CN106167788A - Laying hen magnum tubae uterinae epithelial cell is cultivated and oxidative stress method for establishing model - Google Patents
Laying hen magnum tubae uterinae epithelial cell is cultivated and oxidative stress method for establishing model Download PDFInfo
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Abstract
The invention discloses a kind of laying hen magnum tubae uterinae epithelial cell to cultivate and oxidative stress method for establishing model, described cell culture processes is: takes whole section of fallopian tube of laying hen and is soaked in PBS, and removes mesentery, connective tissue and blood；Oviducal bulb is cut, after cleaning, shreds into piece of tissue；Piece of tissue after cleaning is transferred in collagenase IV digest；Being filtered by Digestive system, filtrate is centrifuged, and abandons supernatant, adds complete medium, makes cell resuspended, is finally transferred to cell in culture bottle cultivate.Described oxidative stress method for establishing model is: the heavy metal of test variable concentrations and the cell viability under different action time, determines some action time；With the heavy metal concentration gradient that the design of this time point is different, measure the situations such as apoptosis, set up oxidative stress model.The present invention is to probe into the concrete mechanism that external environment causes Ovum Gallus domesticus album quality to decline further, it is established that laying hen magnum tubae uterinae epithelial cell oxidative stress model.
Description
Technical field
The invention belongs to cell toxicity technical field, specifically, relate to a kind of laying hen magnum tubae uterinae epithelial cell
Cultural method, further relate to the method for building up of a kind of laying hen magnum tubae uterinae epithelial cell oxidative stress model.
Background technology
Egg is as the main source of necessary for human protein.The change of egg quality, may ask safely along with egg
Topic.Along with living standard improves, 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 the most whole to laying hen industry
Animal husbandry has havoc, and human health also produces certain impact.Egg is in addition to egg yolk, and remaining is all at fallopian tube position
Formed.Laying hen fallopian tube is divided into infundibulum, bulb, isthmus, uterus and portio vaginalis, wherein infundibulum conveying yolk, bulb
Forming Ovum Gallus domesticus album, isthmus forms egg shell membrane, and uterus forms eggshell, and portio vaginalis conveying egg, to cloaca, finally excretes.Wherein
Ovum Gallus domesticus album mainly endoplasmic reticulum by magnum tubae uterinae epithelial cell synthesize and stores, and being secreted into official jargon, being wrapped in outside egg yolk
Face, forms white of an egg layer.Magnum tubae uterinae epithelial cell is distributed in bulb mucosa fold.The security quality of egg, mainly
Comprising egg size, eggshell strength, thickness, albumen height, breathe out not unit etc., wherein Ovum Gallus domesticus album quality is easily by external environmental effect, mainly
Showing as albumen height to reduce, egg white solution is thinning, breathes out not unit and declines.And Ovum Gallus domesticus album is mainly by magnum tubae uterinae epithelial cell
Endoplasmic reticulum synthesize and stores, and being secreted into official jargon, being wrapped in outside egg yolk, forming white of an egg layer.Magnum tubae uterinae epithelium is thin
Born of the same parents are distributed in bulb mucosa fold.At present, in laying hen industry production, laying hen is easily affected by the external environment, in feedstuff
Mycotoxin (aflatoxin, fusarium toxin), heavy metal (vanadium, copper, cadmium) and feeding environment deteriorate (temperature in house,
Environmental stress) etc., all may affect in white of an egg forming process by affecting the form (crimp height) of magnum tubae uterinae
In motion, and impact decision Ovum Gallus domesticus album quality (white of an egg height and HU) the dense white of an egg, ovomucin (Ovomucin, OVW) divides
Secrete, thus cause the reduction of white of an egg quality.Oxidative stress is due to the too much free radical of internal generation, it is impossible to removes in time, makes
Become body oxidative and anti-oxidative unbalance.Many researchs also demonstrate that this is likely due to laying hen body by external environmental stimulus, make
Laying hen magnum tubae uterinae epithelial cell produces oxidative stress, in turn results in the disorder of 26S Proteasome Structure and Function, affects the Ovum Gallus domesticus album of secretion,
But this research is only limited on laying hen live body, adds vanadium, and whether vanadium can reach defeated ovum by the way of diet or drinking-water
Pipe bulb, and the effective dose affecting fallopian tube 26S Proteasome Structure and Function also lacks unified conclusion, and laying hen fallopian tube expands
The original cuiture of portion's epithelial cell rarely has report both at home and abroad, and isolation and culture of cell method is immature, and the cell of cultivation is easily subject to
Fibroblast pollutes, and the most identified success mostly, therefore proves the direct modeling of its mechanism on Epithelium Cells
Evidence seems necessity very.
