CN108034631A - A kind of method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model - Google Patents
A kind of method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model Download PDFInfo
- Publication number
- CN108034631A CN108034631A CN201711280433.7A CN201711280433A CN108034631A CN 108034631 A CN108034631 A CN 108034631A CN 201711280433 A CN201711280433 A CN 201711280433A CN 108034631 A CN108034631 A CN 108034631A
- Authority
- CN
- China
- Prior art keywords
- cell
- hydrogen peroxide
- culture fluid
- induction
- cell culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Dermatology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a kind of method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model, includes the following steps：Prepare cell culture fluid；Chitterlings epithelial cell is seeded to after being cultivated in cell culture fluid, carries out Trypsin Induced processing, inducing cell is treated in acquisition；To cell culture fluid of the addition containing hydrogen peroxide in inducing cell is treated, hydrogen peroxide induction is carried out.In technical solution provided by the invention, first model of cell apoptosis is established using chitterlings epithelial cell as model cell, and using hydrogen peroxide as derivant, have the advantages that induction time is short, easy to operate, of low cost and effect stability is reliable, the model of cell apoptosis be established as research chitling road damage mechanisms and its regulation measure provide an important channel.
Description
Technical field
The present invention relates to model of cell apoptosis constructing technology field, more particularly to a kind of hydrogen peroxide induction chitterlings epithelium is thin
The method for building up of born of the same parents' Apoptosis Model.
Background technology
China is the first in the world pig raising big country, and pig raising becomes the important industry for concerning national economy already.The weight of pig breeding industry
One of hang-up is intestine of young pigs health problem, it has also become restrict pig breeding industry rapid and healthy " bottleneck ", China it is annual because
Economic loss is up to 30,000,000,000 yuan caused by intestine of young pigs health problem (such as indigestion, diarrhea, retarded growth or even death).Have
Research shows that the disorder of enterocyte apoptosis can cause the damage of chitling mucosa structure and dysfunction, and Radical Metabolism is abnormal
It is closely related with apoptosis disorder, therefore chitterlings Epithelial Cell Apoptosis model is built to research chitling road damage mechanisms and its regulation and control
Measure has important theory and practice meaning.
Structure model of cell apoptosis needs Selection Model cell and derivant, wherein, structure cell is commonly applied at present to wither
The cell for dying model is mostly liver cell, cancer cell, mammary glandular cell, neutrophil leucocyte and haemocyte etc., and derivant includes fusion
Albumen, monoclonal antibody, lipopolysaccharides etc., these derivants are in actual application, and there are preparation process complexity, induction operation
Numerous and diverse, the shortcomings of apoptosis rate is not high.Therefore, it is not suitable for building chitterlings Epithelial Cell Apoptosis mould in the prior art
The method of type.
The content of the invention
The main object of the present invention is to propose a kind of method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model,
It is intended to make that the structure of chitterlings Epithelial Cell Apoptosis model is easy to operate and effect stability is reliable.
To achieve the above object, the present invention proposes a kind of foundation side of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model
Method, includes the following steps：
Prepare cell culture fluid；
Chitterlings epithelial cell is seeded to after being cultivated in cell culture fluid, carries out Trypsin Induced processing, treated
Inducing cell；
To cell culture fluid of the addition containing hydrogen peroxide in inducing cell is treated, hydrogen peroxide induction is carried out.
Preferably, the step of preparing cell culture fluid, specifically includes：
5% hyclone, 1% mycillin mixed liquor, 1% glutamy is added into DMEM/F-12 culture mediums
The epithelical cell growth factor of amine, 0.1% transferrins and 5 μ g/L, is made cell culture fluid after mixing.
Preferably, chitterlings epithelial cell is seeded to after being cultivated in cell culture fluid, carries out Trypsin Induced processing,
Acquisition treats the step of inducing cell, including：
Chitterlings epithelial cell is seeded in the container containing cell culture fluid, is placed in incubator and cultivates；
Cultivate in the incubator after reaching 70~80% to cell confluency rate, carry out secondary culture；
Trypsin Induced processing is carried out to the chitterlings epithelial cell by secondary culture, inducing cell is treated in acquisition.
Preferably, chitterlings epithelial cell is seeded in the container containing cell culture fluid, is placed in incubator and cultivates
The step of, specifically include：
After taking the chitterlings epithelial cell frozen to recover, it is seeded in the container containing cell culture fluid, is placed in incubator
Middle culture, wherein, the condition of culture of the incubator is：CO2Volumetric concentration be 4.5~5.5%, cultivation temperature be 36.5~
37.5 DEG C, culture when a length of 2~3 days, every 20~26h replace a nutrient solution.
Preferably, in the step of cultivating in the incubator after reaching 70~80% to cell confluency rate, carrying out secondary culture：
The incubation time of the secondary culture is 7~8 days.
