CN106167788B - Laying hen magnum tubae uterinae epithelial cell culture and oxidative stress method for establishing model - Google Patents
Laying hen magnum tubae uterinae epithelial cell culture and oxidative stress method for establishing model Download PDFInfo
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- G01N2800/7009—Oxidative stress
Abstract
The invention discloses a kind of laying hen magnum tubae uterinae epithelial cell culture and oxidative stress method for establishing model, the cell culture processes are as follows: takes whole section of fallopian tubal of laying hen to be soaked in PBS, and removes mesentery, connective tissue and blood；The bulb of fallopian tubal is cut, tissue block is shredded into after cleaning；Tissue block after cleaning is transferred in clostridiopetidase A IV and is digested；Digestive juice is filtered, filtrate centrifugation abandons supernatant, complete medium is added, cell is resuspended, finally cell is transferred in culture bottle and is cultivated.The oxidative stress method for establishing model are as follows: test the cell viability under the heavy metal and different action time of various concentration, determine action time point；With the different heavy metal concentration gradient of this time point design, situations such as measuring Apoptosis, oxidative stress model is established.The present invention is the specific mechanism that external environment causes egg white quality decline of further probing into, it is established that laying hen magnum tubae uterinae epithelial cell oxidative stress model.
Description
Technical field
The invention belongs to cell toxicity technical fields, specifically, being related to a kind of laying hen magnum tubae uterinae epithelial cell
Cultural method, further relate to a kind of method for building up of laying hen magnum tubae uterinae epithelial cell oxidative stress model.
Background technique
Main source of the egg as necessary for human protein.The change of egg quality may ask safely along with egg
Topic.With the improvement of living standards, people increasingly pay attention to the Quality Safety problem of egg, and the fast development of modern industry, add
The acute pollution of environment, the safety of mankind's conventional food such as egg is on the hazard therewith.This is not only even entire to laying hen industry
Animal husbandry has havoc, also generates certain influence to human health.Egg is other than yolk, remaining is at fallopian tubal position
It is formed.Laying hen fallopian tubal is divided into pars infundibularis, bulb, isthmus, uterus and portio vaginalis, and wherein pars infundibularis conveys yolk, bulb
Egg white is formed, isthmus forms egg shell membrane, and uterus forms eggshell, and portio vaginalis conveys egg to cloaca, finally excretes.Wherein
Egg white is mainly synthesized and is stored in the endoplasmic reticulum by magnum tubae uterinae epithelial cell, and is secreted into official jargon, is wrapped in outside yolk
Face forms albumen layer.Magnum tubae uterinae epithelial cell is distributed in bulb mucous membrane fold.The security quality of egg, mainly
Comprising egg size, eggshell strength, thickness, albumen height breathes out not unit etc., and wherein egg white quality is influenced vulnerable to external environment, mainly
Albumen height reduction is shown as, egg white solution is thinning, breathes out not unit and declines.And egg white is mainly by magnum tubae uterinae epithelial cell
It synthesizes and stores in endoplasmic reticulum, and be secreted into official jargon, be wrapped in outside yolk, form albumen layer.Magnum tubae uterinae epithelium is thin
Born of the same parents are distributed in bulb mucous membrane fold.Currently, laying hen is easily affected by the external environment in laying hen industry production, in feed
Mycotoxin (aflatoxin, fusarium toxin), heavy metal (vanadium, copper, cadmium) and feeding environment deteriorate (temperature in giving up,
Environmental stress) etc., it may all be influenced in albumen forming process by influencing the form (crimp height) of magnum tubae uterinae
Movement, and influence to determine point of ovomucin (Ovomucin, OVW) in egg white quality (albumen height and HU) dense albumen
It secretes, so as to cause the reduction of albumen quality.Oxidative stress cannot be removed, be made in time due to generating excessive free radical in vivo
It is unbalance at body oxidative and anti-oxidative.Many researchs also turn out that this may be to make since laying hen body is by external environmental stimulus
Laying hen magnum tubae uterinae epithelial cell generates oxidative stress, in turn results in the disorder of structure and function, influences the egg white of secretion,
But this research is only limited on laying hen living body, and vanadium is added by way of diet or drinking-water, and whether vanadium can reach defeated ovum
Pipe bulb, and its effective dose of influence fallopian tubal structure and function also lack unified conclusion, and laying hen fallopian tubal expands
The originally culture of portion's epithelial cell is rarely reported both at home and abroad, and isolation and culture of cell method is immature, the cell of culture vulnerable to
Fibroblast pollutes, and not identified success mostly, therefore the direct modeling of its mechanism is proved on Epithelium Cells
Evidence seems very necessary.
