CN109593708A - A kind of beef cattle muscle cell model of oxidative construction method and application - Google Patents
A kind of beef cattle muscle cell model of oxidative construction method and application Download PDFInfo
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Abstract
The present invention provides a kind of beef cattle muscle cell model of oxidative construction method and applications, belong to technical field of cell biology.Its technical solution are as follows: a kind of beef cattle muscle cell model of oxidative construction method, comprising the following steps: prepare the acquisition of cell culture fluid, separation beef cattle musculature, culture beef cattle muscle cell and model of oxidative building parameter.The invention has the benefit that the present invention is using isolated beef cattle muscle cell as research object, beef cattle muscle cell model of oxidative is established using hydrogen peroxide, material is provided for the reduction and nutrition regulation mechanism study of beef quality, reduces the research and development cost of follow-up test；The beef cattle muscle cell model of oxidative of foundation can provide mechanism study material for the Regulation Mechanism research of beef quality and the exploitation of feed anti-oxidation active substance, directly substantially reduce research and development cost and time using this model.
Description
Technical field
The present invention relates to cell biology and technical field more particularly to a kind of beef cattle muscle cell model of oxidative structures
Construction method and application.
Background technique
As the improvement of people's living standards, beef has entered huge numbers of families, but quality is multifarious.Beef cattle is attributed to exist
During raising, butcher etc., because manage, transport it is improper caused by stress caused by beef quality reduce.Currently, the problem is
It attracts extensive attention, but most researchs also only reside within production level.In addition, many scholar experts intend adding in cattle feed
Add anti-oxidation active substance, to improve beef quality, but the mechanism study due to lacking molecular cell level, leads to additive
Amount blindly, and feed counter productive it is difficult to predict.Currently, the domestic model of oxidative about beef cattle muscle cell is not built also
It is vertical.
Summary of the invention
The purpose of the present invention is to provide a kind of beef cattle muscle cell model of oxidative construction method and applications.
In order to achieve the above-mentioned object of the invention, the present invention provides a kind of beef cattle muscle cell model of oxidative building sides
Method, comprising the following steps:
Step 1: cell culture fluid is prepared:
50-60 parts of (volume parts) fetal calf serums (FBS), 2-3 portions of (volume parts) mycillin mixed liquors are dissolved in
In 450-500 parts of (volume parts) cell culture fluids, by 0.22 micron membrane filter filtering after be dispensed into sterilizing centrifuge tube in, often
40-45 milliliters of pipe, 4 DEG C of preservations are placed, culture solution was placed in 37 DEG C of water-baths in 0.5-1 hours before cell culture and is preheated, obtained carefully
Born of the same parents' culture solution；
Step 2: separation beef cattle musculature:
Clip mature beef veutro musculature 10g-20g is rinsed 2-3 times with sterile saline, is placed on 100mL rapidly
In the sample bottle of the phosphate buffer (PBS) of the liquid of mycillin containing 1-2%, low-temperature transport after sealed membrane sealing, at 1-2 hours
Inside take back laboratory；Musculature is placed in sterilized petri dishes after taking out in sample bottle in sterile super-clean bench, utilizes body formula
Microscope rejects fascia and superabundant fats tissue, then rinses 3-5 times with PBS, later with eye scissors by musculature shred to
Chyle shape；
Step 3: culture beef cattle muscle cell:
Chyle even tissue is drawn with suction pipe and is placed on culture bottle bottom wall, afterwards turns over culture bottle and prepared cell training is added
Nutrient solution lays flat culture bottle in incubator, overturns culture bottle after 30-40 minutes, and culture solution is allowed slowly to submerge culture bottle bottom wall
Chyle tissue, and again by culture bottle put back to incubator continue culture 38-48 hours after cell adherent growth, have after 5-6 days
80%-90% cell is adherent；Obtain muscle cell；
Step 4: the acquisition of model of oxidative building parameter:
Cell is prepared after isolated adherent muscle cell is digested and poises liquid, and adjustment cell poises the cell concentration of liquid
It is 5 × 104A/mL's, cell is poised into liquid and is inoculated in 96 orifice plates, cultivates 24 hours in the incubator, is then changed to H2O2
(0,50,100,150,200,250,300,350,400,450 μM) culture solution is handled 24 hours, utilizes Cell Counting
Kit-8 kit detects cell survival rate, works as H2O2When rising to 300 μM, cell survival rate is down to 60% or so, continues to improve dense
It spends cell and mortality occurs；Separately by 5 × 104The cell of a/mL poises liquid and is inoculated in 25cm2In culture bottle, extremely to cell confluency
When 80%, it is changed to H2O2(0,50,100,150,200,250,300,350,400,450 μM) culture solution is handled 24 hours, is collected
Cell, detection superoxide dismutase (SOD), catalase (CAT), malonaldehyde (MDA), protein carbonyl group (PC) and 8- hydroxyl
Base deoxyguanosine (8-OHG), discovery SDO and CAT activity are first increased and are reduced afterwards, and MDA, PC and 8-OHG gradually rise；Obtain 300 μ
M H2O2For the useful effect concentration of model of oxidative.