Preferably, to carrying out Trypsin Induced processing by the chitterlings epithelial cell of secondary culture, obtain and wait to induce
The step of cell, specifically include：
Add trypsin solution in by the chitterlings epithelial cell of secondary culture, pancreas egg is discarded after digesting 1~3min
White enzyme solutions, and add cell culture fluid and terminate digestion, inducing cell is treated in acquisition.
Preferably, to the step for the treatment of to add the cell culture fluid containing hydrogen peroxide in inducing cell, carry out hydrogen peroxide induction
In：The concentration of hydrogen peroxide is 0.2~1.0mM in the cell culture fluid containing hydrogen peroxide, a length of 1~6h during induction.
Preferably, to treat in inducing cell add the cell culture fluid containing hydrogen peroxide, carry out hydrogen peroxide induction the step of,
Including：
It will treat that inducing cell is seeded in orifice plate, and add cell culture fluid and continue to cultivate；
After cultivating to cell attachment and growing to 70~80%, absorb the cell culture fluid in orifice plate and use phosphate buffer
Cleaned along hole wall；
The cell culture fluid containing hydrogen peroxide is added into the orifice plate after cleaning, carries out hydrogen peroxide induction.
Preferably, it will treat that inducing cell is seeded in orifice plate, add in the step of cell culture fluid continues culture：It is described to treat
The inoculum concentration that inducing cell is seeded in the orifice plate is 1 × 104~1 × 106A/mL, the cell culture fluid is in the orifice plate
In additive amount be 0.1~1mL/ holes.
In technical solution provided by the invention, Apoptosis mould is established by model cell of chitterlings epithelial cell first
Type, and using hydrogen peroxide as derivant, have the advantages that induction time is short, easy to operate, of low cost and effect stability is reliable, should
Model of cell apoptosis be established as research chitling road damage mechanisms and its regulation measure provide an important channel.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is attached drawing needed in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, without creative efforts, can be with
Other relevant attached drawings are obtained according to these attached drawings.
Fig. 1 is the test result figure of the IPEC-1 cell viabilities of each group in the embodiment of the present invention 1；
Fig. 2 is the test result figure of the IPEC-1 cell supernatant LDH activities of each group in the embodiment of the present invention 2；
Fig. 3 is the survey of the IPEC-1 Apoptosis correlation molecule caspase3 protein levels of each group in the embodiment of the present invention 3
Test result figure；
Fig. 4 is the IPEC-1 Apoptosis correlation molecule p-p38 protein levels and p- of each group in the embodiment of the present invention 3
The test result figure of p38/t-p38 ratios；
Fig. 5 is the test of the IPEC-1 Apoptosis correlation molecule t-p38 expressing quantities of each group in the embodiment of the present invention 3
Result figure；
Fig. 6 is the IPEC-1 Apoptosis correlation molecule p-JNK protein levels and p- of each group in the embodiment of the present invention 3
The test result figure of JNK/t-JNK ratios；
Fig. 7 is the test of the IPEC-1 Apoptosis correlation molecule t-JNK expressing quantities of each group in the embodiment of the present invention 3
Result figure；
Fig. 8 is that the fluorescence of the IPEC-1 Apoptosis correlation molecule NF- κ B p65 albumen of each group in the embodiment of the present invention 4 is shown
Micro mirror test result figure.
Embodiment
, below will be in the embodiment of the present invention to make the purpose, technical scheme and advantage of the embodiment of the present invention clearer
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, builds according to normal condition or manufacturer
The condition of view carries out.Reagents or instruments used without specified manufacturer, is the conventional production that can be obtained by commercially available purchase
Product.
The present invention proposes a kind of method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model, and the hydrogen peroxide lures
The method for building up for leading chitterlings Epithelial Cell Apoptosis model includes the following steps：
Step S10, cell culture fluid is prepared；
Wherein, in the technical solution of the present invention, the cell culture fluid is to be directed to the culture of intestinal epithelial cell and prepare
's.In one embodiment of this invention, step S10 is specifically included：Into DMEM/F-12 culture mediums (HyClone companies of the U.S.)
Add 5% hyclone (Gibco companies of the U.S.), 1% mycillin mixed liquor (HyClone companies of the U.S.), 1% paddy
The epidermal growth of glutamine (Gibco companies of the U.S.), 0.1% transferrins (Gibco companies of the U.S.) and 5 μ g/L because
Sub (Gibco companies of the U.S.), is made cell culture fluid after mixing.During concrete operations, each component of such as aforementioned proportion can be mixed
Conjunction is uniformly placed on 75cm2Blake bottle in, and dispense spare.
Step S20, chitterlings epithelial cell is seeded to after being cultivated in cell culture fluid, carried out at Trypsin Induced
Inducing cell is treated in reason, acquisition；
Enteron aisle is not only the major organs of nutrient digestion absorption, and the immune place of body maximum, is that body is prevented
Imperial primary barrier, plays an important role in terms of the invasion of the harmful substance such as endogenous and external source venereal bacteria is resisted.For
For feeding piglet, the disorder of enterocyte apoptosis can cause the damage of chitling mucosa structure and dysfunction, seriously affect piglet
Health, therefore using chitterlings epithelial cell as model cell build model of cell apoptosis have great importance.In general,
Meeting adherent growth during intestinal cell is cultivated in vitro, therefore the injury caused by cell is blown and beaten repeatedly in order to reduce,
In technical solution of the present invention, trypsin digestion and cell is used after cell culture, inducing cell is treated to obtain.