Meanwhile the foundation of the model of oxidative stress, and research need the powerful measure of Other diseases mechanism.Many diseases
Related with oxidative stress, obese patient's vivo oxidation stress level can increase；Generating excessive oxygen radical (ROS) in vivo can be straight
Impaired isle element β cell is connect, diabetes are caused；Blood vessel generates excessive ROS and causes low-density lipoprotein (LDL) in endarterium
Deposition, cause atherosclerosis；With age, antioxidant ability of organism decrease causes alzheimer's disease etc..Mesh
Before, have through many precedents added the modes such as heavy metals, hydrogen peroxide and establish oxidative stress model, but vanadium, change
Price is closed between -1 to+5 valences, is the inhibitor of a kind of strong oxidizer and many phosphokinases, is medically also used for pancreas
The substitute of island element, but research in recent years gradually finds its toxicity to liver, lung cell, therefore one is established with vanadium
Oxidative stress model and very necessary.
Therefore, in view of the foregoing, in order to further probe into the specific mechanism that oxidative stress causes egg white quality decline, have
Necessity establishes the external oxidative stress model of laying hen magnum tubae uterinae epithelial cell.
Summary of the invention
In view of this, the present invention is directed to the laying hen magnum tubae uterinae epithelial cell of existing method culture vulnerable at fiber finer
The problem of born of the same parents pollute, and in order to further probe into the specific mechanism that external environment causes egg white quality decline, provide a hatching egg
Chicken salpingo bulb epithelial cell culture and oxidative stress method for establishing model.
In order to solve the above-mentioned technical problem, the invention discloses a kind of culture sides of laying hen magnum tubae uterinae epithelial cell
Method, specifically includes the following steps:
Step 1, after laying hen being put to death, aseptically take out whole section of fallopian tubal and be soaked in PBS, and remove mesentery,
Connective tissue and blood；
Step 2, the bulb of fallopian tubal is cut, shreds into tissue block after being cleaned with PBS, and with PBS cleansing tissue block；
Step 3, the tissue block after cleaning is transferred in clostridiopetidase A IV, in 37 DEG C of digestion certain times；
Step 4, postdigestive digestive juice is filtered, filtrate is centrifuged, and supernatant is abandoned after centrifugation and obtains cell, into cell
Complete medium is added, cell is resuspended, finally cell is transferred in culture bottle, is placed in the titanium dioxide that volumetric concentration is 5%
Carbon is cultivated under conditions of 37 DEG C.
Further, further includes: after cultivating 2.5~3h, using difference adherent method, the liquid in culture bottle is transferred to
In new culture bottle, this operation 1~2 time is repeated.
Further, the concentration of the clostridiopetidase A IV is 1mg/ml；The time of the digestion is 70min.
Further, the size of the tissue block is 1mm3；The revolving speed of the centrifugation is 700rpm, time 20min；Institute
State the time preferred 72h of culture.
Further, the complete medium composition are as follows: 91% DMEM/F12 basal medium, 5% fetal calf serum,
2% chicken serum, 1% nonessential amino acid, 5ug/ml insulin, the heparin of 20IU/ml, the L-Glutamine of 2mM, 15ng/ml
Epidermal growth factor, 1% mycillin.
The invention also discloses a kind of method for building up of laying hen magnum tubae uterinae epithelial cell oxidative stress model, specifically
The following steps are included:
Step 1, laying hen magnum tubae uterinae epithelial cell is cultivated；
Step 1.1, it after laying hen being put to death, aseptically takes out whole section of fallopian tubal and is soaked in PBS, and remove and be
Film, connective tissue and blood；
Step 1.2, the bulb of fallopian tubal is cut, shreds into tissue block after being cleaned with PBS, and with PBS cleansing tissue
Block；
Step 1.3, the tissue block after cleaning is transferred in clostridiopetidase A IV, in 37 DEG C of digestion certain times；
Step 1.4, postdigestive digestive juice is filtered, filtrate is centrifuged, and supernatant is abandoned after centrifugation and obtains cell, to cell
Middle addition complete medium, is resuspended cell, finally cell is transferred in culture bottle, is placed in the dioxy that volumetric concentration is 5%
Change carbon, cultivate under conditions of 37 DEG C.