Meanwhile, the foundation of the model of oxidative stress, is also the powerful measure studied and need Other diseases mechanism.Numerous disease
Relevant with oxidative stress, obese patient's vivo oxidation stress level can raise；The too much oxygen-derived free radicals (ROS) of internal generation can be straight
Connect impaired isle element β cell, cause diabetes；Blood vessel produces too much ROS and causes low density lipoprotein, LDL (LDL) at endarterium
Deposition, cause atherosclerosis；With age, antioxidant ability of organism weakens and causes alzheimer's disease etc..Mesh
Before, have and added the mode such as heavy metal, hydrogen peroxide by many and set up the precedent of oxidative stress model, but vanadium, it is changed
Conjunction price, between-1 to+5 valencys, is a kind of strong oxidizer, is also the inhibitor of many phosphokinases, is medically also used for pancreas
The substitute of island element, but research in recent years progressively finds that it, to liver, the toxicity of lung cell, therefore sets up one with vanadium
Oxidative stress model, is also the most necessary.
Therefore, in view of the foregoing, in order to probe into the concrete mechanism that oxidative stress causes Ovum Gallus domesticus album quality to decline further, have
Necessity sets up the external oxidative stress model of laying hen magnum tubae uterinae epithelial cell.
Summary of the invention
In view of this, the laying hen magnum tubae uterinae epithelial cell that the present invention is directed to the cultivation of existing method is easily become fiber finer
The problem that born of the same parents pollute, and in order to probe into the concrete mechanism that external environment causes Ovum Gallus domesticus album quality to decline further, it is provided that a hatching egg
Chicken salpingo bulb epithelial cell is cultivated and oxidative stress method for establishing model.
In order to solve above-mentioned technical problem, the invention discloses the cultivation side of a kind of laying hen magnum tubae uterinae epithelial cell
Method, specifically includes following steps:
Step 1, by laying hen after death, aseptically take out whole section of fallopian tube and be soaked in PBS, and remove mesentery,
Connective tissue and blood；
Step 2, cuts oviducal bulb, with shredding into piece of tissue after PBS, and uses PBS piece of tissue；
Step 3, the piece of tissue after cleaning is transferred in collagenase IV, digests certain time in 37 DEG C；
Step 4, filters postdigestive Digestive system, and filtrate is centrifuged, and abandons supernatant and obtain cell, in cell after being centrifuged
Add complete medium, make cell resuspended, finally cell is transferred in culture bottle, be placed in the titanium dioxide that volumetric concentration is 5%
Carbon, cultivate under conditions of 37 DEG C.
Further, also include: after cultivating 2.5～3h, use difference adherent method, the liquid in culture bottle is transferred to
In new culture bottle, repeat this operation 1～2 time.
Further, the concentration of described collagenase IV is 1mg/ml；The time of described digestion is 70min.
Further, the size of described piece of tissue is 1mm3；Described centrifugal rotating speed is 700rpm, the time is 20min；Institute
State the time preferred 72h of cultivation.
Further, described complete medium consists of: the DMEM/F12 basal medium of 91%, 5% hyclone,
2% chicken serum, 1% non essential amino acid, 5ug/ml insulin, the heparin of 20IU/ml, the L-glutaminate of 2mM, 15ng/ml
Epidermal growth factor, 1% mycillin.
The invention also discloses the method for building up of a kind of laying hen magnum tubae uterinae epithelial cell oxidative stress model, specifically
Comprise the following steps:
Step 1, cultivates laying hen magnum tubae uterinae epithelial cell；
Step 1.1, by laying hen after death, aseptically take out whole section of fallopian tube and be soaked in PBS, and remove system
Film, connective tissue and blood；
Step 1.2, cuts oviducal bulb, with shredding into piece of tissue after PBS, and organizes with PBS
Block；
Step 1.3, the piece of tissue after cleaning is transferred in collagenase IV, digests certain time in 37 DEG C；
Step 1.4, filters postdigestive Digestive system, and filtrate is centrifuged, and abandons supernatant and obtain cell, to cell after being centrifuged
Middle addition complete medium, makes cell resuspended, is finally transferred in culture bottle by cell, is placed in the dioxy that volumetric concentration is 5%
Change carbon, cultivate under conditions of 37 DEG C.