Based on the above results, using 300 μM of H2O2To beef cattle muscle cell processing 45min, 1.5h, 3h, 6h, 12h, for 24 hours,
Cell survival rate, SOD, CAT, MDA, PC and 8-OHG are measured, discovery is gradually dropped with the extension for handling the time, cell survival rate
It is low, handle that 6 hours survival rates are down to 61.2%, SOD and CAT activity first increases reduces afterwards, MDA, PC and 8-OHG gradually rise
It is high.Comprehensive indices, obtain 300 μM of H2O2Action time is advisable for 6h.
Cell culture fluid described in step 1 is (DMEM/F12).
The adjustment cell concentration that cell described in step 4 poises liquid is 5 × 104A/mL.
300 μM of H of useful effect concentration of the model of oxidative obtained in step 42O2, with 300 μM of H2O2To beef cattle
Muscle cell handles 45min, 1.5h, 3h, 6h, 12h, for 24 hours, measures cell survival rate, SOD, CAT, MDA, PC and 8-OHG, with
The extension of processing time, cell survival rate gradually decreases, and handles 6 hours survival rates and is down to 61.2%, SOD and CAT activity first
It is reduced after raising, MDA, PC and 8-OHG gradually rise.
The suitable parameter that the model of oxidative of beef cattle muscle cell described in step 4 is established is 300 μM of H2O2Handle the time
For 6h.
The isolated adherent muscle cell establishes model of oxidative using this model parameter, on the basis of model
Different product can be studied to the regulating effect of beef cattle oxidative damage, this model is exactly to provide place mat for the exploitation of new product.
The exploitation that obtained beef cattle muscle cell model of oxidative is used for beef quality regulation product is provided to mechanism to grind
Study carefully material.
The in vitro beef cattle muscle cell, hydrogen peroxide (H2O2, Sigma company, 323381), (Gibco is public by DMEM/F12
Department, 8117245), fetal calf serum (BI company, 04-001-1A), mycillin mixed liquor (Beijing Suo Laibao Science and Technology Ltd,
P1400), pancreatin (Gibco company, 25200072), Cell Counting Kit-8 (CCK-8, MCE company), antioxidase examination
Agent box, superoxide dismutase (SOD, abcam company, ab65354), catalase (CAT, abcam company, ab83464),
Oxidation product kit: malonaldehyde (MDA, Jiangsu Kai Ji Biotechnology Ltd., KGT004-1), protein carbonyl group
(8-OHG, Cayman are public for (PC, Beijing Suo Laibao Science and Technology Ltd, BC1275), 8-OhdG Elisa kit
Department, 589320)；Incubator (Thermo company, model 371), super-clean bench (Su Jing is safe and sound, model SW-CJ-2F).