Step 2, using the laying hen magnum tubae uterinae epithelial cell of culture, various concentration is tested by mtt assay respectively
Cell viability under heavy metal and different action time determines heavy metal action time point according to test result；
Step 3, at the time point determined with step 2, different heavy metal concentration gradients is designed, it is measurement Apoptosis, intracellular
The generation of active oxygen, cellular superoxide dismutase, lactic dehydrogenase release conditions, choose most suitable heavy metal concentration,
To establish cellular oxidation Stress model.
Further, the step 1 further include:, will be in culture bottle using difference adherent method after cultivating 2.5~3h
Liquid is transferred in new culture bottle, repeats this operation 1~2 time.
Further, the concentration of the clostridiopetidase A IV is 1mg/ml；The time of the digestion is 70min.
Further, the size of the tissue block is 1mm3；The revolving speed of the centrifugation is 700rpm, time 20min；Institute
State the time preferred 72h of culture.
Further, the complete medium composition are as follows: 91% DMEM/F12 basal medium, 5% fetal calf serum,
2% chicken serum, 1% nonessential amino acid, 5ug/ml insulin, the heparin of 20IU/ml, the L-Glutamine of 2mM, 15ng/ml
Epidermal growth factor, 1% mycillin.
Compared with prior art, the present invention can be obtained including following technical effect:
(1) present invention passes through culture laying hen magnum tubae uterinae epithelial cell；Then test various concentration heavy metal and
Cell viability under different action time determines action time point；The heavy metal concentration ladder different with this time point design again
Situations such as degree, measurement Apoptosis, to establish cellular oxidation Stress model, cause egg white product further to probe into external environment
The specific mechanism of matter decline；
(2) present invention establishes bulb epithelial cell oxidative stress by culture laying hen magnum tubae uterinae epithelial cell
Model, the specific mechanism for research egg white quality decline later provide basis, reduce laying hen industry since laying hen autoxidation is answered
Swash loss caused by causing Egg Quality decline；
(3) laying hen magnum tubae uterinae epithelial cell cultural method of the invention is scientific and reasonable, overcomes existing method training
The problem of feeding laying hen magnum tubae uterinae epithelial cell is polluted vulnerable to fibroblast, primitive cell culture success, cell are pure
Degree reaches 95% or more, and has been successfully established laying hen magnum tubae uterinae epithelial cell oxidative stress model.
Certainly, it implements any of the products of the present invention it is not absolutely required to while reaching all the above technical effect.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes a part of the invention, this hair
Bright illustrative embodiments and their description are used to explain the present invention, and are not constituted improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is laying hen magnum tubae uterinae epithelial cell picture of the embodiment of the present invention, wherein (a) and (b) is that cell exists
After 72h under fluorescence inverted microscope (200 ×) picture；
Fig. 2 is laying hen magnum tubae uterinae epithelial cell immunofluorescence dyeing picture of the embodiment of the present invention, wherein (a) is anti-
CK18 picture (b) is anti-ESR1 picture, (c) is anti ova picture, (d) is anti-PGR picture, (e) is anti-Vimentin picture；
Fig. 3 be the embodiment of the present invention under different time cell viability with vanadium concentration change curve；
Fig. 4 is that cell picture after 12h is handled under difference vanadium concentration of the embodiment of the present invention, wherein 0 μm of ol/ml of (a) vanadium concentration,
(b) 25 μm of ol/ml of vanadium concentration, (c) 50 μm of ol/ml of vanadium concentration, (d) 100 μm of ol/ml of vanadium concentration, (e) 250 μm of ol/ of vanadium concentration
Ml, (f) 1000 μm of ol/ml of vanadium concentration；
The apoptosis figure of cell under the different vanadium concentration that Fig. 5, which is the embodiment of the present invention, to be tested using flow cytometer,
In 0 μm of ol/ml of (a) vanadium concentration, (b) 25 μm of ol/ml of vanadium concentration, (c) 50 μm of ol/ml of vanadium concentration, (d) 100 μm of ol/ of vanadium concentration
Ml, (e) 250 μm of ol/ml of vanadium concentration, (f) 1000 μm of ol/ml of vanadium concentration；
Fig. 6 is the apoptosis rate figure of cell under difference vanadium concentration of the embodiment of the present invention；
Intracellular FITC fluorescence is strong under the different vanadium concentration that Fig. 7, which is the embodiment of the present invention, to be tested using flow cytometer
Degree figure；
Fig. 8 is intracellular FITC mean value of fluorescence intensity figure under difference vanadium concentration of the embodiment of the present invention；
Fig. 9 is cellular superoxide dismutase figure under difference vanadium concentration of the embodiment of the present invention；
Figure 10 is cytolipin peroxide figure under difference vanadium concentration of the embodiment of the present invention；
Figure 11 is the activity figure of cellular superoxide hydrogen enzyme under difference vanadium concentration of the embodiment of the present invention；
Figure 12 is cell lactic dehydrogenase enzyme r e lease figure under difference vanadium concentration of the embodiment of the present invention.