Step 2, is used the laying hen magnum tubae uterinae epithelial cell cultivated, is tested variable concentrations by mtt assay respectively
Heavy metal and the cell viability under different action time, determine heavy metal point action time according to test result；
Step 3, the time point determined with step 2, designs different heavy metal concentration gradients, measures apoptosis, intracellular
The generation of active oxygen, cellular superoxide dismutase, the release conditions of lactic acid dehydrogenase, choose optimal heavy metal concentration,
Thus set up cellular oxidation Stress model.
Further, described step 1 also includes: after cultivating 2.5～3h, uses difference adherent method, by culture bottle
Liquid is transferred in new culture bottle, repeats this operation 1～2 time.
Further, the concentration of described collagenase IV is 1mg/ml；The time of described digestion is 70min.
Further, the size of described piece of tissue is 1mm3；Described centrifugal rotating speed is 700rpm, the time is 20min；Institute
State the time preferred 72h of cultivation.
Further, described complete medium consists of: the DMEM/F12 basal medium of 91%, 5% hyclone,
2% chicken serum, 1% non essential amino acid, 5ug/ml insulin, the heparin of 20IU/ml, the L-glutaminate of 2mM, 15ng/ml
Epidermal growth factor, 1% mycillin.
Compared with prior art, the present invention can obtain and include techniques below effect:
(1) present invention is by cultivating laying hen magnum tubae uterinae epithelial cell；Then test variable concentrations heavy metal and
The different cell viabilities under action time, determines some action time；Different heavy metal concentration ladders is designed again with this time point
Degree, measures the situation such as apoptosis, thus sets up cellular oxidation Stress model, causes Ovum Gallus domesticus album product probing into external environment further
The concrete mechanism that matter declines；
(2) present invention is by cultivating laying hen magnum tubae uterinae epithelial cell, sets up bulb epithelial cell oxidative stress
Model, the concrete mechanism for research Ovum Gallus domesticus album quality decline later provides basis, and minimizing laying hen industry should due to laying hen autoxidation
Swash and cause Egg Quality to decline the loss caused；
(3) the laying hen magnum tubae uterinae epithelial cell cultural method of the present invention is scientific and reasonable, overcomes the training of existing method
The problem that the laying hen magnum tubae uterinae epithelial cell supported easily is polluted by fibroblast, primitive cell culture success, cell is pure
Degree reaches more than 95%, and has been successfully established laying hen magnum tubae uterinae epithelial cell oxidative stress model.
Certainly, the arbitrary product implementing the present invention it is not absolutely required to reach all the above technique effect simultaneously.
