CN108865979A - Application of the chitosan selenium in terms of inhibiting pig endometrial epithelial cell ER beta gene expression reduction caused by F-2 toxin - Google Patents
Application of the chitosan selenium in terms of inhibiting pig endometrial epithelial cell ER beta gene expression reduction caused by F-2 toxin Download PDFInfo
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Abstract
The invention discloses application of the chitosan selenium in terms of inhibiting pig endometrial epithelial cell ER beta gene expression reduction caused by F-2 toxin.Experimental result is shown：For endometrial epithelial cell, the chitosan selenium of 0.8-3.0 μm of ol/L is used（Final concentration in culture solution, in terms of selenium）In F-2 toxin concentration within the scope of 10-30 μ g/mL, it can inhibit the reduction of pig endometrial epithelial cell ER beta gene expression caused by F-2 toxin, reached with this and weaken F-2 toxin to the toxic effect of the pig endometrial epithelial cell of in vitro culture, be chitosan selenium in terms of preventing and treating pig F-2 toxin poisoning using providing in vitro study model and theories integration.
Description
Technical field
The present invention relates to the cell engineerings of field of biotechnology, and in particular to chitosan selenium is inhibiting F-2 toxin institute
Pig endometrial epithelial cell ER beta gene expression is caused to reduce the application of aspect.
Background technique
F-2 toxin, also referred to as zearalenone are the mycotoxins generated by Fusarium, are resorcylic acid lactones
Compound, it is similar to estradiol structure, there is estrogenic activity, be widely present in the crops and animal feed of mildew,
It is one of the main fungal toxin of grain and food pollution.Domestic and international numerous studies prove, F-2 toxin has genotoxicity, thin
Cellular toxicity, liver renal toxicity, immunotoxicity and genetoxic.The feed of Long-term Feeding F-2 endotoxin contamination will lead to growth of animals or poultry
Performance decline, immunologic hypofunction and reproductive capability obstacle.
F-2 toxin is similar to estrin structure, competitive binding estrogen receptor（ER）, cause its configuration to change,
F-2 toxin in conjunction with ER after be transferred to nucleus, in conjunction with DNA profiling, to adjust the transcription of target gene and the conjunction of protein
At.ER has two hypotypes of ER α and ER β, their very high homologies, but N-terminal area different with the function in C-terminal area.F-2 toxin can
To activate ER α completely, but ER β cannot be activated completely；F-2 toxin can stimulate the transcription of ER α and ER β, activation ER α gene expression,
Inhibit ER beta gene expression.Therefore the height of ER beta gene expression amount can reflect that F-2 endotoxins on cells toxicity is made to a certain extent
Power.Studies have shown that F-2 toxin can lead to Mouse Leydig Cells, Mouse Uterus tissue, pig uterus and ovary tissue
And pig endometrial stromal cell ER β mrna expression significantly reduces, and embodies it to corresponding cell with this
Toxic effect.
Selenium is microelement necessary to animal and people.The anti-oxidant of selenium, raising immunity of organisms and premunition, adjust machine
Body metabolism, influences animal and the breeding function of people, antitumor, delays senescence, the multiple biological functions such as antagonism toxic element are
For well known to people.Chitosan be from low biological mushroom, algae cell, save branch animal shrimp crab insect shell in extract
Natural polymers, have anti-aging, anti-oxidant, anticancer, antirheumatic, antiviral and antitumor, enhancing immunity of organism living
The effects of property, Cell differentiation inducing activity.Chitosan selenium is the organic compound of selenium and chitosan, can play organic selenium simultaneously and shell is poly-
The resistance oxidation of sugar, protects cell membrane, inhibits peroxide, improves the effect that body resists oxidative damage；In production practice,
Chitosan selenium can also improve the production performance, immunity, oxidation resistance of livestock and poultry in various degree, reduce heavy metal to the storage of body
Product toxicity, can also resist tumour, induce cell apoptosis, hypoglycemic and blood lipid.