Specific embodiment
Carry out the embodiment that the present invention will be described in detail below in conjunction with embodiment, whereby to the present invention how application technology hand
Section solves technical problem and reaches the realization process of technical effect to fully understand and implement.
The cultural method of laying hen magnum tubae uterinae epithelial cell of the present invention, uses Roman egghen for experimental animal, specifically
The following steps are included:
Step 1, it after laying hen being put to death, aseptically takes out whole section of fallopian tubal and is soaked in PBS, then pacify in biology
Mesentery, connective tissue and blood are removed in full cabinet；
Step 2, the position of expanding of fallopian tubal is cut, the phosphate buffer solution (PBS) (Hyclone) preheated with 37 DEG C
Twice of inside and outside cleaning, then shreds bulb to 1mm with eye scissors3The tissue block of left and right, the PBS during which preheated with 37 DEG C are cleaned
Tissue block, until the liquid after cleaning becomes clarification；
Step 3, the tissue block after cleaning is transferred in the clostridiopetidase A IV of 1mg/ml, is placed in 37 DEG C of water-bath digestion 70min；
Selection this time be because largely epithelial cell can be digested, overlong time may digest get off at fiber
Cell is relatively more.
Wherein, clostridiopetidase A IV (Sigma) forms individual cells for digesting aim cell from tissue.Selection
The clostridiopetidase A IV of 1mg/ml, be due under this concentration enzyme effect, in the case where can be by under cell dissociation, to cellular damage compared with
It is small, it digests the cell to get off and is easier to adherent and growth.
Step 4, filtrate 700r is centrifuged 20min, abandons supernatant by postdigestive digestive juice 40nm nylon filter net filtration
Cell is obtained, complete medium is added into cell, gently cell is resuspended in piping and druming, and finally cell is transferred in culture bottle, is set
In volumetric concentration be 5% carbon dioxide, cultivate 72h under the conditions of 37 DEG C, cell picture is as shown in Figure 1.
Cell can be further purified in unicellular removal by 700r centrifugation 20min.
After cell culture 72h, it can be paved with 80% or more bottom, cell viability is stronger at this time, can be used as at latter step test
Reason.
Further, the complete medium composition are as follows: 91% DMEM/F12 basal medium (Hyclone), 5% tire
Cow's serum (Gibco), 2% chicken serum (Gibco), 1% nonessential amino acid (Sigma), 5ug/ml insulin (Gibco),
The heparin of 20IU/ml, the L-Glutamine (Ameresco) of 2mM, 15ng/ml epidermal growth factor (Peprotech), 1% is green
Streptomysin (Hyclone).
Wherein, addition nonessential amino acid can either promote cultivate cell growth, and can extend culture cell survival when
Between, and reduce the external biological resulting load of cell；The fetal calf serum (5%) for selecting low concentration, can inhibit into a certain degree
Fibroblast growth；Insulin and epidermal growth factor can promote the adherent of epithelial cell；Heparin can inhibit fibroblastic
Proliferation；L-Glutamine is the important raw material of cell nucleic acid and protein, is conducive to the proliferation of cell.
Further, adherent using difference after cultivating 2.5~3h in order to be further reduced fibroblastic pollution
Liquid in culture bottle is transferred in new culture bottle by method, repeats this operation 1~2 time, aim cell can reach 95% or more
Purity.
