CN108588005A - A kind of isolated culture method of duck folliculus ovarii film layer cell - Google Patents
A kind of isolated culture method of duck folliculus ovarii film layer cell Download PDFInfo
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- CN108588005A CN108588005A CN201810394679.5A CN201810394679A CN108588005A CN 108588005 A CN108588005 A CN 108588005A CN 201810394679 A CN201810394679 A CN 201810394679A CN 108588005 A CN108588005 A CN 108588005A
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Abstract
The invention discloses a kind of isolation and culture method of duck folliculus ovarii film layer cell, isolation and culture method is as follows：By duck neck portion sacrificed by exsanguination, ovary is taken out, blood stains are rinsed with phosphate buffer；Remove ovarian follicle outer layer connective tissue and rete vasculosum；Ovarian follicle is scratched in ovarian follicle handle opposite avascular area domain, pours out, clean yolk and granular cell, only retain film layer cell；Trypsase water-bath digests；Fetal calf serum is added and terminates digestion, phosphate buffer is used in combination to clean repeatedly；Tissue is shredded, is digested with the digestive juices water-bath such as clostridiopetidase A；It is rushed using icecold phosphate salt buffer and terminates digestion；Cell liquid is resuspended using the improvement Du Shi Eagle's mediums containing 10% fetal calf serum in filtering, centrifugation；It is seeded in Tissue Culture Dish；Culture 6h changes liquid, is placed in incubator and continues to cultivate.The duck folliculus ovarii film layer cell purity that the present invention obtains is high, and adherent close, cellular morphology is normal, and activity is good, and cultural method is reliable and stable, and repeatability is strong.
Description
Technical field
The invention belongs to biotechnologies, are related to gonad cell separation and Vitro Culture Techniques more particularly to a kind of duck
The isolation and culture method of folliculus ovarii film layer cell.
Background technology
The big country that China cultivates and consumes as duck, egg, meat duck cultivation and duck's egg processing are China's traditional advantage herding productions
Industry, overall development is steady nearly ten years for duck aquaculture industry.The study found that duck ovarian follicular growth developmental state is to its egg laying performance
And its Egg Quality has direct influence.The ovarian follicle of duck is ten hundreds of, and with sexually matured arrival, ovarian follicle is developed rapidly successively,
And it is in the different stages of development, and then form stringent hierarchical system.The growth course of ovarian follicle be generally divided into primordial follicle, just
Grade ovarian follicle, secondary follicle and graaffian follicle.
Theca layer cell originates from ovary fibroblastoid stromal cell line, including thecal cells and pericyte, is
The important component of ovarian follicle.Theca layer cell and follicular development are closely related, and the appearance of theca layer indicates secondary ovum
The formation of bubble.Theca layer morphological feature changes with the variation in follicular development stage, is the weight for differentiating the follicular development stage
Indicate.In addition, in terms of physiology, film layer cell is by regulating and controlling a variety of growth factors and granular cell, egg mother cell interaction, into
And regulate and control the growth and development of ovarian follicle jointly.Steroid hormone plays important role in Ovarian Follicles, studies have shown that
Before grade in ovarian follicle, granular cell does not synthesize the ability of progesterone, at this point, all steroid hormones needed for ovarian follicle are nearly all
It is synthesized by film layer cell；Even if in grade ovarian follicle androgen and estrogen can be provided for ovarian follicle if film layer cell.It is comprehensive
On, we are it can be found that theca layer cell is all of great significance entire Ovarian Follicles.
But it is extremely limited to the research of theca layer cell injuring model and report at present, especially on birds, not
Tangible film layer cell injuring model model reliable at specification.Therefore, a reliable duck theca layer cell separation is established,
Identification, culture model specify it in the status of follicular development and specific mechanism of action, in turn to probing into the characteristic of film layer cell
Improve birds egg laying performances, improve its Egg Quality and have great significance.
Invention content
The present invention is directed to be directed to the prior art on birds without reliable film layer cell injuring model model, one is established
The isolation and culture method of kind duck folliculus ovarii film layer cell compensates for blank on this direction, the duck that the method for the present invention obtains
Folliculus ovarii film layer cell has the characteristics that purity is high, repeatability is strong.