Currently, having not seen chitosan selenium in terms of alleviating F-2 toxin to endometrial epithelial cell toxic effect both at home and abroad
Patent or report, not seen in chitosan selenium inhibit F-2 toxin caused by pig endometrial epithelial cell ER beta gene expression reduce
The patent or report of aspect.
Summary of the invention
The invention discloses chitosan seleniums, and pig endometrial epithelial cell ER beta gene expression caused by F-2 toxin to be inhibited to drop
The application of low aspect.Experimental result is shown：For pig endometrial epithelial cell, the chitosan selenium of 0.8-3.0 μm of ol/L is used
（Final concentration in culture solution, in terms of selenium）, in F-2 toxin concentration within the scope of 10-30 μ g/mL, can inhibit F-2 toxin institute
It causes pig endometrial epithelial cell ER beta gene expression to reduce, is reached with this and weaken F-2 toxin to the pig endometrium of in vitro culture
The application that the toxic effect of epithelial cell is chitosan selenium in terms of preventing and treating pig F-2 toxin poisoning provides in vitro study
Model and theories integration.
The present invention further discloses chitosan seleniums to inhibit pig endometrial epithelial cell ER β gene caused by F-2 toxin
Express reduced method, it is characterised in that carry out by following step：
（1）The isolation and purification of pig endometrial cell:
Conventional separation pig endometrial cell, general purification pig endometrial epithelial cell.
（2）The culture of pig endometrial epithelial cell and packet transaction:
Endometrial epithelial cell after purification is diluted to 1 × 105A/mL, is inoculated in 6 orifice plates.Test can be divided into control
Group, F-2 toxin attack malicious group and chitosan selenium group.After cell inoculation 5-7d, culture solution is replaced：Control group and F-2 toxin attack malicious group
Replace Nostoc commune Vanch liquid；Chitosan selenium group replaces the culture solution containing chitosan selenium, makes 0.8-3.0 μm of ol/ of its selenium concentration
L；Continue to take out tissue culture plate after cultivating 12-36h, chitosan selenium group and F-2 toxin, which attack poison group addition F-2 toxin, keeps its dense
Degree is 10-30 μ g/mL, is continued after cultivating 3-12h, culture terminates；
（3）The change of qPCR reaction assay ER beta gene expression amount is carried out using quantitative PCR apparatus：
Cell total rna is extracted, conventional reverse transcription RNA is cDNA, and conventional design synthesizes the internal reference base of pig endometrial epithelial cell
The primer of cause and ER β gene determines reaction system and reaction condition, surveys on quantitative PCR apparatus to ER beta gene expression amount
It is fixed, it is embodied with this and weakens F-2 toxin to the toxic effect of the pig endometrial epithelial cell of in vitro culture.
Present invention mainly solves how using pig endometrial epithelial cell female two caused by chitosan selenium inhibition F-2 toxin
The method and application that alcohol acceptor gene ER beta gene expression amount changes；High spot reviews selenomethionine, selenide of carragheen, chitosan,
Astragalus polyose, Co-Q10, chitosan selenium etc. are able to suppress Apoptosis or there is the substance of protective effect to subtract cellular damage
In terms of weak F-2 toxin is to pig endometrial epithelial cell toxic effect, especially inhibit pig endometrial epithelium caused by F-2 toxin
Cell estradiol receptor gene ER beta gene expression amount reduces the function and effect of aspect；Main difficult point is, in above-mentioned substance
It determines to F-2 toxin to pig endometrial epithelial cell toxic effect（Especially estradiol receptor gene ER beta gene expression amount changes
Become）The best applications condition of substance and the substance with best protection effect；Various substances have successively been investigated thus
Weakening, F-2 toxin is thin to pig endometrial epithelium to the effect in terms of pig endometrial epithelial cell toxic effect, F-2 toxin
The influence of intracellular growth, attacks the conditions such as malicious time, the application dose of chitosan selenium, application time at the toxin dose for attacking poison；It is last true
Fixed scheme is：Pig endometrial epithelial cell culture 5-7 d, when in growth logarithmic phase, using the training containing chitosan selenium
Nutrient solution（0.8-3.0 μm of ol/L of final concentration in culture solution, in terms of selenium）Cell is protected；Continue to cultivate 12-36h,
After chitosan selenium plays one's part to the full, the F-2 toxin that 10-30 μ g/mL is added carries out attacking poison, attacks poison and carries out 3-12h, herein
Under the conditions of, chitosan selenium, which inhibits pig endometrial epithelial cell ER beta gene expression amount caused by F-2 toxin to reduce, has optimal work
Use effect.