Step S30, to cell culture fluid of the addition containing hydrogen peroxide in inducing cell is treated, hydrogen peroxide induction is carried out.
Wherein, hydrogen peroxide can react the free radical to form high activity with intracellular iron ion, lure by producing active oxygen
Guided cell damages, and is usually used in studying the oxidative damage of cell, hydrogen peroxide not only convenient sources are cheap, and property is opposite
Stablize, it is obvious to the damaging action of cell.In the technical solution of the present invention, to treat in inducing cell add containing the thin of hydrogen peroxide
After born of the same parents' nutrient solution, treat that inducing cell in the case where the induction of hydrogen peroxide stimulates, proceeds by and completes Apoptosis, so as to be successfully established
Chitterlings Epithelial Cell Apoptosis model.
Alternatively, in step s 30：In the cell culture fluid containing hydrogen peroxide the concentration of hydrogen peroxide for 0.2~
1.0mM, a length of 1~6h during induction.Wherein, in embodiments of the present invention, using hydrogen peroxide concentration as 0.5mM, a length of 3h during induction
Condition be optimal.During concrete operations, certain density hydrogen peroxide storing liquid can be prepared in advance, then adds hydrogen peroxide storing liquid
Enter in the cell culture fluid prepared in step S10, form the cell culture fluid containing hydrogen peroxide, such as, first compound concentration is
The hydrogen peroxide storing liquid of 50mM, then adds the hydrogen peroxide storing liquid of 10 μ L, you can obtain in the cell culture fluid of every 990 μ L
Hydrogen peroxide concentration is the cell culture fluid of 0.5mM.
In technical solution provided by the invention, Apoptosis mould is established by model cell of chitterlings epithelial cell first
Type, and using hydrogen peroxide as derivant, have the advantages that induction time is short, easy to operate, of low cost and effect stability is reliable, should
Model of cell apoptosis be established as research chitling road damage mechanisms and its regulation measure provide an important channel.
Alternatively, step S20 includes：
Step S21, chitterlings epithelial cell is seeded in the container containing cell culture fluid, is placed in incubator and trains
Support；
Wherein, step S21 is specifically included：After taking the chitterlings epithelial cell frozen to recover, it is seeded to containing cell culture
In the container of liquid, it is placed in incubator and cultivates, wherein, the condition of culture of the incubator is：CO2Volumetric concentration for 4.5~
5.5%th, cultivation temperature for 36.5~37.5 DEG C, culture when a length of 2~3 days, every 20~26h replace a nutrient solution.Specifically
Can be with the following method during operation：By chitterlings epithelial cell (the Intestinal porcine epithelial of liquid nitrogen cryopreservation
Cells, IPEC-1, by Texas A＆M University provide) in 37 DEG C of water-bath recover after, by 3 × 105A/mL connects
In kind to the culture dish (100mm) containing cell culture fluid, it is subsequently placed in incubator and is cultivated, is replaced once every 24h
Nutrient solution.
Step S22, cultivate in the incubator after reaching 70~80% to cell confluency rate, carry out secondary culture；
Wherein, in the step s 21：The incubation time of the secondary culture is 7~8 days.When chitterlings epithelial cell is in perseverance
After being cultivated 2~3 days in incubator, cell confluency rate reaches 70~80%, then carries out secondary culture to chitterlings epithelial cell, through 7
Cell was passaged to for 2~3 generations after~8 days.
Step S23, to carrying out Trypsin Induced processing by the chitterlings epithelial cell of secondary culture, obtain and wait to induce
Cell.
Wherein, step S23 is specifically included：Add trypsin solution in by the chitterlings epithelial cell of secondary culture,
Trypsin solution is discarded after 1~3min of digestion, and adds cell culture fluid and terminates digestion, inducing cell is treated in acquisition.Specific behaviour
Can be with the following method when making：Trypsin solution is added into the chitterlings epithelial cell after secondary culture, and (U.S. Gibco is public
Department, the concentration of trypsase is 0.25%, the EDTA containing 0.9mM), trypsin solution is fully infiltrated attached cell, disappear
Trypsin solution is discarded after changing 1~3min, suitable cell culture fluid is then added and terminates digestion, treats that induction is thin so as to obtain
Born of the same parents.
Alternatively, step S30 includes：
Step S31, it will treat that inducing cell is seeded in orifice plate, and add cell culture fluid and continue to cultivate；
Wherein, in step S31：The inoculum concentration for treating that inducing cell is seeded in the orifice plate is 1 × 104~1 ×
106A/hole, additive amount of the cell culture fluid in the orifice plate are 0.1~1mL/ holes.It can be used during concrete operations as follows
Method：It will treat that inducing cell is inoculated into 96 orifice plates, the cell culture fluid of 100 μ L is added per hole, slight wobble makes cell uniform
Disperse in the orifice plate, to continue culture 2~3 days.