The present invention is realized especially by following technical scheme：
A kind of isolation and culture method of duck folliculus ovarii film layer cell disclosed by the invention, specifically includes following steps：
1) the laying period Liancheng white duck duck neck sacrificed by exsanguination for choosing health, with being cut off after 75% alcohol swab wiped clean
Skin of abdomen takes out ovary and ovarian follicle, then washes away blood stains with PBS buffer solution, and clean folliculus ovarii is put into and is buffered equipped with PBS
In the beaker of liquid, it is transferred in cell culture chamber rapidly；
2) ovary tissue is taken out, grade ovarian follicle is taken out, the connective tissue and rete vasculosum of outer layer is peeled off, uses PBS buffer solution
Clean blood stains are put into the culture dish equipped with PBS buffer solution；
3) avascular area along ovarian follicle surface handle opposite scratches 1~2cm osculums, removes ovarian follicle after yolk and granular cell
Film layer inverts, and is cleaned repeatedly in PBS buffer solution, until yolk and granular cell are washed to the greatest extent；
4) tryptic digestive juice is added, in 37 DEG C of water-baths after oscillation incubation 10min, fetal calf serum termination is added and disappears
Change, tryptic digestive juice is cleaned with PBS buffer solution；
5) morphology is shredded, enzymic digestion liquid is added, is transferred to oscillation incubation 20min in 37 DEG C of water-baths；
6) PBS buffer solution is used to dilute enzymic digestion liquid, the enzymic digestion liquid of transfer cell containing film layer crosses stainless steel cell mesh screen,
It is used in combination PBS buffer solution to rinse mesh screen；
7) cell liquid is transferred to centrifuge tube, 10min is centrifuged with 800g, discards supernatant liquid, use improvement DMEM culture solutions
It is dispensed after cell is resuspended, is put into incubator and cultivates；
8) culture plate wiped clean is placed in 37 DEG C and contains 5%CO with 75% alcohol swab2Incubator constant temperature incubation, after 6h
The supernatant containing haemocyte is discarded, incubator is placed in and continues to cultivate.
Further, the PBS buffer solution is：The NaCl of 8g, the KCl of 0.2g are sequentially added in 500mL deionized waters,
1.56g Na2HPO4·2H2The KH of O, 0.2g2PO4, adjust pH value to 7.2~7.4 with the NaOH of 1mol/L after dissolving, spend from
Sub- water is settled to 1L, and sterilize 30min under the conditions of 121 DEG C of 100kpa.
Further, the tryptic digestive juice is dissolved in 1 liter of PBS by 2.5g trypsase and 0.2g EDTA and obtains.
Further, the enzymic digestion liquid presses mass percentage by 0.3% clostridiopetidase A I types, 0.1%DNase, 4%BSA
Serum-free DMEM is dissolved in be formulated.
Further, the stainless steel filter screen is 200 mesh stainless steel filter screens.
Further, the improvement DMEM culture solutions are：Contain the DMEM of 10% fetal calf serum by percent by volume.
Beneficial effects of the present invention are：
The isolation and culture method of duck folliculus ovarii film layer cell provided by the invention, the reagent and culture medium of use
Commercially available acquisition, it is cheap, economical and practical and easy to operation；The duck folliculus ovarii film layer obtained using the method for the present invention
Cell purity is high, and adherent close, cellular morphology is normal, and activity is good, and cultural method is reliable and stable, and repeatability is strong, is suitble to big
Scale is promoted the use of.
Description of the drawings
Fig. 1 be 1 duck theca layer cell injuring model of the embodiment of the present invention for 24 hours, 48h, 72h, 96h, 120h, 144h and
The microphoto of 168h；
Fig. 2 be 1 duck theca layer cell injuring model of the embodiment of the present invention for 24 hours, 48h, 72h, 96h, 120h, 144h and
The MTT cell growth curves of 168h；
Fig. 3 is duck folliculus ovarii film layer cellular immunofluorescence (FITC) mirror at the time point corresponding with Fig. 1 of the embodiment of the present invention 1
Determine picture.