Detailed description of the invention
Fig. 1 is influence of the chitosan selenium to endometrial epithelial cell ER β gene mRNA expression caused by F-2 toxin;
Note：Mark different capitalization（Or small letter）Letter indicates significant difference P>0.01（Or P<0.05）, similarly hereinafter.
Fig. 2 is influence of the chitosan selenium to endometrial epithelial cell ER β gene mRNA expression caused by F-2 toxin
Fig. 3 is influence of the chitosan selenium to endometrial epithelial cell ER β gene mRNA expression caused by F-2 toxin.
Specific embodiment
Illustrate the present invention below with reference to embodiment, the scheme of embodiment described here does not limit the present invention, this field it is special
Industry personnel spirit according to the invention can make improvements and change, and the such modifications and variations are regarded as at this
In the range of invention, the scope of the present invention and essence are defined by the claims.Wherein used various reagents are commercially available.
The purchase of DMEM/F12 cell culture fluid is bought in GIBCO company, F-2 toxin in Shanghai Yuan Ye Biotechnology Co., Ltd.Shell is poly-
Sugared selenium purchase is by TanJin Agricultural College Clinical Veterinary Medicine laboratory reference literature（Kong Tao, Qu Yunsheng, Zhu Lianqin wait chitosan selenium
Synthesis and its physicochemical property [J] Agriculture of Anhui science, 2007,35（1）：21）Method prepared, prepare raw material and buy in shell
Glycan purchase studies carefully institute purchased from Tianjin chemical reagent in Yi Long Biotechnology Co., Ltd, sodium selenite；The chitosan selenium of preparation
Middle Se content is 2.73 g/kg.
Embodiment 1
The isolation and purification of 1.1 pig endometrial cells
（1）The separation of endometrial cell
It is sterile to remove entire uterus immediately by after the ligation of uterus both ends after pig is butchered, adipose tissue is removed, with containing 200 IU
The normal saline flushing of dual anti-pre-cooling be placed on containing in dual anti-normal saline solution, sent as early as possible to laboratory.Use haemostatic clamp
Uterus both ends are clamped, uterus is taken out in superclean bench, is soaked in alcohol about 60s, PBS is rinsed after taking-up；Cut off uterus in longitudinal direction
（Close to cornua uteri position）, rinsed 3-4 times containing dual anti-PBS, no bloodstain, clip endometrium to 1 mm3What is controlled is granular, presses
0.5 cm every being homogeneously disposed in tissue culture plate, after tissue block is attached at tissue culture plate, adds a small amount of cell culture fluid between left and right,
CO2Enough culture solutions are added after 24 h of incubator culture.48h discards tissue block after cell is moved out, and collects cell suspension,
Low-speed centrifugal abandons supernatant, is resuspended, and stands 10 min, pipettes suspension centrifugation and abandons supernatant, collects bottom cell, culture solution is added to be made carefully
Born of the same parents' suspension.
（2）Cytoactive detection
The blue cellifugal activity of method detection point for refusing dye is expected using platform, and a small amount of cell suspension and trypan blue is taken to mix（Trypan blue
Final concentration 0.04%）, stand dyeing 3min.Microscopically observation cell dyeing situation（Dead cell is blue, and normal cell is refused
Dye）.Calculate the activity of cell.
Cell activity=viable count/（Viable count+dead cell number）*100%.
（3）Pig endometrial cell originally culture
1×105A cell/mL is inoculated in tissue culture plate, is placed in CO2Incubator changes liquid after 24 h, later according to the life of cell
Long situation replaces culture solution, the adherent situation of routine observation cell and upgrowth situation.