Accompanying drawing explanation
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes the part of the present invention, this
Bright schematic description and description is used for explaining the present invention, is not intended that inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 is embodiment of the present invention laying hen magnum tubae uterinae epithelial cell picture, and wherein (a) and (b) are cell and exist
After 72h under fluorescence inverted microscope the picture of (200 ×)；
Fig. 2 is embodiment of the present invention laying hen magnum tubae uterinae epithelial cell immunofluorescence dyeing picture, and wherein (a) is anti-
CK18 picture, (b) is anti-ESR1 picture, and (c) is anti ova picture, and (d) is anti-PGR picture, and (e) is anti-Vimentin picture；
Fig. 3 be the embodiment of the present invention under different time cell viability with the change curve of vanadium concentration；
Fig. 4 is to process cell picture, wherein (a) vanadium concentration 0 μm ol/ml after 12h under embodiment of the present invention difference vanadium concentration,
(b) vanadium concentration 25 μm ol/ml, (c) vanadium concentration 50 μm ol/ml, (d) vanadium concentration 100 μm ol/ml, (e) vanadium concentration 250 μm ol/
Ml, (f) vanadium concentration 1000 μm ol/ml；
Fig. 5 is the apoptosis figure of cell under the different vanadium concentration that the embodiment of the present invention utilizes flow cytometer test to obtain, its
In (a) vanadium concentration 0 μm ol/ml, (b) vanadium concentration 25 μm ol/ml, (c) vanadium concentration 50 μm ol/ml, (d) vanadium concentration 100 μm ol/
Ml, (e) vanadium concentration 250 μm ol/ml, (f) vanadium concentration 1000 μm ol/ml；
Fig. 6 is the apoptosis rate figure of cell under embodiment of the present invention difference vanadium concentration；
Fig. 7 is that under the different vanadium concentration that the embodiment of the present invention utilizes flow cytometer test to obtain, intracellular FITC fluorescence is strong
Degree figure；
Fig. 8 is intracellular FITC fluorescence intensity meansigma methods figure under embodiment of the present invention difference vanadium concentration；
Fig. 9 is cellular superoxide dismutase figure under embodiment of the present invention difference vanadium concentration；
Figure 10 is cytolipin peroxide figure under embodiment of the present invention difference vanadium concentration；
Figure 11 is the activity figure of cellular superoxide hydrogen enzyme under embodiment of the present invention difference vanadium concentration；
Figure 12 is cell lactic dehydrogenase enzyme r e lease figure under embodiment of the present invention difference vanadium concentration.
Detailed description of the invention
Embodiments of the present invention are described in detail, thereby to the present invention how application technology hands below in conjunction with embodiment
Section solves technical problem and reaches the process that realizes of technology effect and can fully understand and implement according to this.
The cultural method of laying hen magnum tubae uterinae epithelial cell of the present invention, employing Roman egghen is experimental animal, specifically
Comprise the following steps:
Step 1, by laying hen after death, aseptically take out whole section of fallopian tube and be soaked in PBS, then in biology pacify
Full cabinet removes mesentery, connective tissue and blood；
Step 2, cuts oviducal position of expanding, by the phosphate buffered solution (PBS) (Hyclone) of 37 DEG C of preheatings
Inside and outside clean twice, shred bulb to 1mm with eye scissors subsequently3Left and right piece of tissue, period with 37 DEG C preheat PBS
Piece of tissue, until the liquid after Qing Xiing becomes clarification；
Step 3, the piece of tissue after cleaning is transferred in the collagenase IV of 1mg/ml, is placed in 37 DEG C of water-bath digestion 70min；
This time is selected to be because being digested by epithelial cell largely, the one-tenth fiber that overlong time may digest
Cell is the most.
Wherein, collagenase IV (Sigma), for being got off from tissue digestion by purpose cell, forms individual cells.Select
The collagenase IV of 1mg/ml, is due under this concentration enzyme effect, in the case of can be by under cell dissociation, to cell injury relatively
Little, the cell digested is easier to adherent and growth.
Step 4, postdigestive Digestive system 40nm nylon filter net filtration, filtrate 700r is centrifuged 20min, abandons supernatant
Obtaining cell, add complete medium in cell, piping and druming makes cell resuspended gently, is finally transferred in culture bottle by cell, puts
In the carbon dioxide that volumetric concentration is 5%, cultivating 72h under the conditions of 37 DEG C, cell picture is as shown in Figure 1.
700r is centrifuged 20min can be further purified cell by unicellular removal.
After cell cultivates 72h, can be paved with bottom more than 80%, now cell viability is relatively strong, after can be used as at a step test
Reason.
Further, described complete medium consists of: the DMEM/F12 basal medium (Hyclone) of 91%, 5% tire
Ox blood serum (Gibco), 2% chicken serum (Gibco), 1% non essential amino acid (Sigma), 5ug/ml insulin (Gibco),
The heparin of 20IU/ml, the L-glutaminate (Ameresco) of 2mM, 15ng/ml epidermal growth factor (Peprotech), 1% is blue or green
Streptomycin (Hyclone).
Wherein, add non essential amino acid can either promote cultivate cell growth, again can with Extending culture cell survival time
Between, and reduce the external biological resulting load of cell；Select the hyclone (5%) of low concentration, it is possible to a certain degree suppress into
Fibroblast growth；Insulin and epidermal growth factor can promote the adherent of epithelial cell；Heparin can suppress fibroblastic
Propagation；L-glutaminate is the propagation of the important raw and processed materials of cell nucleic acid and protein, beneficially cell.
