium replacement: oxygen in dissolved oxygen and anaerobic environment equilibrated in anaerobic environment of the oxygen content lower than 21% is prepared
Partial pressure reaches the culture medium of balance, and the culture medium in S2 step in culture vessel is changed to and above-mentioned has been put down in anaerobic environment
The culture medium to weigh adds the diluted drug of various concentration simultaneously when carrying out anti-anoxic medicine screening；
S4: cell hypoxia culture: covering one layer of hydrone on culture vessel cannot pass through, and gas molecule can be with free exchange
Culture vessel, is then placed in and is able to maintain fixation by microporous polyethylene film, fluorocarbon film or polyethylene terephthalate film
Gas componant, airtightness good modularization culture cell, then by culture cell be put into 37 DEG C of room temperature, 5% CO2 content
It is cultivated in incubator；
S5: cell detection: the hypoxic exposure of the cell in step S4 as needed is cultivated, and cell training is taken out after the completion of culture
Container is supported, the oxidative damage parameters of the change level of gene, albumen, metabolite that detection cell generates in anaerobic environment.
Specific embodiments of the present invention are as follows:
Embodiment one, with reference to attached drawing 2-3.
Rat liver cells (BRL) hypoxia model is established
1, BRL cell culture condition:
BRL cell culture (contains 1 × 105IU/L of penicillin and streptomysin 1 in the DMEM culture medium containing 10% fetal calf serum
× 105IU/L), it 37 DEG C, 5% CO2, is cultivated in incubator.
2, BRL cell hypoxia treatment conditions:
By BRL cell culture in the DMEM high glucose medium containing 10% fetal calf serum, anaerobic environment be 1% O2,5% CO2,
95% N2, the same normal cell of remaining condition of culture, anoxia-induced apoptosis time are carried out by experimental design requirement.
3, BRL cell hypoxia injury experiment:
Experimental design three groupings, i.e. control group, anoxic treatment group 1 and anoxic treatment group 2.
0.1% trypsin solution of BRL cell is digested, cell suspension is formulated as, is inoculated in three 96 respectively
In orifice plate, adjustment density is 1.0 × 106/mL, and every hole adds 1.0 × 104 cells, i.e. 10 microlitres of cell suspensions, it is micro- to add 90
Complete medium is risen to 100 microlitres, for 24 hours by cell culture, original culture medium, PBS washing two are sucked after cell is completely adherent
Time.
Control group: after 96 orifice plates are washed with PBS, 50 microlitres of fresh complete cultures placed under normoxic condition are added in every hole
Base covers 96 orifice plate lids.
Anoxic group 1: after cells rinsed with PBS, every hole adds 50 microlitres in advance in 1% O2,5% CO2, the module of 95% N2
Change equilibrated culture medium for 24 hours in culture cell, covers 96 orifice plate lids.
Anoxic group 2: cell adherent growth on 96 orifice plates, after being washed with PBS, with anoxic group 1, every hole is added 50 microlitres and is mentioned
The preceding equilibrated culture medium for 24 hours in 1% O2, the modularization culture cell of 5% CO2,95% N2 removes 96 orifice plate lids, sticks
Microporous polyethylene film, the film features are that gas can be with free exchange, and vapor cannot be penetrated with microorganism, therefore it is thoroughly
Gas is high-efficient, and cell peripheral partial pressure of oxygen can reach rapidly balance between environment.
Anoxic group 1 and anoxic group 2 are put into filled with 1% O2,5% CO2, in the modularization culture cell of 95% N2, then will
Culture cell and cellular control unit are put into 37 DEG C simultaneously, in 5% CO2 incubator, anoxic culture 24 hours.
4, influence of the CCK-8 method detection anoxic to BRL cell Proliferation
After 96 orifice plates cultivate 24 hours in the incubator, 10 microlitres of CCK-8 solution are added in every hole, and culture plate is placed in incubator
It is incubated for 2 hours, then measures the absorbance at 450nm with microplate reader, as a result analyzed using SPSS.
Cell Counting Kit-8 (CCK-8) is for measuring viable count in cell Proliferation or cell toxicity test
A kind of high sensitivity of purpose, "dead" colorimetric detection method, water-soluble tetrazolium salts-WST-8 (2- methoxyl group -4- nitro
Benzene) -3- (4- nitrobenzene) -5- (2,4- disulfobenzene) -2H- tetrazolium monosodium salt by intracellular dehydrogenase oxidoreductase restore after generate can
It is dissolved in the orange-yellow formazan dyestuff of culture medium, therefore it is directly proportional to living cells quantity to generate formazan amount.As a result such as attached drawing 2-3.