Step S32, after cultivating to cell attachment and growing to 70~80%, absorb the cell culture fluid in orifice plate and use phosphoric acid
Salt buffer is cleaned along hole wall；
It will be absorbed wherein after inducing cell cultivates 2~3 days in the orifice plate, when cell attachment and when growing to 70~80%
Old culture medium, then with phosphate buffer (pH value 7.0, HyClone companies of the U.S.) along hole wall clean twice it is rear spare.
Step S33, the cell culture fluid containing hydrogen peroxide is added into the orifice plate after cleaning, carries out hydrogen peroxide induction.
The cell culture fluid that prepared hydrogen peroxide concentration is 0.5mM is added in orifice plate, per 100 μ L of hole, induction stimulates
3h, you can complete the foundation of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model.
Technical scheme is described in further detail below in conjunction with specific embodiments and the drawings, it will be appreciated that
Following embodiments only to explain the present invention, are not intended to limit the present invention.
Influence of 1 hydrogen peroxide concentration of embodiment to IPEC-1 cell viabilities
(1) by 1 × 105A/hole treats that inducing cell is inoculated into 96 orifice plates, and the cell culture fluid of 100 μ L is added per hole,
Slight wobble makes cell dispersed in the orifice plate, when cell attachment and when growing to 70%-80% after culture 3 days, absorbs culture
Liquid, is cleaned twice with phosphate buffer along hole wall.
(2) cell culture fluid will be prepared successively to be added in orifice plate, per 100 μ L of hole, wherein, cell culture fluid is divided into pair
According to group and experiment A, B group, the concentration of hydrogen peroxide is respectively 0mM, 0.2mM and 0.5mM in cell culture fluid in three groups, every group of repetition
8.
(3) after hydrogen peroxide stimulates 3h, nutrient solution is absorbed, after cleaning twice along hole wall with phosphate buffer, test
IPEC-1 cell viabilities.Test method is as follows：(the Cell Counting of reagent containing 10%CCK8 of 100 μ L are added into each hole
Kit-8, Wuhan cloves circle Science and Technology Ltd.) cell culture fluid, be placed on 5%CO2, 1h is incubated in 37 DEG C of incubator,
It is then taken out, quickly measures absorbance under 450nm wavelength with microplate reader (Bio-Rad iMark, Bio-Rad, the U.S.)
(OD values), test result is as shown in table 1 and Fig. 1.
Influence of 1 hydrogen peroxide concentration of table to IPEC-1 cell viabilities
From table 1 and Fig. 1, the OD values of the IPEC-1 cells of control group, experiment A groups and experiment B groups reduce successively, and with
Control group is compared, and is tested A groups and is tested the P values respectively less than 0.05 of B groups, it is respectively the hydrogen peroxide of 0.2mM and 0.5mM to illustrate concentration
Stimulation can cause IPEC-1 cell viabilities to significantly reduce.
Influence of 2 hydrogen peroxide concentration of embodiment to IPEC-1 cell supernatant lactic acid dehydrogenase activities
(1) by 1 × 105A/hole treats that inducing cell is inoculated into 12 orifice plates, the cell culture fluid of 1mL is added per hole, gently
Micro- rock makes cell dispersed in the orifice plate, when cell attachment and when growing to 70%-80% after culture 3 days, absorbs nutrient solution,
Cleaned twice along hole wall with phosphate buffer.
(2) cell culture fluid will be prepared successively to be added in orifice plate, per hole 1mL, wherein, cell culture fluid is divided into control
Group and experiment A, B group, the concentration of hydrogen peroxide is respectively 0mM, 0.2mM and 0.5mM in cell culture fluid in three groups, every group of repetition 4
It is a.
(3) after hydrogen peroxide stimulates 3h, cell culture supernatant is drawn, is transferred in the sterile centrifugation tube of 1.5mL, uses microplate
Method lactic dehydrogenase (LDH) assay kit (builds up Bioengineering Research Institute to provide) measure cell supernatant LDH's by Nanjing
Vigor.Test method is as follows：Sample sets blank sample, standard sample, measure sample and control sample respectively.Blank sample add 25 μ l distilled waters and
25 μ l Matrix buffers；Standard sample adds 5 μ l distilled waters, 20 μ l titers (0.2mmpl/L) and 25 μ l Matrix buffers；Treat test sample
This adds 20 μ l prepare liquids, 25 μ l Matrix buffers and 5 μ l Coenzyme I；Control sample adds 5 μ l distilled waters, 20 μ l to treat test sample and 25 μ l bases
Matter buffer solution Matrix buffer；After above-mentioned sample is mixed respectively at a temperature of 37 DEG C water-bath 15min, then add the 2 of 25 μ l,
4- dinitrophenylhydrazines, are further continued for water-bath 15min after mixing, be eventually adding the NaOH solution (0.4mol/L) of 25 μ l, quiet in room temperature
After putting 5min, absorbance (OD values) is measured under 450nm wavelength with microplate reader (Bio-Rad iMark, Bio-Rad, USA), so
IPEC-1 cell supernatant LDH activities are calculated according to OD values afterwards, as a result as shown in table 2 and Fig. 2.(wherein, 1000ml serum/slurry
15min is acted on matrix at a temperature of 37 DEG C, it is 1 unit U to produce 1 μm of ol pyruvic acid in the reaction system.)