（4）The purifying of pig endometrial epithelial cell
Epithelial cell is strong compared with the adherent ability of stroma cell, purifies endometrium chrotoplast using differential digestion method.Cell is long extremely
Single layer trypsase（0.25%）Cell culture fluid termination is added immediately and disappears when stroma cell, which starts to shrink, to be rounded for digestion
To change, liquid-transfering gun, which is gently blown and beaten, makes stroma cell fall off, and epithelial cell is still adherent, discards culture solution, it cleans culture plate 2 times,
Suitable cell culture fluid is added to continue to cultivate, if still there is stroma cell mixed growth, the above-mentioned purification process of repetition obtains pure
Endometrial epithelial cell.
The culture of pig endometrial epithelial cell and packet transaction
Endometrial epithelial cell is diluted to 1 × 105A/mL is inoculated in 6 orifice plates, slowly repeatedly to blow and beat before being loaded fabric swatch.Examination
It tests and is divided into 4 groups, i.e., control C group, F-2 toxin attack malicious group（20 μ g/mL of F-2 toxin）, 1 group of chitosan selenium（1.5 μmol/LSe
20 μ g/mL of+F-2 toxin）With 2 groups of chitosan selenium（3 μm of 20 μ g/mL of ol/LSe+F-2 toxin）, every group of 6 repetitions.
After cell inoculation 5d, discards culture solution and clean 2 times, replace culture solution：Control group and F-2 toxin attack poison group replacement Nostoc commune Vanch
Liquid；1,2 group of replacement of chitosan selenium contains 1.5 μm of ol/L and 3 μm of ol/L（In terms of selenium）Chitosan selenium culture solution.Continue to cultivate
Tissue culture plate is taken out after 12h, 1 group of chitosan selenium, 2 groups of chitosan selenium and F-2 toxin group are added F-2 toxin, make its concentration
For 20 μ g/mL, to continue after cultivating 4 h, culture terminates.
Cell total rna extracts
Culture plate is taken out, culture solution is abandoned and is cleaned 3 times, endometrial epithelial cell is collected and is placed in no RNA enzyme pipe,（1）It is added 1
ML Trizol reagent sufficiently blows and beats 5 min of stand at low temperature, is centrifuged 12 000 r/min, 4oC, 10min, pipettes supernatant and be placed in
1.5 mL are without in the centrifuge tube of RNA enzyme.（2）Adding 200 μ L chloroforms, oscillation mixes, 12 000 r/min, 4oC, 10min are centrifuged,
Take its supernatant in another 1.5 mL without in RNA enzyme centrifuge tube.（3）500 μ L isopropanols are added, are centrifuged 12 000 r/min, 4oC,
10min abandons clear liquid,（4）400 μ L are added to enter 75% ethyl alcohol of pre-cooling, oscillation mixes, centrifugation 7500 r/min, 4oC, 5min, in abandoning
Clearly.It repeats（4）Dry 5 min in super-clean bench after 3 times, are added 25 μ L DEPC water in pipe, and RNA precipitate is dissolved in 60oC water-bath,
RNA concentration is measured using nucleic acid concentration analyzer.- 80oC refrigerator is placed in save backup.
Reverse transcription is at cDNA
1-1,1-2 are shown in sample reverse transcription
Table 1-1 sample RNA reverse transcription system
Reaction condition：10 min of 65oC, taking-up are placed on ice.
Table 1-2 sample RNA reverse transcription system
Reaction condition：37 oC 60min, 85 oC react 5min.Above-mentioned cDNA -20oC refrigerator is placed in after reverse transcription to protect
It deposits.
Design of primers
The primer of the DAPDH and ER β gene of pig endometrial epithelial cell, is designed by Primer 3, Shanghai JaRa bioengineering
Company's synthesis, is shown in Table 1-3.
Table 1-3 DAPDH and ER β primer sequence
1.6 qPCR reaction
Reaction system and reaction condition are shown in Table 1-4,1-5.
Table 1-4 qPCR reaction system
Table 1-5 qPCR amplification program
The measurement of 1.7 pig endometrial epithelial cell ER β gene mRNA expression amounts
The measurement result of pig endometrial epithelial cell ER β gene mRNA expression is shown in Fig. 1.