The result shows that: anoxic treatment will affect cell survival, use the anoxic group 2 of microporous polyethylene film, cell survival
Number is less, and compared with the anoxic group 1 of conventional method culture, using the anoxic group 2 of microporous polyethylene film, cell survival rate is more
Low, cell is higher to the sensibility of anoxic, is more suitable for establishing hypoxic cell model.
Embodiment two, referring to attached drawing 4-5.
The measurement of rat liver cells (BRL) hypoxia inducible factor (HIF-1 α) protein expression
1, BRL cell culture condition:
With described in specific embodiment 1.
2, BRL cell hypoxia treatment conditions:
With described in specific embodiment 1.
3, BRL cell hypoxia injury experiment:
Experimental design three groupings, i.e. control group, anoxic treatment group 1 and anoxic treatment group 2.
0.1% trypsin solution of BRL cell is digested, cell suspension is formulated as, is inoculated in three six holes respectively
In plate, adjustment density is 1.0 × 106/mL, and every hole adds 3.0 × 105 cells, i.e. 33 microlitres of cell suspensions, it is micro- to add 167
Complete medium is risen to 200 microlitres, for 24 hours by cell culture, original culture medium, PBS washing two are sucked after cell is completely adherent
Time.
Control group: after six orifice plates are washed with PBS, 200 microlitres of fresh complete trainings placed under normoxic condition are added in every hole
Base is supported, six orifice plate lids are covered.
Anoxic group 1: after cells rinsed with PBS, every hole adds 200 microlitres in advance in 1% O2,5% CO2, the mould of 95% N2
Equilibrated culture medium for 24 hours in block culture cell, covers six orifice plate lids.
Anoxic group 2: cell adherent growth on orifice plate, after being washed with PBS, with anoxic group 1, every hole is added 200 microlitres and is mentioned
The preceding equilibrated culture medium for 24 hours in 1% O2, the modularization culture cell of 5% CO2,95% N2 removes six orifice plate lids, sticks
Microporous polyethylene film.
Anoxic group 1 and anoxic group 2 are put into 1% O2, in the modularization culture cell of 5% CO2,95% N2, then will culture
Cell and cellular control unit are put into 37 DEG C simultaneously, in 5% CO2 incubator, anoxic culture 24 hours.
After anoxic treatment, three groups of cells are taken out from incubator, it is quick that 200 microlitres of RIPA cell tissues are added in every hole
Lysate extracts three groups of cell protein samples, carries out next step operation.
4, protein immunoblotting method (Western Blot, WB) detects BRL cell hypoxia inducible factor (HIF-1 α) egg
White expression:
The expression of hypoxia-inducible factor-1 alpha (HIF-1 α) and active height depend on oxygen supply, under normoxic conditions, HIF-1
It is general then to carry out poly by tumor suppressor by the quick hydroxylating of protein of enzyme domains containing representing prolyl hydroxylase (PHD) by α
Elementization is degraded by proteasome pathway.Under anoxic conditions, the HIF-1 α degradation that pVHL is mediated is suppressed, and leads to HIF-1 α
Nucleus is accumulated and be transferred to, forms heterodimer with HIF- β wherein, it can be with the anoxic response in anoxic promoter
Element (HREs), which combines, adjusts downstream target gene.
Therefore HIF-1 α is the key protein that molecular biology level investigates physiological anoxic.Utilize protein immunoblotting
Method (western blot, WB) detects normal oxygen group, the protein expression of HIF-1 α in 2 cell of anoxic group 1 and anoxic group, and utilizes
Image-J analyzes WB result, carries out sxemiquantitative to the protein expression of HIF-1 α.As a result such as attached drawing 4-5.
The results showed that 96 orifice plates are directly put into anaerobic environment by anoxic group 1 cultivates anoxia model caused by cell,
There was no significant difference with normal oxygen group for the generation of its HIF-1 α.According to this method, anoxic group 2 is used caused by microporous polyethylene film
Anoxia model can significantly induce the generation of HIF-1 α albumen under same anaerobic environment, show what this method was made
Anoxia model susceptibility is higher, can preferably simulate cell hypoxia damage, reflects that cell is to anoxic in molecular biology level
Stress adjusting.
Embodiment three, referring to attached drawing 6.
Anti-anoxic medicine is screened and verified using hypoxia model
1, BRL cell culture condition:
With described in specific embodiment 1.
2, BRL cell hypoxia treatment conditions:
With described in specific embodiment 1.
3, the oxygen lack resistant function of BRL cell hypoxia model verifying acetazolamide:
BRL cell suspension is inoculated in respectively in 96 orifice plates, adjustment density is 1.0 × 106/mL, and every hole adds 1.0 × 104
Cell, i.e. 10 microlitres of cell suspensions, add 90 microlitres of complete mediums to 100 microlitres, for 24 hours by cell culture, complete to cell
Original culture medium is sucked after adherent, PBS is washed twice.