In order to preferably realize foregoing invention purpose, the present invention also provides the examinations of beef cattle muscle cell model of oxidative
Proved recipe method and testing index:
(1), various concentration H2O2Influence to beef cattle muscle cell proliferation and redox state:
In super-clean bench, cell is prepared with basic culture solution and poises liquid, adjustment cell concentration is 5 × 104A/mL.
Cell proliferative conditions detection: cell is poised into liquid and is inoculated in 96 orifice plates (100 hole μ L/), is cultivated in the incubator
For 24 hours, then it is changed to H2O2(0,50,100,150,200,250,300,350,400,450 μM) culture solution, each time point set 6
A repetition, processing are changed to basic culture solution afterwards for 24 hours, the CCK-8 solution of 10 μ L are added in every hole, culture plate is placed in 37
1-4h is incubated in DEG C incubator, with the absorbance at microplate reader measurement 450nm.
Cellular morphology and redox state detection: cell is poised into liquid and is inoculated in 25cm2Culture bottle, extremely to cell confluency
When 80%, it is changed to H2O2(0,50,100,150,200,250,300,350,400,450 μM) culture solution, each time point set 6
Culture plate is placed in 37 DEG C of incubators after handling for 24 hours by a repetition, then om observation cellular morphology collects group of cells, is surveyed
Determine antioxidase (SOD, CAT), oxidation product (MDA, PC, 8-OHG).
Influence of the 1 various concentration H2O2 of table to beef cattle muscle cell activities of antioxidant enzymes:
Note: colleague's data shoulder mark is without letter or has same letter to indicate that difference is not significant (P > 0.05), and different letters indicate
Significant difference (P < 0.05).Following table is same.
(2)H2O2Influence of the different disposal time to beef cattle muscle cell proliferation and redox state:
2 various concentration H of table2O2Influence to beef cattle muscle cell oxidation product content:
According to the H of above-mentioned determination2O2Useful effect concentration, to beef cattle muscle cell processing 45min, 1.5h, 3h, 6h,
12h, for 24 hours, collects raji cell assay Raji cell Proliferation and redox state, and measuring method and index are with shown in (1), with determination
H2O2Effective acting time.
(3) various concentration H2O2Influence to beef cattle muscle cell form, proliferation and oxidative damage:
Fig. 1 is the results show that with H2O2Concentration by 0 μM improve to 450 μM, processing for 24 hours after, visual field inner cell quantity by
It gradually reduces, and form changes, the obvious phenomena of mortality occur for cell when to 450 μM, illustrate H2O2The growth of cell is caused to bear
Face is rung.
From Figure 2 it can be seen that with H2O2The raising of concentration, beef cattle muscle cell survival rate significantly reduce (P < 0.05), illustrate outer
Source H2O2Negative effect is caused to cell survival, works as H2O2When rising to 300 μM, cell survival rate is down to 60% or so, continues to mention
Mortality occurs for cell when high concentration, and when to 450 μM, survival rate is down to 6.69%.
In terms of antioxidase, SOD, CAT activity are in the changing rule (table 1, P < 0.05) for first increasing and reducing afterwards.SOD and
CAT is intracellular important antioxidase, and the height of enzyme activity can reflect the redox state of cell.The result shows that low dense
Spend H2O2The Antioxidative Defense System of cell is had activated, so that SOD and CAT vigor improves, but works as H2O2Concentration persistently increases, and surpasses
Out after cellular anti-oxidant capacity, cause oxidative damage, activities of antioxidant enzymes reduces, in terms of oxidation product, MDA and PC content with
H2O2 concentration increases significant raising, and 8-OHG content first increases reduces (table 2, P < 0.05) afterwards.MDA is lipid oxidation products, reflection
The height of lipid oxidation degree, PC can reflect the height of protein oxidation degree, and 8-OHG is the mark of nucleic acid oxidation product
Object, triplicity reflect the degree of cell itself oxidative damage.MDA, PC and 8-OHG content increase show cell lipid,
The oxidation level of protein and nucleic acid increases, and shows H2O2The raising of concentration causes cell oxidative damage, but the content of 8-OHG exists
300μM H2O2It is reduced after group, this and high concentration H2O2Cause cell death related；Comprehensive indices, determine with 300 μM
H2O2For the suitable concentration for inducing beef cattle muscle cell oxidative damage.
