CN109112105A - A kind of method for building up and verification method of human hepatoma cell line HepG2's oxidative stress model of oleic acid induction - Google Patents
A kind of method for building up and verification method of human hepatoma cell line HepG2's oxidative stress model of oleic acid induction Download PDFInfo
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Abstract
The present invention relates to a kind of method for building up of human hepatoma cell line HepG2's oxidative stress model of oleic acid induction, the method uses the external evoked processing human hepatoma cell line HepG2 of oleic acid.The method of the present invention uses the external evoked processing HepG2 of oleic acid, and oleic acid optimum concentration is 1mmol/L, and compared with prior art, the method for the present invention has been abandoned uses H in the past2O2Inducing cell generates oxidative stress, establishes induced oxidation Stress model using oleic acid for the first time, by investigating cell survival rate and intracellular ROS level, it is determined that the optimal use concentration of oleic acid and cell incubation time.The method operation is easy, oxidation effectiveness is preferable, cost is relatively low, it can help to establish oxidative stress model, some theoretical reference foundations are provided for follow-up study Antioxidation Mechanism, cell culture can be helped and establish the researcher of oxidative stress model than more smoothly completing the culture of HepG2 cell and the foundation of oxidative stress model.
Description
Technical field
The invention belongs to liver cancer cell lines HepG2 oxidative stress modelling technique field, especially a kind of people of oleic acid induction
The method for building up of liver cancer cell lines HepG2 oxidative stress model.
Background technique
With the continuous improvement of China's economic development and living standard, huge change is had occurred in the diet structure of urban and rural residents
Change, shows as diet structure and tend to high-energy density, is i.e. fat intake is excessive.The increase of Energy intaking causes energy i (in vivo) generation
Thank vigorous, by-product active oxygen (reactive oxygen species, ROS) is consequently increased, and then can induce body oxygen
Change stress.And oxidative stress is a kind of unbalance between free radical and antioxidant ability of organism, is physiology most complicated in human body
One of with pathological reaction, the environmental stimulus such as high temperature, ionising radiation, environmental contaminants object can induce oxidative stress, and direct shadow
Survival and normal function of the body in extreme environment are rung, the generation of inflammation and cancer is eventually led to.Free radical is to weight in cell
It wants biomolecule lipid, protein and nucleic acid to generate oxidative damage, forms various corresponding oxidation products, eventually lead to cell function
It can be impaired.Therefore, in order to illustrate, intracellular environment caused by oxidative stress changes and oxidative stress is to tumour cell and normally
The regulatory mechanism of cell should establish reliable cell model.
Oleic acid very permeable cell membrane enters cell, and the unsaturated chain of wherein fatty acid is made to disconnect forming peroxide, breaks
Bad cell structure acts on HepG2 and causes significant oxidative damage, and process is easily obtained and property is relatively stable, is become
Study the important tool of cell oxidative damage.
HepG2 cell strain belongs to Bel7402, has similar biological activity with liver cell, and liver is human body
Metabolism center, be also to carry out the vitals of drug and exogenous toxicant metabolism, while being also that oxidative stress occurs in vivo
Main organs.Therefore, the HepG2 pathogenesis and its clinic diseases related to research oxidative stress as experimental subjects are chosen
Using significant.
By retrieval, patent publication us relevant to present patent application is not yet found.
Summary of the invention
It is an object of the invention in place of overcome the deficiencies in the prior art, provide a kind of Bel7402 of oleic acid induction
The method for building up of HepG2 oxidative stress model, this method operation is easy, cost is relatively low, and this method can be subsequent further investigation
Antioxidation Mechanism provides theoretical reference.
The present invention solves its technical problem and adopts the following technical solutions to achieve:
A kind of method for building up of human hepatoma cell line HepG2's oxidative stress model of oleic acid induction, the method is using oil
The external evoked processing human hepatoma cell line HepG2 of acid.
Moreover, the concentration of the oleic acid is 1mmol/L.