Influence of 2 hydrogen peroxide concentration of table to IPEC-1 cell supernatant lactic acid dehydrogenase activities
LDH is a kind of desmoenzyme of stabilization, and when cell is damaged, intracellular LDH can be discharged into nutrient solution,
Therefore, in the present embodiment, in cell culture supernatant LDH vigor enhancing, illustrate that hydrogen peroxide causes cell to generate
Damage.From table 2 and Fig. 2, compared with control group, the IPEC-1 cell supernatant LDH activities of experiment A groups decrease, and P
Value reaches 0.683；And test B group IPEC-1 cell supernatant LDH vigor and significantly raise, and P values are less than 0.042.Illustrate dioxygen
The concentration of water can't improve the activity of IPEC-1 cell supernatants LDH when being 0.2mM, and when the concentration of hydrogen peroxide is 0.5mM
IPEC-1 cell supernatant LDH activities can be caused to significantly improve, is i.e. cause cell to damage after the hydrogen peroxide induction 3h of 0.5nM concentration
Wound.Therefore, the concentration of hydrogen peroxide is set to 0.5mM in the following embodiments of the present invention.
3 hydrogen peroxide concentration of embodiment is to IPEC-1 Apoptosis correlation molecule caspase3, p38 and JNK protein expressions
Influence
(1) by 1 × 106The cell inoculation of a/ml adds the cell of 4mL into the culture dish of 60mm, in each culture dish
Nutrient solution slight wobble makes cell be dispersed in culture dish, when cell attachment and when growing to 70%-80% after culture 3 days,
Nutrient solution is absorbed, is cleaned twice with phosphate buffer.
(2) cell culture fluid will be prepared successively to be added in orifice plate, per hole 4mL, wherein, cell culture fluid is divided into control
Group and experiment B groups, the concentration of hydrogen peroxide is respectively 0mM and 0.5mM in cell culture fluid in two groups, every group of repetition 4.
(3) after hydrogen peroxide stimulates 3h, nutrient solution is absorbed, after cleaning twice along hole wall with phosphate buffer, to each
After the cell lysis buffer solution (Cell Signaling Technology companies) of 100 μ L is added in hole, with cell scraper by albumen
Lysate is smeared uniformly, and firmly scrapes cell at one in union, then is drawn in the centrifuge tube for being transferred to 1.5mL, and concussion is split
Solution；Then 4 DEG C, under the conditions of 12000g, centrifuge 15min, Aspirate supernatant is transferred in the centrifuge tube of new 1.5mL, is used
Western blot methods measure expression of cellular proteins amount.
Wherein, the specific test method of Western blot methods is as follows：The bovine serum albumin for being first 1mg/ml with concentration
White liquor (BSA, the green skies biotech company in Jiangsu) quantifies the protein sample in centrifuge tube；Then take 10 μ L's respectively
Protein sample boils 5~8min in 100 DEG C of boiling water makes albuminous degeneration, is carried out with sodium dodecyl sulphate-polyacrylamide gel
After electrophoretic separation, go on polyvinylidene fluoride film (Hangzhou Lian Ke Biotechnology Ltd.), add 5% skimmed milk power
(Cell Signaling Technology companies) afterwards room temperature closing 1 it is small when；Tris-HCl buffer solutions (0.1%Tween- is used again
20) wash film 3 times, each 10min, after incubate primary antibody, in 4 DEG C overnight.Primary antibody is respectively caspase3 (1：1000 dilutions, the U.S.
Invitrogen companies), total p38 (t-p38) (1：1000 dilution, Cell Signaling Technology companies of the U.S.), phosphorus
It is acidified p38 (p-p38) (1：1000 dilution, Cell Signaling Technology companies of the U.S.), total JNK (t-JNK) (1：
1000 dilution, Cell Signaling Technology companies of the U.S.), phosphorylation JNK (p-JNK) (1：1000 dilutions, the U.S.
Cell Signaling Technology companies), β-actin (1：1000 dilutions, Sigma Co., USA).Wash secondary antibody after film
(Wuhan An Tejie Bioisystech Co., Ltd) is incubated 2h at room temperature, and secondary antibody is respectively HRP-Goat Anti-Mouse IgG (1：
5000 dilutions, Wuhan An Tejie biotech companies) and HRP-Goat Anti-Rabbit IgG (1：5000 dilutions, Wuhan peace
Te Jie biotech companies).Film 3 times, each 10min finally is washed with Tris-HCl buffer solutions (0.1%Tween-20), then is used
ECL reagents (Pierce companies of the U.S.) fluorescence developing, in Alpha Innotech imaging systems, (U.S. ProteinSimple is public
Department) in detection and analysis band intensity (Caspase3 protein levels are with the protein band intensity rate of Caspase3 and β-actin
Represent, phosphorylated p38 and JNK protein levels are represented with its total protein intensity rate respectively), as a result such as table 3 and Fig. 3 to Fig. 7
It is shown.