The result shows that the F-2 toxin of 20 μ g/mL can significantly reduce the table of ER β gene in pig endometrial epithelial cell
Up to amount, and 1.5 μm of ol/L（In terms of selenium）With 3.0 μm of ol/L（In terms of selenium）Chitosan selenium can be different degrees of raising F-
The reduction of ER beta gene expression amount in pig endometrial epithelial cell caused by 2 toxin.
Embodiment 2
The isolation and purification of 2.1 pig endometrial cells
（1）The separation of endometrial cell
It is sterile to remove entire uterus immediately by after the ligation of uterus both ends after pig is butchered, adipose tissue is removed, with containing 200 IU
The normal saline flushing of dual anti-pre-cooling be placed on containing in dual anti-normal saline solution, sent as early as possible to laboratory.Use haemostatic clamp
Uterus both ends are clamped, uterus is taken out in superclean bench, is soaked in alcohol about 60s, PBS is rinsed after taking-up；Cut off uterus in longitudinal direction
（Close to cornua uteri position）, rinsed 3-4 times containing dual anti-PBS, no bloodstain, clip endometrium to 1 mm3What is controlled is granular, presses
0.5 cm every being homogeneously disposed in tissue culture plate, after tissue block is attached at tissue culture plate, adds a small amount of cell culture fluid between left and right,
CO2Enough culture solutions are added after 24 h of incubator culture.48h discards tissue block after cell is moved out, and collects cell suspension,
Low-speed centrifugal abandons supernatant, is resuspended, and stands 10 min, pipettes suspension centrifugation and abandons supernatant, collects bottom cell, culture solution is added to be made carefully
Born of the same parents' suspension.
（2）Cytoactive detection
The blue cellifugal activity of method detection point for refusing dye is expected using platform, and a small amount of cell suspension and trypan blue is taken to mix（Trypan blue
Final concentration 0.04%）, stand dyeing 3min.Microscopically observation cell dyeing situation（Dead cell is blue, and normal cell is refused
Dye）.Calculate the activity of cell.
Cell activity=viable count/（Viable count+dead cell number）*100%.
（3）Pig endometrial cell originally culture
1×105A cell/mL is inoculated in tissue culture plate, is placed in CO2Incubator changes liquid after 24 h, later according to the life of cell
Long situation replaces culture solution, the adherent situation of routine observation cell and upgrowth situation.
（4）The purifying of pig endometrial epithelial cell
Epithelial cell is strong compared with the adherent ability of stroma cell, purifies endometrium chrotoplast using differential digestion method.Cell is long extremely
Single layer trypsase（0.25%）Cell culture fluid termination is added immediately and disappears when stroma cell, which starts to shrink, to be rounded for digestion
To change, liquid-transfering gun, which is gently blown and beaten, makes stroma cell fall off, and epithelial cell is still adherent, discards culture solution, it cleans culture plate 2 times,
Suitable cell culture fluid is added to continue to cultivate, if still there is stroma cell mixed growth, the above-mentioned purification process of repetition obtains pure
Endometrial epithelial cell.
The culture of pig endometrial epithelial cell and packet transaction
Endometrial epithelial cell is diluted to 1 × 105A/mL is inoculated in 6 orifice plates, slowly repeatedly to blow and beat before being loaded fabric swatch.Examination
It tests and is divided into 4 groups, is i.e. control C group, F-2 toxin group（30 μ g/mL of F-2 toxin）, chitosan selenium A group（1.25 μmol/LSe +
30 μ g/mL of F-2 toxin）With chitosan selenium B group（2.5 μm of 30 μ g/mL of ol/LSe+F-2 toxin）, every group of 4 repetitions.Carefully
Born of the same parents are inoculated with after 7d in growth logarithmic phase, discard culture solution and clean 2 times, replace culture solution：Control group and the replacement of F-2 toxin group
Nostoc commune Vanch liquid；Chitosan selenium A group and the replacement of B group contain 1.25 μm of ol/L and 2.5 μm of ol/L（In terms of selenium）Chitosan selenium
Culture solution.Continue culture and take out tissue culture plate afterwards for 24 hours, F- is added in chitosan selenium A group, chitosan selenium B group and F-2 toxin group
2 toxin make 30 μ g/mL of its concentration, continue after cultivating 3 h, culture terminates.