Specific implementation mode
Technical solution of the present invention is further described with reference to embodiment, it is as described below, only it is to the present invention
Preferred embodiment not limits the present invention, any person skilled in the art possibly also with
The technology contents of the disclosure above are changed or are modified as the equivalent embodiment changed on an equal basis.It is every without departing from the present invention program
Hold, according to the technical essence of the invention any simple modification, equivalent change and modification made to the above embodiment, all falls within this
In the protection domain of invention.
Unless otherwise specified, biochemical reagents as used in the following examples are commercial reagent, skill used in embodiment
The conventional means that art means are well known to those skilled in the art.
Instrument and reagent：
Constant temperature cell incubator (WTC Binder, Germany), 6 hole Tissue Culture Dish (corning, America), bar
This moral suction pipe, superclean bench, water-bath, the stainless steel filter screen (Suo Laibao, China) of 200 mesh, syringe filters
(corning, America), Nikon ECLIPSE 90i fluorescence microscopes (Nikon, Japan), OLYMPUS IX 70 are inverted
Microscope (Nikon, Japan), centrifuge (Thermo, Germany), ultra-pure water instrument.
DMEM-F12 (Hyclone, USA), fetal calf serum (hyclone, USA), PBS (the green skies, China), trypsase
(Gibco, USA), DNase (Coolaber, China), BSA (Gibco, USA), Collagenase I types (Gibco, USA), MTT
(Amresco, USA), DMSO (Solarbio, China) DAPI (the green skies, China), paraformaldehyde (Solarbio, China),
TritonX-100, confining liquid (Amresco, USA), Tween-20 (the green skies, China), Anti-CYP17Rabbit
Ployclonal (Bioss, China), Anti-CYP19Rabbit ployclonal (Bioss, China), FITC-Goat
Anti-Rabbit Antibody(EARTHOX,USA)。
Film layer cellular immunofluorescence is identified：
The film layer cell of culture for 24 hours is taken out, PBS oscillations washing 3 times, each 3min are used.PBS is outwelled, 4% poly is added
Formaldehyde outwells paraformaldehyde after fixing 30min, with PBS oscillations washing 3 times, 2min/ times, during which keeps being protected from light.Penetrate liquid
(0.05% TritonX-100 and 1% Tween-20 are dissolved in PBS solution) room temperature, which is protected from light, is incubated 30min, discards and penetrates
Liquid, with PBS oscillations washing 2 times, 2min/ times.Confining liquid is added, 37 DEG C of closing 1h discard confining liquid, and PBS oscillations are washed 3 times,
2min/ times.
By primary antibody dilution (according to 1：50 volume ratios are diluted) be separately added into theca cell (CYP17, CYP19,
FSHR) then PBS is used to replace primary antibody with corresponding granular cell (FSHR), control group, be incubated overnight under 4 degree of light protected environments, PBS
Oscillation washing 3 times, 2min/ times.Primary antibody Incubating Solution is discarded, PBS is cleaned 3 times, and 3min/ times, secondary antibody diluent (1 is added:200 bodies
Product ratio) 37 DEG C be incubated 1h.Secondary antibody diluent is discarded, PBS is cleaned 3 times, 3min/ times, during which paid attention to keeping being protected from light.It is added and uses
500 μ L of PBS diluted DAPI (1ng/mL) solution, room temperature, which is protected from light, is incubated 10min.DAPI solution is discarded, PBS is cleaned 3 times,
2min/ times.
To prevent from quenching, microscopy is observed as early as possible after dyeing, acquires image.The cell of the corresponding albumen of expression can show that green is glimmering
Light, and karyon can then be displayed in blue fluorescence.