Influence of 3 hydrogen peroxide concentration of table to IPEC-1 Apoptosis correlation molecule caspase3, p38 and JNK protein expressions
From table 3 and Fig. 3 to Fig. 7, compared with control group, 0.5mM hydrogen peroxide, which stimulates, causes IPEC-1 Apoptosis
Correlation molecule Caspase3 expressing quantities significantly raise, and also result in p-p38/t-p38 and p-JNK/t-JNK ratios and significantly rise
It is high.Since Caspase3 is the significant albumen of Apoptosis, and p38 and JNK are apoptosis pathway key molecules.
Therefore, after the above results show the hydrogen peroxide induction 3h of 0.5mM, apoptosis pathway is have activated, so as to promote cell
Apoptosis.
Influence of 4 hydrogen peroxide concentration of embodiment to IPEC-1 Apoptosis correlation molecule NF- κ B p65 albumen
(1) by 1 × 105The cell inoculation in a/hole is on 12 orifice plate circle creep plates (Corning companies of the U.S.), each
The cell culture fluid of 1mL is added on creep plate, slight wobble makes cell be dispersed on creep plate, and culture treats cell attachment after 3 days
And when growing to 70%-80%, nutrient solution is absorbed, is cleaned twice with phosphate buffer.
(2) cell culture fluid will be prepared successively to be added in orifice plate, per hole 1mL, wherein, cell culture fluid is divided into control
Group and experiment B groups, the concentration of hydrogen peroxide is respectively 0mM and 0.5mM in cell culture fluid in two groups, every group of repetition 4.
(3) after hydrogen peroxide stimulates 3h, nutrient solution is absorbed, is cleaned twice with phosphate buffer.Core is activated according to NF- κ B
Transport detection kit (the green skies biotech company in Jiangsu) step and make cell climbing sheet, with fluorescence inverted microscope (IX73
Research grade, Japanese Olympus companies) observation cell climbing sheet, wherein, fluorescent dye selects DAPI (i.e. 4', 6- diamidino -2- benzene
Base indoles (4', 6-diamidino-2-phenylindole), is a kind of fluorescent dye that can be combined with DNA strengths, commonly uses
In fluorescence microscopy) (wherein blue-fluorescence contaminates for core, and red fluorescence is NF- κ B p65 protein moleculars), as a result such as Fig. 8 institutes
Show.In Fig. 8, figure (a) is the karyomorphism figure that IPEC-1 cells use DAPI cores dye, and figure (b) is NF- κ B in IPEC-1 cells
The aspect graph of p65 protein moleculars, figure (c) are the nucleus that NF- κ B p65 protein moleculars enter after nucleus in IPEC-1 cells
Aspect graph.
As shown in Figure 8, compared with control group, the fluorescence of experiment B groups is more, shows NF- κ B p65 eggs in IPEC-1 cells
Enter core increase in vain.Usually, NF- κ B and I κ B are often present in cytoplasm in the form of compound, and phosphorylation occurs for I κ B
After being degraded with ubiquitination, NF- κ B are dissociateed from cytoplasm to be come and is transferred in nucleus, is specifically bound with corresponding site
And regulate and control the transcript and expression of its target gene (such as inflammation, apoptosis-related genes).Therefore, the above results show, 0.5mM's is double
After the induction of oxygen water stimulates 3h, NF- κ B p65 albumen in IPEC-1 cells is caused to enter core increase, i.e. hydrogen peroxide activation NF- κ B signals,
The apoptosis of inducing cell.
In conclusion in embodiments of the present invention, the hydrogen peroxide of 0.5mM, which acts on IPEC-1 cells, to be caused under cytoactive
Drop, apoptosis correlation molecule expressing quantity significantly rise, when being successfully established the Apoptosis Model of IPEC-1 cells, and having had induction
Between short, easy to operate, of low cost and effect stability it is reliable the advantages of, for research chitling road damage mechanisms and its regulation measure carry
Important channel is supplied.
It these are only the preferred embodiment of the present invention, be not intended to limit the scope of the invention, for this area
For technical staff, the invention may be variously modified and varied.It is all in the spirit and principles in the present invention etc, that is made is any
Modification, equivalent substitution, improvement etc., should all be included within the scope of the present invention.
Claims (9)
1. a kind of method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model, it is characterised in that include the following steps：
Prepare cell culture fluid；
Chitterlings epithelial cell is seeded to after being cultivated in cell culture fluid, carries out Trypsin Induced processing, obtain and wait to induce
Cell；
To cell culture fluid of the addition containing hydrogen peroxide in inducing cell is treated, hydrogen peroxide induction is carried out.