Further, in order to reduce fibroblastic pollution further, after cultivating 2.5～3h, use difference adherent
Method, is transferred to the liquid in culture bottle in new culture bottle, repeats this operation 1～2 time, and purpose cell can reach more than 95%
Purity.
The method for building up of laying hen magnum tubae uterinae epithelial cell oxidative stress model of the present invention, comprises the following steps:
Step 1, cultivates laying hen magnum tubae uterinae epithelial cell；
Step 1.1, by laying hen after death, aseptically take out whole section of fallopian tube and be soaked in PBS, then in biology
Safety cabinet is removed mesentery, connective tissue and blood；
Step 1.2, cuts oviducal position of expanding, by the phosphate buffered solution (PBS) of 37 DEG C of preheatings
(Hyclone) inside and outside clean twice, shred bulb to 1mm with eye scissors subsequently3The piece of tissue of left and right, period is with 37 DEG C of preheatings
PBS piece of tissue, until liquid after Qing Xiing becomes clarification；
Step 1.3, the piece of tissue after cleaning is transferred in the collagenase IV of 1mg/ml, is placed in 37 DEG C of water-bath digestion
70min；
Wherein, collagenase IV (Sigma), for being got off from tissue digestion by purpose cell, forms individual cells.
Step 1.4, postdigestive Digestive system 40nm nylon filter net filtration, filtrate 700r is centrifuged 20min, abandons supernatant
Liquid obtains cell, adds complete medium in cell, and piping and druming makes cell resuspended gently, is finally transferred in culture bottle by cell,
Being placed in carbon dioxide that volumetric concentration is 5%, cultivate 72h under the conditions of 37 DEG C, cell picture is as shown in Figure 1；
Further, described complete medium consists of: the DMEM/F12 basal medium (Hyclone) of 91%, 5% tire
Ox blood serum (Gibco), 2% chicken serum (Gibco), 1% non essential amino acid (Sigma), 5ug/ml insulin (Gibco),
The heparin of 20IU/ml, the L-glutaminate (Ameresco) of 2mM, 15ng/ml epidermal growth factor (Peprotech), 1% is blue or green
Streptomycin (Hyclone).
Further, in order to reduce fibroblastic pollution further, after cultivating 2.5～3h, use difference adherent
Method, is transferred to the liquid in culture bottle in new culture bottle, repeats this operation 1～2 time, and purpose cell can reach more than 95%
Purity.
Step 2, is used the laying hen magnum tubae uterinae epithelial cell cultivated, is tested variable concentrations by mtt assay respectively
Heavy metal and the cell viability under different action time, determine heavy metal point action time according to test result；
Described heavy metal forms compound with different valent states, and high-valence state metal is to lower valency, such as ferrum, copper, chromium, vanadium
Produce ROS by oxidoreduction, cause oxidative stress.Vanadium is trace element necessary to animal growth, common chemical combination
Valency has+2 ,+3 ,+4 and+5, and the high-valence state vanadium of+5 valencys is as a kind of reducing agent, has strong oxidisability, easily makes cell produce
Oxidative stress.It is therefore preferable that the heavy metal vanadium of+5 valencys of employing.
Step 3, the time point determined with step 2, designs different heavy metal vanadium Concentraton gradient, utilizes flow cytometer to survey
Determine apoptosis and the generation of intracellular reactive oxygen species generation (ROS), cellular superoxide dismutase (SOD), lipid peroxide (MDA),
The activity of catalase (CAT) and the release conditions of lactic acid dehydrogenase (LDH), choose optimal heavy metal vanadium concentration, from
And set up cellular oxidation Stress model.