3 H of table2O2Influence of the different disposal time to beef cattle muscle cell activities of antioxidant enzymes:
(4)H2O2Influence of the different disposal time to beef cattle muscle cell proliferation and oxidative damage:
According to above-mentioned test result, select with 300 μM of H2O2For processing means, beef cattle muscle cell is acted on respectively
45min, 1.5h, 3h, 6h, 12h, for 24 hours, and be control with normal cell；The result shows that with the extension of processing time, cell
Survival rate is in first reducing the changing rule (Fig. 3, P < 0.05) gradually risen afterwards, and survival rate is down to 61.2% when 6h, until when 12h,
Survival rate is minimum (47.4%), and when extending to for 24 hours the time, survival rate starts to increase again, this may be with H2O2Had by cell disintegration
It closes.
In terms of antioxidase, SOD is consistent with the active changing rule of CAT, show as first increasing and reduce afterwards (table 3, P <
0.05), SOD starts to reduce after handling 6h, and CAT starts to reduce after handling 3h.In terms of oxidation product, MDA, PC and 8-OHG
Content extends with the processing time significantly improves (table 4, P < 0.05), and wherein MDA is significantly increased after handling 1.5h, and PC is being handled
It is significantly increased after 45min, 8-OHG is significantly increased after handling 6h.Comprehensive indices, beef cattle muscle cell Oxidative Damage In Vitro
Model is suitable for H2O2Concentration and action time are respectively 300 μM and 6h.
4 H of table2O2Influence of the different disposal time to beef cattle muscle cell oxidation product content:
The invention has the benefit that the present invention is using isolated beef cattle muscle cell as research object, using hydrogen peroxide
Beef cattle muscle cell model of oxidative is established, material is provided for the reduction of beef quality and nutrition regulation, reduces follow-up test
Research and development cost；The beef cattle muscle cell model of oxidative of foundation is anti-oxidant for the Regulation Mechanism research of beef quality and feed
The exploitation of active material provides research material, directly substantially reduces research and development cost and time using this model.
Detailed description of the invention
Fig. 1 is various concentration (0,50,100,150,200,250,300,350,400,450 μM) in the embodiment of the present invention
H2O2To the structural schematic diagram of beef cattle muscle cell morphology influence.
Fig. 2 is various concentration H in the embodiment of the present invention2O2The structural schematic diagram of influence to beef cattle muscle cell survival rate.
Fig. 3 is H in the embodiment of the present invention2O2The different disposal time structure of the influence to beef cattle muscle cell survival rate is shown
It is intended to.
Specific embodiment
In order to clarify the technical characteristics of the invention, being illustrated below by specific embodiment to this programme.