The method for building up of laying hen magnum tubae uterinae epithelial cell oxidative stress model of the present invention, comprising the following steps:
Step 1, laying hen magnum tubae uterinae epithelial cell is cultivated；
Step 1.1, it after laying hen being put to death, aseptically takes out whole section of fallopian tubal and is soaked in PBS, then in biology
Mesentery, connective tissue and blood are removed in safety cabinet；
Step 1.2, the position of expanding of fallopian tubal is cut, the phosphate buffer solution (PBS) preheated with 37 DEG C
(Hyclone) twice of inside and outside cleaning, then shreds bulb to 1mm with eye scissors3During which the tissue block of left and right is preheated with 37 DEG C
PBS cleansing tissue block, until cleaning after liquid become clarification；
Step 1.3, the tissue block after cleaning is transferred in the clostridiopetidase A IV of 1mg/ml, is placed in 37 DEG C of water-bath digestion
70min；
Wherein, clostridiopetidase A IV (Sigma) forms individual cells for digesting aim cell from tissue.
Step 1.4, filtrate 700r is centrifuged 20min, abandons supernatant by postdigestive digestive juice 40nm nylon filter net filtration
Liquid obtains cell, and complete medium is added into cell, and gently cell is resuspended in piping and druming, and finally cell is transferred in culture bottle,
It is placed in carbon dioxide that volumetric concentration is 5%, cultivates 72h under the conditions of 37 DEG C, cell picture is as shown in Figure 1；
Further, the complete medium composition are as follows: 91% DMEM/F12 basal medium (Hyclone), 5% tire
Cow's serum (Gibco), 2% chicken serum (Gibco), 1% nonessential amino acid (Sigma), 5ug/ml insulin (Gibco),
The heparin of 20IU/ml, the L-Glutamine (Ameresco) of 2mM, 15ng/ml epidermal growth factor (Peprotech), 1% is green
Streptomysin (Hyclone).
Further, adherent using difference after cultivating 2.5~3h in order to be further reduced fibroblastic pollution
Liquid in culture bottle is transferred in new culture bottle by method, repeats this operation 1~2 time, aim cell can reach 95% or more
Purity.
Step 2, using the laying hen magnum tubae uterinae epithelial cell of culture, various concentration is tested by mtt assay respectively
Cell viability under heavy metal and different action time determines heavy metal action time point according to test result；
The heavy metal forms compound with different valent state, and high-valence state metal is to lower valency, such as iron, copper, chromium, vanadium
ROS is generated by redox, causes oxidative stress.Vanadium is microelement necessary to animal growth, common chemical combination
Valence has+2 ,+3 ,+4 and+5, and the high-valence state vanadium of+5 valences has strong oxidisability as a kind of reducing agent, easily generates cell
Oxidative stress.It is therefore preferable that using the heavy metal vanadium of+5 valences.
Step 3, at the time point determined with step 2, different heavy metal vanadium concentration gradients is designed, is surveyed using flow cytometer
Determine the generation of Apoptosis and intracellular reactive oxygen species generation (ROS), cellular superoxide dismutase (SOD), lipid peroxide (MDA),
The activity of catalase (CAT) and the release conditions of lactic dehydrogenase (LDH), choose most suitable heavy metal vanadium concentration, from
And establish cellular oxidation Stress model.
Illustrate beneficial effects of the present invention below with reference to specific experiment:
1, by cellular immunofluorescence technology, identify that cultivated laying hen magnum tubae uterinae epithelial cell and cell are pure
Degree:
(1) test method: by magnum tubae uterinae epithelial cell with 1 × 105Density is inoculated in 6 orifice plates, to cell density
It is cleaned cell 3 times with PBS first when reaching 70%, then fixes 10min, PBS cleaning with the 4% paraformaldehyde room temperature of 1000ul
Cell 3 times, each 5min；0.5% triton X-100 (TritonX-100) that 1000ul is added is placed at room temperature for 20min, PBS
Cleaning cell 3 times, each 5min；The 1% lowlenthal serum albumin confining liquid of 1000ul is added, room temperature closes 1h.Abandon closing
Liquid, respectively be added rabbit-anti cytokeratin -18 (CK-18, Boster), rabbit-anti vimentin (Anti-Vimentin, LSBio),
Antiovalbumin (ovalbumin, OVA, Acis), rabbit-anti estrogen receptor 1 (ESR1, NOVUS) and mouse antiprogestin receptor (PGR,
NOVUS) in 5 different holes, 4 DEG C are incubated overnight 600ul, and PBS is cleaned cell 3 times, each 5min；600ul is added
The goat anti-rabbit igg (CST) of 488nm label and the goat anti-rabbit igg (CST) of 555nm label, 37 DEG C are protected from light incubation 1h, and PBS is clear
Cell 3 times are washed, each 5min.The 4' of 500ul, 6- diamidino -2-phenylindone (DAPI) is added, room temperature redyes 10min, PBS
It washes 6 times, the anti-quencher of 50% glycerol of 1000ul is added, room temperature is protected from light observation coloration result under inverted fluorescence microscope.