Below in conjunction with concrete experiment beneficial effects of the present invention is described:
1, by cellular immunofluorescence technology, identify that the laying hen magnum tubae uterinae epithelial cell cultivated and cell are pure
Degree:
(1) test method: by magnum tubae uterinae epithelial cell with 1 × 105Density is inoculated in 6 orifice plates, treats cell density
Reach first to use PBS cell 3 times when 70%, then fix 10min, PBS by the 4% paraformaldehyde room temperature of 1000ul
Cell 3 times, each 5min；Triton X-100 (TritonX-100) room temperature of the 0.5% of addition 1000ul places 20min, PBS
Clean cell 3 times, each 5min；The lowlenthal serum albumin confining liquid of the 1% of addition 1000ul, room temperature closes 1h.Abandon closing
Liquid, each add rabbit anti-cell keratin-18 (CK-18, Boster), the anti-Vimentin of rabbit (Anti-Vimentin, LSBio),
Antiovalbumin (ovalbumin, OVA, Acis), rabbit estrogen antagonist receptor 1 (ESR1, NOVUS) and mouse-anti progesterone receptor (PGR,
NOVUS) 600ul is in 5 different holes, 4 DEG C of night incubation, PBS cell 3 times, each 5min；Add 600ul
The goat anti-rabbit igg (CST) of 488nm labelling and the goat anti-rabbit igg (CST) of 555nm labelling, it is clear that 37 DEG C of lucifuges hatch 1h, PBS
Wash cell 3 times, each 5min.Adding the 4', 6-diamidino-2-phenylindone (DAPI) of 500ul, room temperature redyes 10min, PBS
Washing 6 times, add the 50% anti-quencher of glycerol of 1000ul, under inverted fluorescence microscope, room temperature lucifuge observes coloration result.
(2) result of the test: shown by immunofluorescence, sees Fig. 2, and cell anti-CK18, OVA, PGR and ESR1 are sun
Property, and anti-vimentin is negative, and shows that the cell cultivated is laying hen magnum tubae uterinae epithelial cell, and purity reaches 95%
Above.
2, utilize the laying hen magnum tubae uterinae epithelial cell cultivated, use 5 × 6 factorial experiment designs, be divided at 30
Reason, design 6 vanadium Concentraton gradient (0,25,50,100,250,1000 μm ol/ml) and 5 time points (1,4,12,24,48h),
Cell viability is surveyed by mtt assay.
Test result: as it is shown on figure 3, under 12h, the vanadium concentration of 50 μm ol/L and 100 μm ol/L, cell viability is 70%
Left and right, illustrates cell creates certain impact, and the vanadium concentration versus cell damage of other times point is too small or reaches irreversible damage
Wound, it is thus determined that vanadium point action time is 12h.
3, selecting vanadium point action time is 12h, uses single factor experiment design, is divided into 6 process, each process corresponding
Individual vanadium Concentraton gradient, each process 6 repetition, utilize flow cytometer to measure apoptosis and the life of intracellular reactive oxygen species generation (ROS)
Becoming, cellular superoxide dismutase (SOD), lipid peroxide (MDA), in the activity of catalase (CAT) and cell
The burst size of lactic acid dehydrogenase (LDH) in clear liquid, result is as follows:
(1) cell state: as shown in Figure 4, cell is after each vanadium concentration processes 12h, and cell is at inverted fluorescence microscope
Under cell state.250,1000 μm ol/L vanadium process 12h to cell damage relatively greatly, and cell is rounded even floats on culture medium
In, and 100 μm ol/L vanadium process 12h, more cell energy maintenance preferably form itself, but still visible floating cell breakage
Thing；25,50 μm ol/L vanadium process 12h, have a small amount of cell breakage thing, but overwhelming majority cell can maintain normal form；0μ
Mol/L vanadium processes 12h, and only visible a small amount of products of cellular metabolism is floating, and cellular morphology is normal.
(2) apoptosis: as shown in Figure 5 and Figure 6, percentage of cerebral apoptosis raises along with increasing of vanadium addition, and
Significant difference；The vanadium of 250 μm ol/L and 1000 μm ol/L makes apoptosis rate reach more than 70%；The vanadocyte of 100 μm ol/L
Apoptosis rate is 38.22%, is significantly higher than matched group.
(3) generation of intracellular reactive oxygen species generation (ROS): by the volume of production of ROS, FITC value in FITC fluorescence intensity reacting cells
The biggest, ROS generates the most, as it is shown in fig. 7, the curve of 100 and 250 μm ol/L vanadium process groups offsets to the right, illustrates that FITC value increases
Greatly, in general cell, the amount of ROS increases；As shown in Figure 8, the FITC value of 100 μm ol/L and 250 μm ol/L vanadium process groups is significantly high
In 0 μm ol/L vanadium process group, the ROS amount produced in general cell is described significantly increases, 25 μm ol/L and 50 μm ol/L vanadium and 0 μ
It is the most notable that mol/L vanadium process group compares difference.Owing to 1000 μm ol/L vanadium process groups cause cell mortality, detect it
The value of FITC is substantially less than 0 μm ol/L vanadium process group.