Cell total rna extracts
Culture plate is taken out, culture solution is abandoned and is cleaned 3 times, endometrial epithelial cell is collected and is placed in no RNA enzyme pipe,（1）It is added 1
ML Trizol reagent sufficiently blows and beats 5 min of stand at low temperature, is centrifuged 12 000 r/min, 4oC, 10min, pipettes supernatant and be placed in
1.5 mL are without in the centrifuge tube of RNA enzyme.（2）Adding 200 μ L chloroforms, oscillation mixes, 12 000 r/min, 4oC, 10min are centrifuged,
Take its supernatant in another 1.5 mL without in RNA enzyme centrifuge tube.（3）500 μ L isopropanols are added, are centrifuged 12 000 r/min, 4oC,
10min abandons clear liquid,（4）400 μ L are added to enter 75% ethyl alcohol of pre-cooling, oscillation mixes, centrifugation 7500 r/min, 4oC, 5min, in abandoning
Clearly.It repeats（4）Dry 5 min in super-clean bench after 3 times, are added 25 μ L DEPC water in pipe, and RNA precipitate is dissolved in 60oC water-bath,
RNA concentration is measured using nucleic acid concentration analyzer.- 80oC refrigerator is placed in save backup.
Reverse transcription is at cDNA
2-1,2-2 are shown in sample reverse transcription
Table 2-1 sample RNA reverse transcription system
Reaction condition：10 min of 65oC, taking-up are placed on ice.
Table 2-2 sample RNA reverse transcription system
Reaction condition：37 oC 60min, 85 oC react 5min.Above-mentioned cDNA -20oC refrigerator is placed in after reverse transcription to protect
It deposits.
Design of primers
The primer of the DAPDH and ER β gene of pig endometrial epithelial cell, is designed by Primer 3, Shanghai JaRa bioengineering
Company's synthesis, is shown in Table 2-3.
Table 2-3 DAPDH and ER β primer sequence
2.6 qPCR reaction
Reaction system and reaction condition are shown in Table 2-4,2-5.
Table 2-4 qPCR reaction system
Table 2-5 qPCR amplification program
The measurement of 2.7 pig endometrial epithelial cell ER β gene mRNA expression amounts
The measurement result of pig endometrial epithelial cell ER β gene mRNA expression amount is shown in Fig. 2.
The result shows that the F-2 toxin of 30 μ g/mL can be extremely significant reduce ER β gene in pig endometrial epithelial cell
Expression quantity, and 1.25 μm of ol/L（In terms of selenium）With 2.5 μm of ol/L（In terms of selenium）Chitosan selenium can be different degrees of
Increase the reduction of ER beta gene expression amount in pig endometrial epithelial cell caused by F-2 toxin.
Embodiment 3
The isolation and purification of 3.1 pig endometrial cells
（1）The separation of endometrial cell
It is sterile to remove entire uterus immediately by after the ligation of uterus both ends after pig is butchered, adipose tissue is removed, with containing 200 IU
The normal saline flushing of dual anti-pre-cooling be placed on containing in dual anti-normal saline solution, sent as early as possible to laboratory.Use haemostatic clamp
Uterus both ends are clamped, uterus is taken out in superclean bench, is soaked in alcohol about 60s, PBS is rinsed after taking-up；Cut off uterus in longitudinal direction
（Close to cornua uteri position）, rinsed 3-4 times containing dual anti-PBS, no bloodstain, clip endometrium to 1 mm3What is controlled is granular, presses
0.5 cm every being homogeneously disposed in tissue culture plate, after tissue block is attached at tissue culture plate, adds a small amount of cell culture fluid between left and right,
CO2Enough culture solutions are added after 24 h of incubator culture.48h discards tissue block after cell is moved out, and collects cell suspension,
Low-speed centrifugal abandons supernatant, is resuspended, and stands 10 min, pipettes suspension centrifugation and abandons supernatant, collects bottom cell, culture solution is added to be made carefully
Born of the same parents' suspension.