(2) test result: being shown by immunofluorescence, and referring to fig. 2, cell anti-CK18, OVA, PGR and ESR1 are in sun
Property, and anti-vimentin is negative, and shows that the cell of culture is laying hen magnum tubae uterinae epithelial cell, and purity reaches 95%
More than.
2, it using the laying hen magnum tubae uterinae epithelial cell of culture, is designed, is divided at 30 using 5 × 6 factorial experiments
Reason, designs 6 vanadium concentration gradients (0,25,50,100,250,1000 μm of ol/ml) and 5 time points (1,4,12,24,48h),
Cell viability is surveyed by mtt assay.
Test result: as shown in figure 3, in 12h, under the vanadium concentration of 50 μm of ol/L and 100 μm of ol/L, cell viability is 70%
Or so, illustrate to produce certain influence to cell, the vanadium concentration versus cell damage of other times is too small or reaches irreversible damage
Wound, it is thus determined that vanadium action time point is 12h.
3, selecting vanadium action time point is 12h, is designed with single factor experiment, and 6 processing, each processing corresponding one are divided into
A vanadium concentration gradient, 6 repetitions of each processing utilize the life of flow cytometer measurement Apoptosis and intracellular reactive oxygen species generation (ROS)
At, cellular superoxide dismutase (SOD), lipid peroxide (MDA), in the activity and cell of catalase (CAT)
The burst size of lactic dehydrogenase (LDH) in clear liquid is as a result as follows:
(1) cell state: as shown in figure 4, cell is after each vanadium concentration handles 12h, cell is in inverted fluorescence microscope
Under cell state.250,1000 μm of ol/L vanadium processing 12h are larger to cell damage, and cell is rounded or even floats on culture medium
In, and 100 μm of ol/L vanadium handle 12h, and more cell can maintain preferable form itself, but still the clasmatosis of visible floating
Object；25,50 μm of ol/L vanadium handle 12h, have a small amount of clasmatosis object, but most cells can maintain normal form；0μ
Mol/L vanadium handles 12h, and only visible a small amount of products of cellular metabolism floating, cellular morphology are normal.
(2) Apoptosis: as shown in Figure 5 and Figure 6, percentage of cerebral apoptosis increases and increases with vanadium additive amount, and
Significant difference；The vanadium of 250 μm of ol/L and 1000 μm of ol/L makes apoptosis rate reach 70% or more；The vanadocyte of 100 μm of ol/L
Apoptosis rate is 38.22%, is significantly higher than control group.
(3) generation of intracellular reactive oxygen species generation (ROS): pass through the output of ROS in FITC fluorescence intensity reacting cells, FITC value
Bigger, ROS generation is more, as shown in fig. 7, the curve of 100 and 250 μm of ol/L vanadium processing groups deviates to the right, illustrates that FITC value increases
Greatly, the amount of ROS increases in general cell；As shown in figure 8, the FITC value of 100 μm of ol/L and 250 μm of ol/L vanadium processing groups is significantly high
In 0 μm of ol/L vanadium processing group, illustrate that the ROS amount generated in general cell significantly increases, 25 μm of ol/L and 50 μm of ol/L vanadium and 0 μ
Mol/L vanadium processing group is not significant compared to difference.Since 1000 μm of ol/L vanadium processing groups cause cell mortality, it is detected
The value of FITC is substantially less than 0 μm of ol/L vanadium processing group.
(4) cellular superoxide dismutase (SOD): as shown in figure 9, intracellular SOD content adding with various concentration vanadium
Add and change greatly, the content of 50 μm of ol/L vanadium SOD is substantially less than control group, and the content of 100 μm of ol/L vanadium SOD is low compared with control group；
The SOD content and control group difference of 50 μm of ol/L and 100 μm of ol/L vanadium be not significant；The SOD of 50 μm of ol/L and 100 μm of ol/L vanadium contains
Amount is substantially less than control group.