Moreover, steps are as follows:
Oleic acid is sub-packed in centrifuge tube, 4 DEG C of preservations are added DMSO and dissolve oleic acid, and DMSO: the volume ratio of oleic acid is 1:
1000,0.22 μm of films excessively are stand-by；
HepG2 control group and oleic acid group is respectively set, is adjusted with the DMEM high glucose medium of the 5%FBS containing mass concentration thin
Born of the same parents' concentration is (4-5) × 105A/mL, 37 DEG C, 5%CO2After cultivating 36-48h in incubator, film process is added in the every hole of experimental group
Oleic acid continue to cultivate, in 4h, 8h, 12h and for 24 hours when, collect the Bel7402 that cell induces to get oleic acid respectively
HepG2 oxidative stress model.
Moreover, human hepatoma cell line HepG2's oxidative stress model of the oleic acid induction detects cell survival using mtt assay
Rate, using fluorescence spectrometry intracellular ROS level.
Moreover, the cell concentration when mtt assay detection cell survival rate is 4 × 105A/mL, oleic acid concentration are respectively
1.5,1.0,0.75,0.5,0mmol/L, cell incubation time are 4h, and absorbance value is measured under 570nm absorbance and calculates cell
Survival rate；
Cell survival rate calculates are as follows: the OD value of each instrument connection is subtracted background OD value, the OD value of each repeating hole is averaged
±SD；Cell survival rate indicates that T is the OD value of oleic acid cell with T/C%, and C is the OD value of control cell；Cell survival rate %=
(dosing cell OD/ control cell OD) × 100, medicine when drug concentration (IC50) and T/C=10% when finding out T/C=50%
Object concentration.
Moreover, the cell concentration of the fluorescence spectrometry intracellular ROS level is 5 × 106A/mL, oleic acid concentration difference
For 1.5,1.0,0.75,0.5,0mmol/L, the cell incubation time is 60min；
Cell precipitate is collected after centrifugation, the cell gathered is resuspended with PBS, and is examined with fluorescent sub-photometer
It surveys.
Moreover, being 500 ± 15nm with the excitation wavelength that fluorescent sub-photometer is detected, launch wavelength is 530 ± 20nm.
The authentication of the method for building up of human hepatoma cell line HepG2's oxidative stress model of oleic acid induction as described above
Method, steps are as follows:
(1) various concentration oleic acid handles the influence to cell survival rate
1. experimental group and processing
It is respectively set HepG2 control group and various concentration gradient oleic acid group, 1.5,1.0,0.75,0.5,0mmol/L, with containing
The DMEM high glucose medium adjustment HepG2 concentration of mass concentration 5%FBS is 4 × 105A/mL, and 96 orifice plates are inoculated in, every hole
100 μ L, 37 DEG C, 5%CO2It is cultivated in incubator；Culture solution is sucked out after 48 hours, the every hole of experimental group is added 100 μ L and passes through without blood
The oleic acid of the diluted various concentration of clear cell culture fluid, cell culture fluid of the blank control group addition equivalent without serum, 37
DEG C, 5%CO2Continue to cultivate in incubator；
2. mtt assay detects cell survival rate
Continue to cultivate 4h, 8h, 12h and for 24 hours when, discard part cell culture fluid, every hole retains 90 μ L, and 10 μ are added
LMTT solution, 37 DEG C, 5%CO24h is incubated in incubator；Liquid is carefully discarded supernatant, 100 μ L DMSO, plate shaker is added in every hole
It shakes 10 minutes, stands 5-10min, with absorbance value at microplate reader measurement 570nm wavelength, calculate cell survival rate；
(2) various concentration oleic acid handles the influence to intracellular ROS level
1. experimental group and processing
Cell controls group and various concentration gradient oleic acid group, 1.5,1.0,0.75,0.5,0mmol/L, with containing matter are set respectively
The DMEM high glucose medium adjustment HepG2 concentration for measuring concentration 5%FBS is 5 × 106A/mL, is inoculated in 6 orifice plates, every hole 1mL, and 37
DEG C, 5%CO2It is cultivated in incubator；Culture solution is sucked out after 48 hours；The every hole of experimental group is added 1mL and passes through the cell training without serum
The cell culture fluid that equivalent is free of serum, 37 DEG C, 5%CO are added in the oleic acid of the diluted various concentration of nutrient solution, blank control group2Training
It supports and continues to cultivate in case；
2. fluorescence spectrometry intracellular ROS level
Continue to cultivate 4h, 8h, 12h and for 24 hours when collect cell, PBS is washed 2 times, and DCFH-DA fluorescence probe is added in every hole
100 μ L, 37 DEG C, 5%CO260min is incubated in incubator；Pancreatin digestion is added culture medium and terminates digestion, cell suspension is made,
It is centrifuged 10min；PBS is washed 2 times, cell precipitate is collected by centrifugation, single cell suspension is made for fluorescence detection；Fluorescence microplate reader is surveyed
Determine absorbance value, 500 ± 15nm of excitation wavelength, 530 ± 20nm of launch wavelength are set.