Embodiment 1
A kind of isolation and culture method of duck folliculus ovarii film layer cell, is completed especially by following steps：
Prepare PBS buffer solution：The NaCl of 8g, the KCl of 0.2g are sequentially added in 500mL deionized waters, 1.56g's
Na2HPO4·2H2The KH of O, 0.2g2PO4, pH value is adjusted to 7.2~7.4 with the NaOH of 1mol/L after dissolving, is determined with deionized water
Hold to 1L, sterilize 30min under the conditions of 121 DEG C of 100kpa, spare.Choose 180 age in days egg-laying peak Liancheng white ducks of health
Duck neck sacrificed by exsanguination takes out ovary and ovarian follicle with abdominal cut skin after 75% alcohol swab wiped clean, then uses phosphate
Buffer solution (phosphate buffer saline, PBS) washes away blood stains, and clean folliculus ovarii is put into equipped with PBS's
In 500mL beakers, it is transferred in cell culture chamber rapidly.
Ovary tissue is taken out, grade ovarian follicle is taken out, the connective tissue and rete vasculosum of outer layer are peeled off, equipped with PBS's
Blood stains are cleaned in 500mL beakers to be put into the culture dish equipped with PBS.Avascular area along ovarian follicle surface handle opposite scratches 1-2cm
Osculum removes yolk and granular cell, then inverts theca layer, cleaned repeatedly in PBS, until yolk and granular cell
It is washed to the greatest extent.Trypsase is added, tryptic digestive juice is dissolved in 1 liter of PBS by 2.5g trypsase and 0.2g EDTA and obtains,
The oscillation incubation 10min in 37 DEG C of water-baths is added fetal calf serum and terminates digestion, trypsase is cleaned with PBS.
Morphology is shredded, be added enzymic digestion liquid, enzymic digestion liquid by mass percentage by 0.3% clostridiopetidase A I types,
0.1%DNase, 4%BSA are dissolved in serum-free DMEM and are formulated, and are transferred to oscillation incubation 20min in 37 DEG C of water-baths.Using ice-cold
(4 DEG C) PBS dilute enzymic digestion liquid, transfer the cell containing film layer enzymic digestion liquid cross 200 mesh stainless steel cell mesh screens, PBS is used in combination
Rinse mesh screen.Cell liquid is transferred to 15ml centrifuge tubes, 10min is centrifuged with 800g after sealing, carefully discards supernatant liquid, is used
Contain improvement Du Shi Eagle's mediums (the dulbecco's modified eagle of 10% serum by percent by volume
Medium, DMEM) cell is resuspended, it dispenses according to demand, is put into incubator and cultivates.Blackboard eraser will be cultivated with 75% alcohol swab
After wiping totally, it is placed in 37 DEG C and contains 5%CO2Incubator constant temperature incubation, 6h change liquid, discard the supernatant containing haemocyte, are placed in culture
Case continues to cultivate.
Cellular morphology is observed：
After cell inoculation, is observed in detail with microscope daily, records cellular morphology and growing state, acquisition image is as schemed
Shown in 1.
As shown in Fig. 2, observing duck folliculus ovarii film layer cellular morphology using inverted microscope, thecal cells are in epithelium
Sample, and theca of follicle of von Baer cell is in threadiness.After the incubation of 1d, film layer cell is closely adherent.The proliferation period of film layer cell
Continue three days, during which proliferation is very rapid, and entire culture dish is paved with substantially in 3d caudacorias confluent monolayer cells.Cell integrally enters flat later
The steady phase, until after 5d, film layer cell total body density starts to become smaller, and in 6~7d, its density then significantly decays.
Film layer cellular immunofluorescence is identified：
As shown in figure 3, granular cell can express FSHR reaction and with its antibody response, show FITC green fluorescences.But film
Confluent monolayer cells can express CYP17/19 albumen, not express FSHR albumen, so can react and show with CYP17/19antibodies
FITC green fluorescences, and cannot be with FSHR antibody reaction solutions.