(5) lipid peroxide (MDA): as shown in Figure 10, compared with 0 μm of ol/L vanadium processing group, 50 μm of ol/L vanadium processing
Group MDA content difference is not significant, but the MDA content of 100 μm of ol/L vanadium processing groups significantly increases, 100 μm of ol/L vanadium processing group MDA
Content is higher than 0 μm 2.4 times of group of ol/L vanadium processing.
(6) activity of catalase (CAT): as shown in figure 11, compared with 0 μm of ol/L vanadium processing group, 50 μm of ol/L and
100 μm of ol/L vanadium processing group cell CAT activity are remarkably decreased.
(7) lactic dehydrogenase (LDH) burst size: as shown in figure 12, each processing group LDH burst size significant difference, with vanadium
The burst size of the increase of concentration, LDH linearly increases；The burst size of 100 μm of ol/L vanadium processing group LDH is 0 μm of ol/L vanadium processing
2.04 times of group.
The above method makes cell generate oxidative stress by the heavy metal vanadium of+5 valences, and primitive cell culture is successful, and cell is pure
Degree reaches 95% or more, acts on 12h using the vanadium of 100 μm of ol/L, and cell viability maintains 65% or so, cause Apoptosis with
And the generation of ROS intracellular dramatically increases, and causes anti-oxidant enzyme activity corresponding change, illustrates to set up the oxidation of bulb epithelial cell
Stress model.
As used some vocabulary in the specification and claims to censure special component or method.Art technology
Personnel are, it is to be appreciated that different regions may call the same ingredient with different nouns.This specification and claims are not
In such a way that the difference of title is as ingredient is distinguished.As the "comprising" mentioned by throughout the specification and claims is
One open language, therefore should be construed to " including but not limited to "." substantially " refer within the acceptable error range, this field
Technical staff can solve the technical problem within a certain error range, basically reach the technical effect.Specification is subsequent
It is described as implementing better embodiment of the invention, so the description is for the purpose of illustrating rule of the invention, not
To limit the scope of the invention.Protection scope of the present invention is as defined by the appended claims.
It should also be noted that, the terms "include", "comprise" or its any other variant are intended to nonexcludability
Include, so that commodity or system including a series of elements not only include those elements, but also including not clear
The other element listed, or further include for this commodity or the intrinsic element of system.In the feelings not limited more
Under condition, the element that is limited by sentence "including a ...", it is not excluded that in the commodity or system for including the element also
There are other identical elements.
Above description has shown and described several preferred embodiments of invention, but as previously described, it should be understood that invention is not
It is confined to form disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other combinations, modification
And environment, and can be carried out within that scope of the inventive concept describe herein by the above teachings or related fields of technology or knowledge
Change.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention, then it all should be in the appended power of invention
In the protection scope that benefit requires.
Claims (8)
1. the cultural method of laying hen magnum tubae uterinae epithelial cell, which is characterized in that specifically includes the following steps:
Step 1, it after laying hen being put to death, aseptically takes out whole section of fallopian tubal and is soaked in PBS, and remove mesentery, connective
Tissue and blood；
Step 2, the bulb of fallopian tubal is cut, shreds into tissue block after being cleaned with PBS, and with PBS cleansing tissue block；
Step 3, the tissue block after cleaning is transferred in clostridiopetidase A IV, in 37 DEG C of digestion certain times；
Step 4, postdigestive digestive juice is filtered, filtrate is centrifuged, and supernatant is abandoned after centrifugation and obtains cell, is added into cell
Cell is resuspended in complete medium, and finally cell is transferred in culture bottle, is placed in carbon dioxide, 37 that volumetric concentration is 5%
It is cultivated under conditions of DEG C；
Further include: after cultivating 2.5~3h, using difference adherent method, the liquid in culture bottle is transferred in new culture bottle,
Repeat this operation 1~2 time；
The concentration of the clostridiopetidase A IV is 1mg/ml；The time of the digestion is 70min.
2. the cultural method of laying hen magnum tubae uterinae epithelial cell as described in claim 1, which is characterized in that the tissue
The size of block is 1mm3；The revolving speed of the centrifugation is 700rpm, time 20min；The time of the culture preferred 72h.