Referring to Fig. 1 to Fig. 3, the present invention is: a kind of beef cattle muscle cell model of oxidative construction method, including following step
It is rapid:
Step 1: cell culture fluid is prepared:
50-60 parts of (volume parts) fetal calf serums (FBS), 2-3 portions of (volume parts) mycillin mixed liquors are dissolved in
In 450-500 parts of (volume parts) cell culture fluids, by 0.22 micron membrane filter filtering after be dispensed into sterilizing centrifuge tube in, often
40-45 milliliters of pipe, 4 DEG C of preservations are placed, culture solution was placed in 37 DEG C of water-baths in 0.5-1 hours before cell culture and is preheated, obtained carefully
Born of the same parents' culture solution；
Step 2: separation beef cattle musculature:
Clip mature beef veutro musculature 10g-20g is rinsed 2-3 times with sterile saline, is placed on 100mL rapidly
In the sample bottle of the phosphate buffer (PBS) of the liquid of mycillin containing 1-2%, low-temperature transport after sealed membrane sealing, at 1-2 hours
Inside take back laboratory；Musculature is placed in sterilized petri dishes after taking out in sample bottle in sterile super-clean bench, utilizes body formula
Microscope rejects fascia and superabundant fats tissue, then rinses 3-5 times with PBS, later with eye scissors by musculature shred to
Chyle shape；
Step 3: culture beef cattle muscle cell:
Chyle even tissue is drawn with suction pipe and is placed on culture bottle bottom wall, afterwards turns over culture bottle and prepared cell training is added
Nutrient solution lays flat culture bottle in incubator, overturns culture bottle after 30-40 minutes, and culture solution is allowed slowly to submerge culture bottle bottom wall
Chyle tissue, and again by culture bottle put back to incubator continue culture 38-48 hours after cell adherent growth, have after 5-6 days
80%-90% cell is adherent；Obtain muscle cell；
Step 4: the acquisition of model of oxidative building parameter:
Cell is prepared after isolated adherent muscle cell is digested and poises liquid, and adjustment cell poises the cell concentration of liquid
It is 5 × 104A/mL's, cell is poised into liquid and is inoculated in 96 orifice plates, cultivates 24 hours in the incubator, is then changed to H2O2
(0,50,100,150,200,250,300,350,400,450 μM) culture solution is handled 24 hours, utilizes Cell Counting
Kit-8 kit detects cell survival rate, works as H2O2When rising to 300 μM, cell survival rate is down to 60% or so, continues to improve dense
It spends cell and mortality occurs；Separately by 5 × 104The cell of a/mL poises liquid and is inoculated in 25cm2In culture bottle, extremely to cell confluency
When 80%, it is changed to H2O2(0,50,100,150,200,250,300,350,400,450 μM) culture solution is handled 24 hours, is collected
Cell, detection superoxide dismutase (SOD), catalase (CAT), malonaldehyde (MDA), protein carbonyl group (PC) and 8- hydroxyl
Base deoxyguanosine (8-OHG), discovery SDO and CAT activity are first increased and are reduced afterwards, and MDA, PC and 8-OHG gradually rise；Obtain 300 μ
M H2O2For the useful effect concentration of model of oxidative.
Cell culture fluid described in step 1 is (DMEM/F12).
The adjustment cell concentration that cell described in step 4 poises liquid is 5 × 104A/mL.
300 μM of H of useful effect concentration of the model of oxidative obtained in step 42O2, with 300 μM of H2O2To beef cattle
Muscle cell handles 45min, 1.5h, 3h, 6h, 12h, for 24 hours, measures cell survival rate, SOD, CAT, MDA, PC and 8-OHG, with
The extension of processing time, cell survival rate gradually decreases, and handles 6 hours survival rates and is down to 61.2%, SOD and CAT activity first
It is reduced after raising, MDA, PC and 8-OHG gradually rise.
The suitable parameter that the model of oxidative of beef cattle muscle cell described in step 4 is established is 300 μM of H2O2Handle the time
For 6h.
The isolated adherent muscle cell establishes model of oxidative using this model parameter, on the basis of model
Different product can be studied to the regulating effect of beef cattle oxidative damage, this model is exactly to provide place mat for the exploitation of new product.
The exploitation that obtained beef cattle muscle cell model of oxidative is used for beef quality regulation product is provided to mechanism to grind
Study carefully material.
The in vitro beef cattle muscle cell, hydrogen peroxide (H2O2, Sigma company, 323381), (Gibco is public by DMEM/F12
Department, 8117245), fetal calf serum (BI company, 04-001-1A), mycillin mixed liquor (Beijing Suo Laibao Science and Technology Ltd,
P1400), pancreatin (Gibco company, 25200072), Cell Counting Kit-8 (CCK-8, MCE company), antioxidase examination
Agent box, superoxide dismutase (SOD, abcam company, ab65354), catalase (CAT, abcam company, ab83464),
Oxidation product kit: malonaldehyde (MDA, Jiangsu Kai Ji Biotechnology Ltd., KGT004-1), protein carbonyl group
(8-OHG, Cayman are public for (PC, Beijing Suo Laibao Science and Technology Ltd, BC1275), 8-OhdG Elisa kit
Department, 589320)；Incubator (Thermo company, model 371), super-clean bench (Su Jing is safe and sound, model SW-CJ-2F).