Anoxia-induced apoptosis cell model, i.e. cell adherent growth on 96 orifice plates are constructed according to the method described in the present invention, are used
After PBS washing, every hole add 50 microlitres in advance in 1% O2, the modularization culture cell of 5% CO2,95% N2 it is equilibrated for 24 hours
The culture medium containing various concentration acetazolamide, remove 96 orifice plate lids, stick microporous polyethylene film.Acetazolamide concentration point
It Wei not 10-3,10-4,10-5,10-6,10-7 mol/L.
96 orifice plates are put into 1% O2, anoxic culture 24 hours in the modularization culture cell of 5% CO2,95% N2.
4, CCK-8 method detection acetazolamide is effectively dense to the protective effect of anoxia-induced apoptosis BRL cell Proliferation and screening drug
Degree.
After 96 orifice plates cultivate 24 hours in the incubator, 10 microlitres of CCK-8 solution are added in every hole, and culture plate is placed in culture
It is incubated for 2 hours in case, then measures the absorbance at 450nm with microplate reader.Result is analyzed with SPSS:
The result shows that: when acetazolamide concentration is 10-5 mol/L, the survival rate of hypoxic cell is apparently higher than other administration groups,
With significant difference compared with anoxic group, show that the acetazolamide of the concentration has protective effect to BRL cell.When drug is dense
When degree is higher than 10-5 mol/L, survival rate is slightly decreased trend, the possible reason is high concentration generates certain cytotoxicity.
Therefore, anti-anoxic medicine effectively can be screened and verify using the anoxia-induced apoptosis cell model that this method is established.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (9)
1. a kind of method for establishing anoxia-induced apoptosis cell model, it is characterised in that: this establishes the method for anoxia-induced apoptosis cell model
Steps are as follows:
S1: cell culture: cell is cultivated in 5% CO2 incubator, 80% is fused to when cell is grown in culture bottle ~
When 90% density, preparing becomes cell suspension；
S2: plating cells: cell suspension is then added in culture vessel, is carried out bed board, is regrowed to cell to 80% ~ 90%
Density；
S3: culture medium replacement: preparation culture medium equilibrated in anaerobic environment, and by the culture in S2 step in culture vessel
Base is changed to above-mentioned culture medium equilibrated in anaerobic environment, adds various concentration when carrying out anti-anoxic medicine screening simultaneously
Diluted drug；
S4: cell hypoxia culture: one layer of ventilated membrane is covered on culture vessel, then culture vessel is placed in containing certain gas
In the modularization culture cell of component, then by culture cell be put into 37 DEG C of room temperature, trained in the incubator of 5% CO2 content
It supports；
S5: cell detection: the hypoxic exposure of the cell in step S4 as needed is cultivated, and cell training is taken out after the completion of culture
Container is supported, oxidative damage parameters are detected.
2. a kind of method for establishing anoxia-induced apoptosis cell model according to claim 1, it is characterised in that: the step S1
In cell can be attached cell, be also possible to suspension growth cell.
3. a kind of method for establishing anoxia-induced apoptosis cell model according to claim 1, it is characterised in that: the step S3
In culture medium be partial pressure of oxygen reaches balance in dissolved oxygen and anaerobic environment culture medium.
4. a kind of method for establishing anoxia-induced apoptosis cell model according to claim 1, it is characterised in that: the step S3
In anaerobic environment be oxygen content be lower than 21% anaerobic environment.
5. a kind of method for establishing anoxia-induced apoptosis cell model according to claim 1, it is characterised in that: the step S2
In culture vessel include the culture dish that can be used for cultivating cell, culture plate, culture bottle.
6. a kind of method for establishing anoxia-induced apoptosis cell model according to claim 1, it is characterised in that: the step S4
In modularization culture cell be to be able to maintain fixed gas componant, airtightness good modularization culture cell.
7. a kind of method for establishing anoxia-induced apoptosis cell model according to claim 1, it is characterised in that: the step S4
In ventilated membrane be that a kind of hydrone cannot pass through, gas molecule can be with the semi-permeable membrane of free exchange.
8. a kind of method for establishing anoxia-induced apoptosis cell model according to claim 7, it is characterised in that: the semi-permeable membrane
It can be microporous polyethylene film, fluorocarbon film or polyethylene terephthalate film.
9. a kind of method for establishing anoxia-induced apoptosis cell model according to claim 1, it is characterised in that: the step S5
In oxidative damage parameters be cell generated in anaerobic environment gene, albumen, metabolite change level.
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