(4) cellular superoxide dismutase (SOD): as it is shown in figure 9, intracellular SOD content adding along with variable concentrations vanadium
Adding and change greatly, the content of 50 μm ol/L vanadium SOD is substantially less than matched group, and the content of 100 μm ol/L vanadium SOD is low compared with matched group；
The SOD content of 50 μm ol/L and 100 μm ol/L vanadium is the most notable with matched group difference；The SOD of 50 μm ol/L and 100 μm ol/L vanadium contains
Amount is substantially less than matched group.
(5) lipid peroxide (MDA): as shown in Figure 10, compared with 0 μm ol/L vanadium process group, 50 μm ol/L vanadium process
Group MDA content difference is not notable, but the MDA content of 100 μm ol/L vanadium process groups significantly raises, 100 μm ol/L vanadium process groups MDA
Content is higher than 0 μm ol/L vanadium process group 2.4 times.
(6) activity of catalase (CAT): as shown in figure 11, compared with 0 μm ol/L vanadium process group, 50 μm ol/L and
100 μm ol/L vanadium process group cell CAT activity are remarkably decreased.
(7) lactic acid dehydrogenase (LDH) burst size: as shown in figure 12, each process group LDH burst size significant difference, along with vanadium
The increase of concentration, the burst size of LDH linearly increases；The burst size of 100 μm ol/L vanadium process groups LDH is that 0 μm ol/L vanadium processes
2.04 times of group.
Said method makes cell produce oxidative stress by the heavy metal vanadium of+5 valencys, and primitive cell culture success, cell is pure
Degree reach more than 95%, utilize the vanadium effect 12h of 100 μm ol/L, cell viability maintains about 65%, cause apoptosis with
And the generation of intracellular ROS dramatically increases, and cause antioxidase respective change alive, illustrate to set up the oxidation of bulb epithelial cell
Stress model.
As employed some vocabulary in the middle of description and claim to censure special component or method.Art technology
Personnel are it is to be appreciated that same composition may be called with different nouns in different regions.This specification and claims are not
In the way of the difference of title is used as distinguishing composition." comprising " as mentioned by the middle of description and claim in the whole text is
One open language, therefore " comprise but be not limited to " should be construed to." substantially " refer in receivable range of error, this area
Technical staff can solve described technical problem in the range of certain error, basically reaches described technique effect.Description is follow-up
It is described as implementing the better embodiment of the present invention, for the purpose of right described description is the rule so that the present invention to be described, not
In order to limit the scope of the present invention.Protection scope of the present invention is when being as the criterion depending on the defined person of claims.
Also, it should be noted term " includes ", " comprising " or its any other variant are intended to nonexcludability
Comprise, so that include that the commodity of a series of key element or system not only include those key elements, but also include the most clearly
Other key elements listed, or also include the key element intrinsic for this commodity or system.In the feelings not having more restriction
Under condition, statement " including ... " key element limited, it is not excluded that in the commodity including described key element or system also
There is other identical element.
Described above illustrate and describes some preferred embodiments of invention, but as previously mentioned, it should be understood that invention is not
It is confined to form disclosed herein, is not to be taken as the eliminating to other embodiments, and can be used for other combinations various, amendment
And environment, and can be carried out by above-mentioned teaching or the technology of association area or knowledge in invention contemplated scope described herein
Change.And the change that those skilled in the art are carried out and change are without departing from the spirit and scope of invention, the most all should weigh appended by invention
In the protection domain that profit requires.
Claims (10)
1. the cultural method of laying hen magnum tubae uterinae epithelial cell, it is characterised in that specifically include following steps:
Step 1, by laying hen after death, aseptically take out whole section of fallopian tube and be soaked in PBS, and remove mesentery, connective
Tissue and blood；
Step 2, cuts oviducal bulb, with shredding into piece of tissue after PBS, and uses PBS piece of tissue；
Step 3, the piece of tissue after cleaning is transferred in collagenase IV, digests certain time in 37 DEG C；
Step 4, filters postdigestive Digestive system, and filtrate is centrifuged, and abandons supernatant and obtain cell after being centrifuged, and adds in cell
Complete medium, makes cell resuspended, is finally transferred in culture bottle by cell, be placed in carbon dioxide that volumetric concentration is 5%, 37
Cultivate under conditions of DEG C.