In order to preferably realize foregoing invention purpose, the present invention also provides the examinations of beef cattle muscle cell model of oxidative
Proved recipe method and testing index:
(1), various concentration H2O2Influence to beef cattle muscle cell proliferation and redox state:
In super-clean bench, cell is prepared with basic culture solution and poises liquid, adjustment cell concentration is 5 × 104A/mL.
Cell proliferative conditions detection: cell is poised into liquid and is inoculated in 96 orifice plates (100 hole μ L/), is cultivated in the incubator
For 24 hours, then it is changed to H2O2(0,50,100,150,200,250,300,350,400,450 μM) culture solution, each time point set 6
A repetition, processing are changed to basic culture solution afterwards for 24 hours, the CCK-8 solution of 10 μ L are added in every hole, culture plate is placed in 37
1-4h is incubated in DEG C incubator, with the absorbance at microplate reader measurement 450nm.
Cellular morphology and redox state detection: cell is poised into liquid and is inoculated in 25cm2Culture bottle, extremely to cell confluency
When 80%, it is changed to H2O2(0,50,100,150,200,250,300,350,400,450 μM) culture solution, each time point set 6
Culture plate is placed in 37 DEG C of incubators after handling for 24 hours by a repetition, then om observation cellular morphology collects group of cells, is surveyed
Determine antioxidase (SOD, CAT), oxidation product (MDA, PC, 8-OHG).
1 various concentration H of table2O2Influence to beef cattle muscle cell activities of antioxidant enzymes:
Note: colleague's data shoulder mark is without letter or has same letter to indicate that difference is not significant (P > 0.05), and different letters indicate
Significant difference (P < 0.05).Following table is same.
(2)H2O2Influence of the different disposal time to beef cattle muscle cell proliferation and redox state:
2 various concentration H of table2O2Influence to beef cattle muscle cell oxidation product content:
According to the H of above-mentioned determination2O2Useful effect concentration, to beef cattle muscle cell processing 45min, 1.5h, 3h, 6h,
12h, for 24 hours, collects raji cell assay Raji cell Proliferation and redox state, and measuring method and index are with shown in (1), with determination
H2O2Effective acting time.
(3) various concentration H2O2Influence to beef cattle muscle cell form, proliferation and oxidative damage:
Fig. 1 is the results show that with H2O2Concentration by 0 μM improve to 450 μM, processing for 24 hours after, visual field inner cell quantity by
It gradually reduces, and form changes, the obvious phenomena of mortality occur for cell when to 450 μM, illustrate H2O2The growth of cell is caused to bear
Face is rung.
From Figure 2 it can be seen that with H2O2The raising of concentration, beef cattle muscle cell survival rate significantly reduce (P < 0.05), illustrate outer
Source H2O2Negative effect is caused to cell survival, works as H2O2When rising to 300 μM, cell survival rate is down to 60% or so, continues to mention
Mortality occurs for cell when high concentration, and when to 450 μM, survival rate is down to 6.69%.
In terms of antioxidase, SOD, CAT activity are in the changing rule (table 1, P < 0.05) for first increasing and reducing afterwards.SOD and
CAT is intracellular important antioxidase, and the height of enzyme activity can reflect the redox state of cell.The result shows that low dense
Spend H2O2The Antioxidative Defense System of cell is had activated, so that SOD and CAT vigor improves, but works as H2O2Concentration persistently increases, and surpasses
Out after cellular anti-oxidant capacity, cause oxidative damage, activities of antioxidant enzymes reduces, in terms of oxidation product, MDA and PC content with
H2O2 concentration increases significant raising, and 8-OHG content first increases reduces (table 2, P < 0.05) afterwards.MDA is lipid oxidation products, reflection
The height of lipid oxidation degree, PC can reflect the height of protein oxidation degree, and 8-OHG is the mark of nucleic acid oxidation product
Object, triplicity reflect the degree of cell itself oxidative damage.MDA, PC and 8-OHG content increase show cell lipid,
The oxidation level of protein and nucleic acid increases, and shows H2O2The raising of concentration causes cell oxidative damage, but the content of 8-OHG exists
300μM H2O2It is reduced after group, this and high concentration H2O2Cause cell death related；Comprehensive indices, determine with 300 μM
H2O2For the suitable concentration for inducing beef cattle muscle cell oxidative damage.