The advantages of present invention obtains and good effect are:
The method of the present invention uses the external evoked processing HepG2 of oleic acid, and oleic acid optimum concentration is 1mmol/L, with the prior art
It compares, the method for the present invention has been abandoned uses H in the past2O2Inducing cell generates oxidative stress, establishes induced oxidation using oleic acid for the first time and answers
Model is swashed, by investigating cell survival rate and intracellular ROS level, it is determined that when the optimal use concentration and cell incubation of oleic acid
Between.The method operation is easy, oxidation effectiveness is preferable, cost is relatively low, can help to establish oxidative stress model, is follow-up study antioxygen
Change mechanism provides some theoretical reference foundations, and the researcher's ratio that can be helped cell culture and establish oxidative stress model is more smoothly complete
At the culture of HepG2 cell and the foundation of oxidative stress model.
Detailed description of the invention
Fig. 1 is the influence diagram that oleic acid handles to HepG2 survival rate in the present invention；
Fig. 2 is the influence diagram that oleic acid handles to ROS level in HepG2 in the present invention.
Specific embodiment
Below with reference to the invention will be further described by specific embodiment, following embodiment be it is descriptive, no
It is restrictive, this does not limit the scope of protection of the present invention.
Raw material used in the present invention is unless otherwise specified conventional commercial product；Used in the present invention
Method is unless otherwise specified the conventional method of this field.
A kind of method for building up of human hepatoma cell line HepG2's oxidative stress model of oleic acid induction, the method is using oil
The external evoked processing human hepatoma cell line HepG2 of acid.
More preferably, the concentration of the oleic acid is 1mmol/L.
More preferably, steps are as follows:
Oleic acid is sub-packed in centrifuge tube, 4 DEG C of preservations are added DMSO and dissolve oleic acid, and DMSO: the volume ratio of oleic acid is 1:
1000,0.22 μm of films excessively are stand-by；
HepG2 control group and oleic acid group is respectively set, is adjusted with the DMEM high glucose medium of the 5%FBS containing mass concentration thin
Born of the same parents' concentration is (4-5) × 105A/mL, 37 DEG C, 5%CO2After cultivating 36-48h in incubator, film process is added in the every hole of experimental group
Oleic acid continue to cultivate, in 4h, 8h, 12h and for 24 hours when, collect the Bel7402 that cell induces to get oleic acid respectively
HepG2 oxidative stress model.
More preferably, human hepatoma cell line HepG2's oxidative stress model of the oleic acid induction is deposited using mtt assay detection cell
Motility rate, using fluorescence spectrometry intracellular ROS level.
More preferably, the cell concentration when mtt assay detection cell survival rate is 4 × 105A/mL, oleic acid concentration difference
For 1.5,1.0,0.75,0.5,0mmol/L, the cell incubation time is 4h, and absorbance value is measured under 570nm absorbance and is calculated thin
Born of the same parents' survival rate；
Cell survival rate calculates are as follows: the OD value of each instrument connection is subtracted background OD value, the OD value of each repeating hole is averaged
±SD；Cell survival rate indicates that T is the OD value of oleic acid cell with T/C%, and C is the OD value of control cell；Cell survival rate %=
(dosing cell OD/ control cell OD) × 100, medicine when drug concentration (IC50) and T/C=10% when finding out T/C=50%
Object concentration.