（2）Cytoactive detection
The blue cellifugal activity of method detection point for refusing dye is expected using platform, and a small amount of cell suspension and trypan blue is taken to mix（Trypan blue
Final concentration 0.04%）, stand dyeing 3min.Microscopically observation cell dyeing situation（Dead cell is blue, and normal cell is refused
Dye）.Calculate the activity of cell.
Cell activity=viable count/（Viable count+dead cell number）*100%.
（3）Pig endometrial cell originally culture
1×105A cell/mL is inoculated in tissue culture plate, is placed in CO2Incubator changes liquid after 24 h, later according to the life of cell
Long situation replaces culture solution, the adherent situation of routine observation cell and upgrowth situation.
（4）The purifying of pig endometrial epithelial cell
Epithelial cell is strong compared with the adherent ability of stroma cell, purifies endometrium chrotoplast using differential digestion method.Cell is long extremely
Single layer trypsase（0.25%）Cell culture fluid termination is added immediately and disappears when stroma cell, which starts to shrink, to be rounded for digestion
To change, liquid-transfering gun, which is gently blown and beaten, makes stroma cell fall off, and epithelial cell is still adherent, discards culture solution, it cleans culture plate 2 times,
Suitable cell culture fluid is added to continue to cultivate, if still there is stroma cell mixed growth, the above-mentioned purification process of repetition obtains pure
Endometrial epithelial cell.
The culture of pig endometrial epithelial cell and packet transaction
Endometrial epithelial cell is diluted to 1 × 105A/mL is inoculated in 6 orifice plates, slowly repeatedly to blow and beat before being loaded fabric swatch.Examination
It tests and is divided into 4 groups, is i.e. control C group, F-2 toxin group（10 μ g/mL of F-2 toxin）, I group of chitosan selenium（0.8 μm of ol/LSe+F-2 poison
10 μ g/mL of element）With II group of chitosan selenium（1.6 μm of 10 μ g/mL of ol/LSe+F-2 toxin）, every group of 4 repetitions.Cell inoculation
In growth logarithmic phase after 6d, discards culture solution and clean 2 times, replace culture solution, II group of I group of chitosan selenium and chitosan selenium are more
It changes containing 0.8 μm of ol/L and 1.6 μm of ol/L（In terms of selenium）Chitosan selenium culture solution.Continue to take out cell culture after cultivating 36h
F-2 toxin is added in plate, I group of chitosan selenium, II group of chitosan selenium and F-2 toxin group, and making its concentration is 10 μ g/mL, continues
After cultivating 12 h, culture terminates.
Cell total rna extracts
Culture plate is taken out, culture solution is abandoned and is cleaned 3 times, endometrial epithelial cell is collected and is placed in no RNA enzyme pipe,（1）It is added 1
ML Trizol reagent sufficiently blows and beats 5 min of stand at low temperature, is centrifuged 12 000 r/min, 4oC, 10min, pipettes supernatant and be placed in
1.5 mL are without in the centrifuge tube of RNA enzyme.（2）Adding 200 μ L chloroforms, oscillation mixes, 12 000 r/min, 4oC, 10min are centrifuged,
Take its supernatant in another 1.5 mL without in RNA enzyme centrifuge tube.（3）500 μ L isopropanols are added, are centrifuged 12 000 r/min, 4oC,
10min abandons clear liquid,（4）400 μ L are added to enter 75% ethyl alcohol of pre-cooling, oscillation mixes, centrifugation 7500 r/min, 4oC, 5min, in abandoning
Clearly.It repeats（4）Dry 5 min in super-clean bench after 3 times, are added 25 μ L DEPC water in pipe, and RNA precipitate is dissolved in 60oC water-bath,
RNA concentration is measured using nucleic acid concentration analyzer.- 80oC refrigerator is placed in save backup.