2. the method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model as claimed in claim 1, it is characterised in that
The step of preparing cell culture fluid, specifically includes：
Into DMEM/F-12 culture mediums add 5% hyclone, 1% mycillin mixed liquor, 1% glutamine,
The epithelical cell growth factor of 0.1% transferrins and 5 μ g/L, is made cell culture fluid after mixing.
3. the method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model as claimed in claim 1, it is characterised in that
Chitterlings epithelial cell is seeded to after being cultivated in cell culture fluid, carries out Trypsin Induced processing, inducing cell is treated in acquisition
The step of, including：
Chitterlings epithelial cell is seeded in the container containing cell culture fluid, is placed in incubator and cultivates；
Cultivate in the incubator after reaching 70~80% to cell confluency rate, carry out secondary culture；
Trypsin Induced processing is carried out to the chitterlings epithelial cell by secondary culture, inducing cell is treated in acquisition.
4. the method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model as claimed in claim 3, it is characterised in that
Chitterlings epithelial cell is seeded in the container containing cell culture fluid, is placed in the step of being cultivated in incubator, is specifically included：
After taking the chitterlings epithelial cell frozen to recover, it is seeded in the container containing cell culture fluid, is placed in incubator and trains
Support, wherein, the condition of culture of the incubator is：CO2Volumetric concentration be 4.5~5.5%, cultivation temperature be 36.5~37.5
DEG C, culture when a length of 2~3 days, every 20~26h replace a nutrient solution.
5. the method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model as claimed in claim 3, it is characterised in that
In the step of cultivating in the incubator after reaching 70~80% to cell confluency rate, carrying out secondary culture：The secondary culture
Incubation time is 7~8 days.
6. the method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model as claimed in claim 3, it is characterised in that
To carrying out Trypsin Induced processing by the chitterlings epithelial cell of secondary culture, the step for the treatment of inducing cell is obtained, specifically
Including：
Add trypsin solution in by the chitterlings epithelial cell of secondary culture, trypsase is discarded after digesting 1~3min
Solution, and add cell culture fluid and terminate digestion, inducing cell is treated in acquisition.
7. the method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model as claimed in claim 1, it is characterised in that
To in the step for the treatment of to add the cell culture fluid containing hydrogen peroxide in inducing cell, carry out hydrogen peroxide induction：It is described to contain dioxygen
The concentration of hydrogen peroxide is 0.2~1.0mM in the cell culture fluid of water, a length of 1~6h during induction.
8. the method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model as claimed in claim 7, it is characterised in that
To treat in inducing cell add the cell culture fluid containing hydrogen peroxide, carry out hydrogen peroxide induction the step of, including：
It will treat that inducing cell is seeded in orifice plate, and add cell culture fluid and continue to cultivate；
Culture absorbs cell culture fluid in orifice plate and with phosphate buffer along hole to cell attachment and after growing to 70~80%
Wall cleans；
The cell culture fluid containing hydrogen peroxide is added into the orifice plate after cleaning, carries out hydrogen peroxide induction.
9. the method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model as claimed in claim 8, it is characterised in that
It will treat that inducing cell is seeded in orifice plate, add in the step of cell culture fluid continues culture：It is described to treat that inducing cell is seeded to
Inoculum concentration in the orifice plate is 1 × 104~1 × 106A/mL, additive amount of the cell culture fluid in the orifice plate are
0.1~1mL/ holes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711280433.7A CN108034631A (en) | 2017-12-04 | 2017-12-04 | A kind of method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711280433.7A CN108034631A (en) | 2017-12-04 | 2017-12-04 | A kind of method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108034631A true CN108034631A (en) | 2018-05-15 |
Family
ID=62095679
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711280433.7A Pending CN108034631A (en) | 2017-12-04 | 2017-12-04 | A kind of method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108034631A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111944738A (en) * | 2020-07-07 | 2020-11-17 | 广东省农业科学院动物科学研究所 | Porcine epithelial cell oxidative stress model and establishing method and application thereof |
| CN111944746A (en) * | 2020-08-04 | 2020-11-17 | 山东中医药大学 | Method for inducing apoptosis by thermal stimulation |
| CN114107172A (en) * | 2021-11-12 | 2022-03-01 | 上海市农业科学院 | Method for constructing non-infectious gastric mucosa injury cell model and application |