3 H of table2O2Influence of the different disposal time to beef cattle muscle cell activities of antioxidant enzymes:
(4)H2O2Influence of the different disposal time to beef cattle muscle cell proliferation and oxidative damage:
According to above-mentioned test result, select with 300 μM of H2O2For processing means, beef cattle muscle cell is acted on respectively
45min, 1.5h, 3h, 6h, 12h, for 24 hours, and be control with normal cell；The result shows that with the extension of processing time, cell
Survival rate is in first reducing the changing rule (Fig. 3, P < 0.05) gradually risen afterwards, and survival rate is down to 61.2% when 6h, until when 12h,
Survival rate is minimum (47.4%), and when extending to for 24 hours the time, survival rate starts to increase again, this may be with H2O2Had by cell disintegration
It closes.
In terms of antioxidase, SOD is consistent with the active changing rule of CAT, show as first increasing and reduce afterwards (table 3, P <
0.05), SOD starts to reduce after handling 6h, and CAT starts to reduce after handling 3h.In terms of oxidation product, MDA, PC and 8-OHG
Content extends with the processing time significantly improves (table 4, P < 0.05), and wherein MDA is significantly increased after handling 1.5h, and PC is being handled
It is significantly increased after 45min, 8-OHG is significantly increased after handling 6h.Comprehensive indices, beef cattle muscle cell Oxidative Damage In Vitro
Model is suitable for H2O2Concentration and action time are respectively 300 μM and 6h.
4 H of table2O2Influence of the different disposal time to beef cattle muscle cell oxidation product content:
Technical characteristic of the present invention without description can realize that details are not described herein by or using the prior art, certainly,
The above description is not a limitation of the present invention, and the present invention is also not limited to the example above, the ordinary skill of the art
The variations, modifications, additions or substitutions that personnel are made within the essential scope of the present invention also should belong to protection model of the invention
It encloses.
Claims (8)
1. a kind of beef cattle muscle cell model of oxidative construction method, which comprises the following steps:
Step 1: cell culture fluid is prepared:
50-60 parts of (volume parts) fetal calf serums (FBS), 2-3 portions of (volume parts) mycillin mixed liquors are dissolved in 450-
In 500 parts of (volume parts) cell culture fluids, by 0.22 micron membrane filter filtering after be dispensed into sterilizing centrifuge tube in, every pipe
40-45 milliliters, 4 DEG C of preservations are placed, culture solution was placed in 37 DEG C of water-baths in 0.5-1 hours before cell culture and is preheated, obtain cell
Culture solution；
Step 2: separation beef cattle musculature:
Clip mature beef veutro musculature 10g-20g is rinsed 2-3 times with sterile saline, is placed on 100mL rapidly containing 1-
In the sample bottle of the phosphate buffer (PBS) of 2% mycillin liquid, low-temperature transport after sealed membrane sealing, the band in 1-2 hours
Go back to laboratory；Musculature is placed in sterilized petri dishes after taking out in sample bottle in sterile super-clean bench, it is micro- using body formula
Mirror rejects fascia and superabundant fats tissue, then is rinsed 3-5 times with PBS, is later shredded musculature to chyle with eye scissors
Shape；
Step 3: culture beef cattle muscle cell:
Chyle even tissue is drawn with suction pipe and is placed on culture bottle bottom wall, afterwards turns over culture bottle and prepared cell culture is added
Liquid lays flat culture bottle in incubator, overturns culture bottle after 30-40 minutes, and culture solution is allowed slowly to submerge culture bottle bottom wall
Chyle tissue, and culture bottle is put back into cell adherent growth after incubator continues culture 38-48 hours again, have after 5-6 days