2. the cultural method of laying hen magnum tubae uterinae epithelial cell as claimed in claim 1, it is characterised in that also include:
After cultivating 2.5～3h, use difference adherent method, the liquid in culture bottle is transferred in new culture bottle, repeat this operation 1
～2 times.
3. the cultural method of laying hen magnum tubae uterinae epithelial cell as claimed in claim 1, it is characterised in that described collagen
The concentration of enzyme IV is 1mg/ml；The time of described digestion is 70min.
4. the cultural method of laying hen magnum tubae uterinae epithelial cell as claimed in claim 1, it is characterised in that described tissue
The size of block is 1mm3；Described centrifugal rotating speed is 700rpm, the time is 20min；The time preferred 72h of described cultivation.
5. the cultural method of laying hen magnum tubae uterinae epithelial cell as claimed in claim 1, it is characterised in that described completely
Culture medium consists of: the DMEM/F12 basal medium of 91%, 5% hyclone, 2% chicken serum, 1% non essential amino acid,
5ug/ml insulin, the heparin of 20IU/ml, the L-glutaminate of 2mM, 15ng/ml epidermal growth factor, 1% mycillin.
6. the method for building up of laying hen magnum tubae uterinae epithelial cell oxidative stress model, it is characterised in that specifically include following
Step:
Step 1, cultivates laying hen magnum tubae uterinae epithelial cell；
Step 1.1, by laying hen after death, aseptically take out whole section of fallopian tube and be soaked in PBS, and remove mesentery, knot
Form tissue and blood；
Step 1.2, cuts oviducal bulb, with shredding into piece of tissue after PBS, and uses PBS piece of tissue；
Step 1.3, the piece of tissue after cleaning is transferred in collagenase IV, digests certain time in 37 DEG C；
Step 1.4, filters postdigestive Digestive system, and filtrate is centrifuged, and abandons supernatant and obtains cell, add in cell after being centrifuged
Enter complete medium, make cell resuspended, finally cell is transferred in culture bottle, be placed in carbon dioxide that volumetric concentration is 5%,
Cultivate under conditions of 37 DEG C.
Step 2, is used the laying hen magnum tubae uterinae epithelial cell cultivated, is tested a huge sum of money for variable concentrations by mtt assay respectively
Belong to and the cell viability under different action time, determine heavy metal point action time 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, the release conditions of lactic acid dehydrogenase, choose optimal heavy metal concentration, thus
Set up cellular oxidation Stress model.
7. the method for building up of laying hen magnum tubae uterinae epithelial cell oxidative stress model as claimed in claim 6, its feature
Being, described step 1 also includes: after cultivating 2.5～3h, uses difference adherent method, is transferred to newly by the liquid in culture bottle
Culture bottle in, repeat this operation 1～2 time.
8. the method for building up of laying hen magnum tubae uterinae epithelial cell oxidative stress model as claimed in claim 6, its feature
Being, the concentration of described collagenase IV is 1mg/ml；The time of described digestion is 70min.
9. the method for building up of laying hen magnum tubae uterinae epithelial cell oxidative stress model as claimed in claim 6, its feature
Being, the size of described piece of tissue is 1mm3；Described centrifugal rotating speed is 700rpm, the time is 20min；The time of described cultivation
Preferably 72h.
10. the method for building up of laying hen magnum tubae uterinae epithelial cell oxidative stress model as claimed in claim 6, its feature
Being, described complete medium consists of: the DMEM/F12 basal medium of 91%, 5% hyclone, 2% chicken serum, and 1%
Non essential amino acid, 5ug/ml insulin, the heparin of 20IU/ml, the L-glutaminate of 2mM, 15ng/ml epidermal growth factor,
1% mycillin.
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| CN111657229A (en) * | 2020-06-19 | 2020-09-15 | 四川农业大学 | Method for establishing laying hen ovary oxidative stress model |
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