More preferably, the cell concentration of the fluorescence spectrometry intracellular ROS level is 5 × 106A/mL, oleic acid concentration point
Not Wei 1.5,1.0,0.75,0.5,0mmol/L, the cell incubation time be 60min；
Cell precipitate is collected after centrifugation, the cell gathered is resuspended with PBS, and is examined with fluorescent sub-photometer
It surveys.Moreover, being 500 ± 15nm with the excitation wavelength that fluorescent sub-photometer is detected, launch wavelength is 530 ± 20nm.
The verification method of the method for building up of human hepatoma cell line HepG2's oxidative stress model of above-mentioned oleic acid induction, step
It is rapid as follows:
One, experimental method
Influence of the 1 various concentration oleic acid processing to cell survival rate
(1) experimental group and processing
HepG2 control group and various concentration gradient oleic acid group (1.5,1.0,0.75,0.5,0mmol/L) is respectively set, uses
The DMEM high glucose medium adjustment HepG2 concentration of the 5%FBS containing mass concentration is 4 × 105A/mL, and 96 orifice plates are inoculated in, often
100 μ L of hole, 37 DEG C, 5%CO2It is cultivated in incubator；Culture solution is sucked out after 48 hours, 100 μ L are added through being free of in the every hole of experimental group
The cell culture fluid that equivalent is free of serum is added in the oleic acid of the diluted various concentration of the cell culture fluid of serum, blank control group,
37 DEG C, 5%CO2Continue to cultivate in incubator.
(2) mtt assay detects cell survival rate
Continue to cultivate 4h, 8h, 12h and for 24 hours when, discard part cell culture fluid, every hole retains 90 μ L, and 10 μ are added
LMTT solution, 37 DEG C, 5%CO24h is incubated in incubator；Liquid is carefully discarded supernatant, 100 μ L DMSO, plate shaker is added in every hole
It shakes 10 minutes, stands 5-10min, with absorbance value at microplate reader measurement 570nm wavelength, calculate cell survival rate.
Influence of the 2 various concentration oleic acid processing to intracellular ROS level
(1) experimental group and processing
Cell controls group and various concentration gradient oleic acid group (1.5,1.0,0.75,0.5,0mmol/L) are set respectively, with containing matter
The DMEM high glucose medium adjustment HepG2 concentration for measuring concentration 5%FBS is 5 × 106A/mL, is inoculated in 6 orifice plates, every hole 1mL, and 37
DEG C, 5%CO2It is cultivated in incubator；Culture solution is sucked out after 48 hours；The every hole of experimental group is added 1mL and passes through the cell training without serum
The cell culture fluid that equivalent is free of serum, 37 DEG C, 5%CO are added in the oleic acid of the diluted various concentration of nutrient solution, blank control group2Training
It supports and continues to cultivate in case.
(2) fluorescence spectrometry intracellular ROS level
Continue to cultivate 4h, 8h, 12h and for 24 hours when collect cell, PBS is washed 2 times, and DCFH-DA fluorescence probe is added in every hole
100 μ L, 37 DEG C, 5%CO260min is incubated in incubator；Pancreatin digestion is added culture medium and terminates digestion, cell suspension is made,
It is centrifuged 10min；PBS is washed 2 times, cell precipitate is collected by centrifugation, single cell suspension is made for fluorescence detection；Fluorescence microplate reader is surveyed
Determine absorbance value.500 ± 15nm of excitation wavelength, 530 ± 20nm of launch wavelength are set.