80%-90% cell is adherent；Obtain muscle cell:
Step 4: the acquisition of model of oxidative building parameter:
Cell is prepared after isolated adherent muscle cell is digested and poises liquid, and the cell concentration that adjustment cell poises liquid is 5
×104Cell is poised liquid and is inoculated in 96 orifice plates by a/mL, is cultivated 24 hours in the incubator, is then changed to H2O2(0,
50,100,150,200,250,300,350,400,450 μM) culture solution processing 24 hours, utilize Cell Counting Kit-8
Kit detects cell survival rate, works as H2O2When rising to 300 μM, cell survival rate is down to 60% or so, continues to improve concentrations of cells
Mortality occurs；Separately by 5 × 104The cell of a/mL poises liquid and is inoculated in 25 cm2In culture bottle, to cell confluency to 80%
When, it is changed to H2O2(0,50,100,150,200,250,300,350,400,450 μM) culture solution is handled 24 hours, is collected thin
Born of the same parents, detection superoxide dismutase (SOD), catalase (CAT), malonaldehyde (MDA), protein carbonyl group (PC) and 8- hydroxyl
Deoxyguanosine (8-OHG), discovery SDO and CAT activity are first increased and are reduced afterwards, and MDA, PC and 8-OHG gradually rise；Obtain 300 μM
H2O2For the useful effect concentration of model of oxidative.
2. beef cattle muscle cell model of oxidative construction method according to claim 1, which is characterized in that in step 1
The cell culture fluid is (DMEM/F12).
3. beef cattle muscle cell model of oxidative construction method according to claim 1 or 2, which is characterized in that step
The adjustment cell concentration that cell described in four poises liquid is 5 × 104A/mL.
4. beef cattle muscle cell model of oxidative construction method according to claim 1-3, which is characterized in that
300 μM of H are used in step 42O245 min, 1.5 h, 3 h, 6 h, 12 h, 24 h are handled to beef cattle muscle cell, measurement is thin
Born of the same parents' survival rate, SOD, CAT, MDA, PC and 8-OHG, discovery are gradually decreased with the extension for handling the time, cell survival rate, are handled
6 hours survival rates are down to 61.2%, SOD and CAT activity first increases reduces afterwards, and MDA, PC and 8-OHG gradually rise；It is comprehensive each
Item index show that 300 μM of H2O2 action times are that 6 h are advisable.
5. beef cattle muscle cell model of oxidative construction method according to claim 1-4, which is characterized in that
300 μM of H of useful effect concentration of the model of oxidative obtained in step 42O2, action time is 6 h.
6. beef cattle muscle cell model of oxidative construction method according to claim 1-5, which is characterized in that
The suitable parameter that the model of oxidative of beef cattle muscle cell described in step 4 is established is 300 μM of H2O2The processing time is 6 h.
7. the application of beef cattle muscle cell model of oxidative as claimed in any one of claims 1 to 6, which is characterized in that described
Isolated adherent muscle cell establishes model of oxidative using this model parameter, and difference can be studied on the basis of model
For product to the regulating effect of beef cattle oxidative damage, this model is exactly to provide place mat for the exploitation of new product.
8. the application of beef cattle muscle cell model of oxidative according to claim 7, which is characterized in that the meat that will be obtained
Exploitation of the ox muscle cell model of oxidative for beef quality regulation product provides mechanism study material.
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| CN106167788A (en) * | 2016-07-15 | 2016-11-30 | 四川农业大学 | Laying hen magnum tubae uterinae epithelial cell is cultivated and oxidative stress method for establishing model |
| CN106479958A (en) * | 2016-10-17 | 2017-03-08 | 广西大学 | The method for building up of the pig spleen cell oxidative stress model of hydrogen peroxide-induced |
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