Reverse transcription is at cDNA
3-1,3-2 are shown in sample reverse transcription
Table 3-1 sample RNA reverse transcription system
Reaction condition：10 min of 65oC, taking-up are placed on ice.
Table 3-2 sample RNA reverse transcription system
Reaction condition：37 oC 60min, 85 oC react 5min.Above-mentioned cDNA -20oC refrigerator is placed in after reverse transcription to protect
It deposits.
Design of primers
The primer of the DAPDH and ER β gene of pig endometrial epithelial cell, is designed by Primer 3, Shanghai JaRa bioengineering
Company's synthesis, is shown in Table 3-3.
Table 3-3 DAPDH and ER β primer sequence
3.6 qPCR reaction
Reaction system and reaction condition are shown in Table 3-4,3-5.
Table 3-4 qPCR reaction system
Table 3-5 qPCR amplification program
The measurement of 3.7 pig endometrial epithelial cell ER β gene mRNA expression amounts
The measurement result of pig endometrial epithelial cell ER β gene mRNA expression is shown in Fig. 3.
The result shows that the F-2 toxin of 10 μ g/mL can significantly reduce the table of ER β gene in pig endometrial epithelial cell
Up to amount, and 0.8 μm of ol/L（In terms of selenium）With 1.6 μm of ol/L（In terms of selenium）Chitosan selenium can be different degrees of raising F-2
The reduction of ER beta gene expression amount in pig endometrial epithelial cell caused by toxin.
SEQUENCE LISTING
<110>TanJin Agricultural College
<120>Chitosan selenium is in terms of inhibiting pig endometrial epithelial cell ER beta gene expression reduction caused by F-2 toxin
Using
<160> 4
<170> PatentIn version 3.5
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<211> 25
<212> DNA
<213>Artificial sequence
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<212> DNA
<213>Artificial sequence
<400> 2
caggaggcat tgctgacaat cttg 24
<210> 3
<211> 20
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<213>Artificial sequence
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gctgaatgcc gtgactgatg 20
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Claims (2)
1. chitosan selenium answering in terms of inhibiting caused pig endometrial epithelial cell ER beta gene expression reduction caused by F-2 toxin
With.
2. chitosan selenium described in claim 1 is inhibiting the reduction of pig endometrial epithelial cell ER beta gene expression caused by F-2 toxin
Method, it is characterised in that carried out by following step：
（1）The isolation and purification of pig endometrial cell:
Conventional separation pig endometrial cell, general purification pig endometrial epithelial cell；
（2）The culture of pig endometrial epithelial cell and packet transaction:
Endometrial epithelial cell after purification is diluted to 1 × 105A/mL, is inoculated in 6 orifice plates, and test can be divided into control
Group, F-2 toxin attack malicious group and chitosan selenium group；After cell inoculation 5-7d, culture solution is replaced：Control group and F-2 toxin attack malicious group
Replace Nostoc commune Vanch liquid；Chitosan selenium group replaces the culture solution containing chitosan selenium, makes 0.8-3.0 μm of ol/ of its selenium concentration
L；Continue to take out tissue culture plate after cultivating 12-36h, chitosan selenium group and F-2 toxin, which attack poison group addition F-2 toxin, keeps its dense
Degree is 10-30 μ g/mL, is continued after cultivating 3-12h, culture terminates；
（3）The change of qPCR reaction assay ER beta gene expression amount is carried out using quantitative PCR apparatus：
Cell total rna is extracted, conventional reverse transcription RNA is cDNA, and conventional design synthesizes the internal reference base of pig endometrial epithelial cell
The primer of cause and ER β gene determines reaction system and reaction condition, surveys on quantitative PCR apparatus to ER beta gene expression amount
It is fixed.
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| CN110951671A (en) * | 2019-09-30 | 2020-04-03 | 天津农学院 | Application of chitosan selenium in reducing pig endometrial epithelial cell CASP3 gene expression increase caused by F-2 toxin |
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| CN110951671A (en) * | 2019-09-30 | 2020-04-03 | 天津农学院 | Application of chitosan selenium in reducing pig endometrial epithelial cell CASP3 gene expression increase caused by F-2 toxin |
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