3 experimental datas carry out one-way analysis of variance (One-WayANOVA) using SSP18.0 statistical software, Duncan group
Between compare, as a result withIt indicates.Shoulder mark a indicates that P < 0.05 compared with the control group, shoulder mark b indicate P < compared with the control group
0.01。
Two, experimental result
Influence of<a>various concentration oleic acid processing to HepG2 survival rate
As a result as shown in Figure 1, with oleic acid concentration raising, cell survival rate gradually decreases, and with the increasing of action time
Add, cell survival rate is also on a declining curve.Wherein 1mmol/L oleic acid acts on HepG2 cell 4h, 8h, 12h, can reduce for 24 hours carefully
Born of the same parents' survival rate (P < 0.05), and effect is most significant (P < 0.01) when for 24 hours.Continue to increase to 36h, cell mortality, no
With theoretical significance.
<b>influence (Fig. 2) of the various concentration oleic acid processing to ROS level in HepG2
As a result as shown in Fig. 2, with oleic acid concentration raising, intracellular reactive oxygen level gradually increases, and when with effect
Between increase, oleic acid processing group intracellular reactive oxygen level gradually decreases.Oleic acid act on HepG2 cell 4h, 8h, 12h, for 24 hours
Intracellular ROS level (P < 0.05) can be significantly increased, and wherein 1mmol/L oleic acid effect when for 24 hours is most significant (P < 0.01).
Although disclosing the embodiment of the present invention and attached drawing for the purpose of illustration, those skilled in the art can be managed
Solution: do not departing from the present invention and spirit and scope of the appended claims in, various substitutions, changes and modifications be all it is possible,
Therefore, the scope of the present invention is not limited to the embodiment and attached drawing disclosure of that.
Claims (8)
1. a kind of method for building up of human hepatoma cell line HepG2's oxidative stress model of oleic acid induction, it is characterised in that: the side
Method uses the external evoked processing human hepatoma cell line HepG2 of oleic acid.
2. the method for building up of human hepatoma cell line HepG2's oxidative stress model of oleic acid induction according to claim 1,
Be characterized in that: the concentration of the oleic acid is 1mmol/L.
3. the foundation side of human hepatoma cell line HepG2's oxidative stress model of oleic acid induction according to claim 1 or 2
Method, it is characterised in that: steps are as follows:
Oleic acid is sub-packed in centrifuge tube, 4 DEG C of preservations are added DMSO and dissolve oleic acid, DMSO: the volume ratio of oleic acid is 1:1000,
0.22 μm of film excessively is stand-by；
HepG2 control group and oleic acid group is respectively set, it is dense with the DMEM high glucose medium adjustment cell of the 5%FBS containing mass concentration
Degree is (4-5) × 105A/mL, 37 DEG C, 5%CO2After cultivating 36-48h in incubator, the oil of film process is added in the every hole of experimental group
Acid continues to cultivate, in 4h, 8h, 12h and for 24 hours when, collect the human hepatoma cell line HepG2 that cell induces to get oleic acid respectively
Oxidative stress model.
4. the method for building up of human hepatoma cell line HepG2's oxidative stress model of oleic acid induction according to claim 3,
Be characterized in that: human hepatoma cell line HepG2's oxidative stress model of the oleic acid induction detects cell survival rate using mtt assay,
Using fluorescence spectrometry intracellular ROS level.
5. the method for building up of human hepatoma cell line HepG2's oxidative stress model of oleic acid induction according to claim 4,
Be characterized in that: the cell concentration when mtt assay detection cell survival rate is 4 × 105A/mL, oleic acid concentration is respectively 1.5,
1.0,0.75,0.5,0mmol/L, cell incubation time are 4h, and absorbance value is measured under 570nm absorbance and calculates cell survival
Rate；
Cell survival rate calculates are as follows: the OD value of each instrument connection is subtracted background OD value, the OD value of each repeating hole is averaged ± SD；
Cell survival rate indicates that T is the OD value of oleic acid cell with T/C%, and C is the OD value of control cell；Cell survival rate %=(dosing
Cell OD/ control cell OD) × 100, drug when drug concentration (IC50) and T/C=10% when finding out T/C=50% is dense
Degree.