| CN114990031A (en) * | 2022-07-18 | 2022-09-02 | 江西省科学院生物资源研究所 | Construction method and application of small intestine cell apoptosis model |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101707877A (en) * | 2007-06-13 | 2010-05-12 | 杰伊·普拉夫达 | Materials and methods for treatment and diagnosis of disorders associated with oxidative stress |
| US20150038674A1 (en) * | 2013-08-05 | 2015-02-05 | Bayrak Bertan Boran | Use of glp-2 analogues in pulmonary diseases for therapeutic purpose |
-
2017
- 2017-12-04 CN CN201711280433.7A patent/CN108034631A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101707877A (en) * | 2007-06-13 | 2010-05-12 | 杰伊·普拉夫达 | Materials and methods for treatment and diagnosis of disorders associated with oxidative stress |
| US20150038674A1 (en) * | 2013-08-05 | 2015-02-05 | Bayrak Bertan Boran | Use of glp-2 analogues in pulmonary diseases for therapeutic purpose |
Non-Patent Citations (6)
| Title |
|---|
| TONY E. HAYNES等: "L-Glutamine or L-alanyl-L-glutamine prevents oxidant- or endotoxin-induced death of neonatal enterocytes", 《AMINO ACIDS》 * |
| YANG WANG等: "Bacillus amyloliquefaciens SC06 alleviates the oxidative stress of IPEC-1 via modulating Nrf2/Keap1 signaling pathway and decreasing ROS production", 《APPL MICROBIOL BIOTECHNOL》 * |
| 刘琳娜等: "唐古特大黄多糖对过氧化氢诱导的肠上皮细胞凋亡的保护作用及其可能机制", 《中国药理学通报》 * |
| 潘銮凤: "《分子生物学技术》", 31 October 2008, 复旦大学出版社 * |
| 王富伟: "《现代常用医学检验技术及临床应用》", 30 June 2017, 吉林科学技术出版社 * |
| 程宝鸾: "《动物细胞培养技术》", 31 March 2000, 华南理工大学出版社 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111944738A (en) * | 2020-07-07 | 2020-11-17 | 广东省农业科学院动物科学研究所 | Porcine epithelial cell oxidative stress model and establishing method and application thereof |
| CN111944746A (en) * | 2020-08-04 | 2020-11-17 | 山东中医药大学 | Method for inducing apoptosis by thermal stimulation |
| CN111944746B (en) * | 2020-08-04 | 2022-05-06 | 山东中医药大学 | Method for inducing apoptosis by thermal stimulation |
| CN114107172A (en) * | 2021-11-12 | 2022-03-01 | 上海市农业科学院 | Method for constructing non-infectious gastric mucosa injury cell model and application |
| CN114990031A (en) * | 2022-07-18 | 2022-09-02 | 江西省科学院生物资源研究所 | Construction method and application of small intestine cell apoptosis model |
| CN114990031B (en) * | 2022-07-18 | 2024-03-29 | 江西省科学院生物资源研究所 | Construction method and application of small intestine cell apoptosis model |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108034631A (en) | A kind of method for building up of hydrogen peroxide induction chitterlings Epithelial Cell Apoptosis model | |
| Sun et al. | In vitro effects of low‐intensity ultrasound stimulation on the bone cells | |
| Smyth | Studies on tapeworm physiology: I. The cultivation of Schistocephalus solidus in vitro | |
| Nippert et al. | Isolation and cultivation of adult rat cardiomyocytes | |
| Neng et al. | Isolation and culture of endothelial cells, pericytes and perivascular resident macrophage-like melanocytes from the young mouse ear | |
| Reed et al. | Single or group culture of mammalian embryos: the verdict of the literature | |
| CN105177044B (en) | The method for obtaining lymthoma miniature pig disease model by knocking out P53 gene | |
| CN105566353A (en) | Compound and preparation method thereof | |
| Rodriguez-Dorta et al. | Effect of coculture with oviduct epithelial cells on viability after transfer of vitrified in vitro produced goat embryos | |
| CN103710299A (en) | In-vitro culture solution for culturing swine parthenogenetic activated embryos and swine in-vitro fertilized embryos | |
| Luo et al. | Isolation of mouse coronary endothelial cells | |
| CN105238738A (en) | Isolated culture method of piglet myocardial fibroblasts | |
| CN106834216A (en) | A kind of in vitro culture liquid and cultural method for the lonely female activation embryo of pig | |
| CN109825470A (en) | The mitochondria of mesenchymal stem cell is improving the application in egg mother cell drug | |
| Cheng et al. | Abnormal lipid metabolism induced apoptosis of spermatogenic cells by increasing testicular HSP60 protein expression | |
| CN105039402A (en) | Method for improving pig meat quality | |
| CN108865985A (en) | A kind of method of the pre- epithelial-mesenchymal conversion of stem cell source excretion soma | |
| Oh et al. | The promise of dog cloning | |
| CN103484425B (en) | Pseudosciaena crocea head kidney cell line and construction method thereof | |
| Rothschild et al. | The ex vivo culture and pattern recognition receptor stimulation of mouse intestinal organoids | |
| CN104694475A (en) | Novel neural stem cell culture additive, screening method and application of additive and culture medium utilizing additive | |
| CN103710300A (en) | In-vitro culture solution for culturing swine parthenogenetic activated embryos and swine in-vitro fertilized embryos | |
| Souza-Fabjan et al. | Reproductive biotechnologies applied to the female sheep and goat | |
| CN103040867A (en) | Application of microvesicles from bone marrow mesenchymal stem cells in preparing drug for treating kidney injury | |
| CN107365801B (en) | The method for handling bovine somatic cells clone's embryo |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180515 |