3. the cultural method of laying hen magnum tubae uterinae epithelial cell as described in claim 1, which is characterized in that described complete
Culture medium composition are as follows: 91% DMEM/F12 basal medium, 5% fetal calf serum, 2% chicken serum, 1% nonessential amino acid,
5ug/ml insulin, the heparin of 20IU/ml, the L-Glutamine of 2mM, 15ng/ml epidermal growth factor, 1% mycillin.
4. the method for building up of laying hen magnum tubae uterinae epithelial cell oxidative stress model, which is characterized in that specifically include following
Step:
Step 1, laying hen magnum tubae uterinae epithelial cell is cultivated；
Step 1.1, it after laying hen being put to death, aseptically takes out whole section of fallopian tubal and is soaked in PBS, and remove mesentery, knot
Form tissue and blood；
Step 1.2, the bulb of fallopian tubal is cut, shreds into tissue block after being cleaned with PBS, and with PBS cleansing tissue block；
Step 1.3, the tissue block after cleaning is transferred in clostridiopetidase A IV, in 37 DEG C of digestion certain times；
Step 1.4, postdigestive digestive juice is filtered, filtrate is centrifuged, and supernatant is abandoned after centrifugation and obtains cell, is added into cell
Enter complete medium, cell be resuspended, finally cell is transferred in culture bottle, be placed in volumetric concentration be 5% carbon dioxide,
It is cultivated under conditions of 37 DEG C；
Step 2, using the laying hen magnum tubae uterinae epithelial cell of culture, a huge sum of money for various concentration is tested respectively by mtt assay
Cell viability under category and different action time, determines heavy metal action time point according to test result；
Step 3, the time point determined with step 2 designs different heavy metal concentration gradients, measures Apoptosis, intracellular activity
The generation of oxygen, cellular superoxide dismutase, lactic dehydrogenase release conditions, choose most suitable heavy metal concentration, thus
Establish cellular oxidation Stress model.
5. the method for building up of laying hen magnum tubae uterinae epithelial cell oxidative stress model as claimed in claim 4, feature
It is, the step 1 further include: after cultivating 2.5~3h, using difference adherent method, the liquid in culture bottle is transferred to newly
Culture bottle in, repeat this operation 1~2 time.
6. the method for building up of laying hen magnum tubae uterinae epithelial cell oxidative stress model as claimed in claim 4, feature
It is, the concentration of the clostridiopetidase A IV is 1mg/ml；The time of the digestion is 70min.
7. the method for building up of laying hen magnum tubae uterinae epithelial cell oxidative stress model as claimed in claim 4, feature
It is, the size of the tissue block is 1mm3；The revolving speed of the centrifugation is 700rpm, time 20min；The time of the culture
It is preferred that 72h.
8. the method for building up of laying hen magnum tubae uterinae epithelial cell oxidative stress model as claimed in claim 4, feature
It is, the complete medium composition are as follows: 91% DMEM/F12 basal medium, 5% fetal calf serum, 2% chicken serum, 1%
Nonessential amino acid, 5ug/ml insulin, the heparin of 20IU/ml, the L-Glutamine of 2mM, 15ng/ml epidermal growth factor,
1% mycillin.
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| CN104818240A (en) * | 2015-04-27 | 2015-08-05 | 山东省农业科学院畜牧兽医研究所 | In-vitro culture method of mouse oviduct epithelial cells |
| CN105695396A (en) * | 2016-05-03 | 2016-06-22 | 河南农业大学 | Method for in-vitro isolated culture of chicken fallopian tube epithelial cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104818240A (en) * | 2015-04-27 | 2015-08-05 | 山东省农业科学院畜牧兽医研究所 | In-vitro culture method of mouse oviduct epithelial cells |
| CN105695396A (en) * | 2016-05-03 | 2016-06-22 | 河南农业大学 | Method for in-vitro isolated culture of chicken fallopian tube epithelial cells |
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| H9N2亚型禽流感病毒对鸡输卵管致病性的研究;王晶钰等;《中国兽医学报》;20130430;500-503 |
| 含钒蛋黄粉对大鼠生长、氧化应激状态及其相关基因表达的影响;崔仁勇等;《动物营养学报》;20160531;1956-1964 |
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