6. the method for building up of human hepatoma cell line HepG2's oxidative stress model of oleic acid induction according to claim 4,
Be characterized in that: the cell concentration of the fluorescence spectrometry intracellular ROS level is 5 × 106A/mL, oleic acid concentration are respectively
1.5,1.0,0.75,0.5,0mmol/L, cell incubation time are 60min；
Cell precipitate is collected after centrifugation, the cell gathered is resuspended with PBS, and is detected with fluorescent sub-photometer.
7. the method for building up of human hepatoma cell line HepG2's oxidative stress model of oleic acid induction according to claim 6,
It is characterized in that: being 500 ± 15nm with the excitation wavelength that fluorescent sub-photometer is detected, launch wavelength is 530 ± 20nm.
8. the foundation of human hepatoma cell line HepG2's oxidative stress model of oleic acid induction as described in any one of claim 1 to 7
The verification method of method, it is characterised in that: steps are as follows:
(1) various concentration oleic acid handles the influence to cell survival rate
1. experimental group and processing
HepG2 control group and various concentration gradient oleic acid group, 1.5,1.0,0.75,0.5,0mmol/L, with containing quality are respectively set
The DMEM high glucose medium adjustment HepG2 concentration of concentration 5%FBS is 4 × 105A/mL, and 96 orifice plates are inoculated in, every 100 μ of hole
L, 37 DEG C, 5%CO2It is cultivated in incubator；Culture solution is sucked out after 48 hours, the every hole of experimental group is added 100 μ L and passes through without serum
The oleic acid of the diluted various concentration of cell culture fluid, cell culture fluid of the blank control group addition equivalent without serum, 37 DEG C,
5%CO2Continue to cultivate in incubator；
2. mtt assay detects cell survival rate
Continue to cultivate 4h, 8h, 12h and for 24 hours when, discard part cell culture fluid, every hole retains 90 μ L, and it is molten that 10 μ LMTT are added
Liquid, 37 DEG C, 5%CO24h is incubated in incubator；Liquid is carefully discarded supernatant, 100 μ L DMSO are added in every hole, and plate shaker shakes 10 points
Clock stands 5-10min, with absorbance value at microplate reader measurement 570nm wavelength, calculates cell survival rate；
(2) various concentration oleic acid handles the influence to intracellular ROS level
1. experimental group and processing
Cell controls group and various concentration gradient oleic acid group are set respectively, and 1.5,1.0,0.75,0.5,0mmol/L, with containing, quality is dense
The DMEM high glucose medium adjustment HepG2 concentration for spending 5%FBS is 5 × 106A/mL, is inoculated in 6 orifice plates, every hole 1mL, 37 DEG C,
5%CO2It is cultivated in incubator；Culture solution is sucked out after 48 hours；The every hole of experimental group is added 1mL and passes through the cell culture fluid without serum
The cell culture fluid that equivalent is free of serum, 37 DEG C, 5%CO are added in the oleic acid of diluted various concentration, blank control group2Incubator
In continue to cultivate；
2. fluorescence spectrometry intracellular ROS level
Continue to cultivate 4h, 8h, 12h and for 24 hours when collect cell, PBS is washed 2 times, and 100 μ L of DCFH-DA fluorescence probe is added in every hole,
37 DEG C, 5%CO260min is incubated in incubator；Pancreatin digestion is added culture medium and terminates digestion, cell suspension is made, is centrifuged
10min；PBS is washed 2 times, cell precipitate is collected by centrifugation, single cell suspension is made for fluorescence detection；Fluorescence microplate reader measurement is inhaled
500 ± 15nm of excitation wavelength, 530 ± 20nm of launch wavelength is arranged in shading value.
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| CN113180240A (en) * | 2021-04-14 | 2021-07-30 | 沈阳农业大学 | Screening and application of agricultural products with effect of resisting oxidative stress reaction |
| CN114134117A (en) * | 2021-11-18 | 2022-03-04 | 广州大学 | Cell strain and preparation method and application thereof |
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