Embodiment 2
The isolation and culture method of duck folliculus ovarii film layer cell, operating procedure are：
Prepare PBS buffer solution：The NaCl of 8g, the KCl of 0.2g are sequentially added in 500mL deionized waters, 1.56g's
Na2HPO4·2H2The KH of O, 0.2g2PO4, pH value is adjusted to 7.2~7.4 with the NaOH of 1mol/L after dissolving, is determined with deionized water
Hold to 1L, sterilize 30min under the conditions of 121 DEG C of 100kpa, spare.Choose 750 age in days laying period Liancheng white duck ducks of health
Neck sacrificed by exsanguination takes out ovary and ovarian follicle with abdominal cut skin after 75% alcohol swab wiped clean, then uses phosphate-buffered
Liquid (phosphate buffer saline, PBS) washes away blood stains, and clean folliculus ovarii, which is put into the 200mL equipped with PBS, to be burnt
In cup, it is transferred in cell culture chamber rapidly.
Ovary tissue is taken out, grade ovarian follicle is taken out, the connective tissue and rete vasculosum of outer layer are peeled off, equipped with PBS's
Blood stains are cleaned in 200mL beakers to be put into the culture dish equipped with PBS.Avascular area along ovarian follicle surface handle opposite scratches 1-2cm
Osculum removes yolk and granular cell, then inverts theca layer, cleaned repeatedly in PBS, until yolk and granular cell
It is washed to the greatest extent.Trypsase is added, tryptic digestive juice is dissolved in 1 liter of PBS by 2.5g trypsase and 0.2g EDTA and obtains,
The oscillation incubation 10min in 37 DEG C of water-baths is added fetal calf serum and terminates digestion, trypsase is cleaned with PBS.
Morphology is shredded, be added enzymic digestion liquid, enzymic digestion liquid by mass percentage by 0.3% clostridiopetidase A I types,
0.1%DNase, 4%BSA are dissolved in serum-free DMEM and are formulated, and are transferred to oscillation incubation 20min in 37 DEG C of water-baths.Using ice-cold
(4 DEG C) PBS dilute enzymic digestion liquid, transfer the cell containing film layer enzymic digestion liquid cross 200 mesh stainless steel cell mesh screens, PBS is used in combination
Rinse mesh screen.Cell liquid is transferred to 15ml centrifuge tubes, 10min is centrifuged with 800g after sealing, carefully discards supernatant liquid, is used
Contain improvement Du Shi Eagle's mediums (the dulbecco's modified eagle of 10% serum by percent by volume
Medium, DMEM) cell is resuspended, it dispenses according to demand, is put into incubator and cultivates.Blackboard eraser will be cultivated with 75% alcohol swab
After wiping totally, it is placed in 37 DEG C and contains 5%CO2Incubator constant temperature incubation, 6h change liquid, discard the supernatant containing haemocyte, are placed in culture
Case continues to cultivate.
Cellular morphology is observed：
After cell inoculation, is observed in detail with microscope daily, records cellular morphology and growing state, acquire image.
Duck folliculus ovarii film layer cellular morphology is observed using inverted microscope, thecal cells are in Epithelial, and ovarian follicle
Pericyte is in threadiness.After the incubation of 1d, film layer cell is closely adherent.The proliferation period of film layer cell continues three days,
Period proliferation is very rapid, and entire culture dish is paved with substantially in 3d caudacorias confluent monolayer cells.Cell integrally enters the stage of stable development later, until
After 5d, film layer cell total body density starts to become smaller, and in 6~7d, its density then significantly decays.
Film layer cellular immunofluorescence is identified：
Granular cell can express FSHR reaction and with its antibody response, show FITC green fluorescences.But film layer cell energy table
Up to CYP17/19 albumen, FSHR albumen is not expressed, so can be reacted with CYP17/19antibodies and show that FITC greens are glimmering
Light, and cannot be with FSHR antibody reaction solutions.
Embodiment 3
Prepare PBS buffer solution：The NaCl of 8g, the KCl of 0.2g are sequentially added in 500mL deionized waters, 1.56g's
Na2HPO4·2H2The KH of O, 0.2g2PO4, pH value is adjusted to 7.2~7.4 with the NaOH of 1mol/L after dissolving, is determined with deionized water
Hold to 1L, sterilize 30min under the conditions of 121 DEG C of 100kpa, spare.The 220 age in days egg-laying peak Beijing ducks for choosing health are female
Duck neck portion sacrificed by exsanguination takes out ovary and ovarian follicle with abdominal cut skin after 75% alcohol swab wiped clean, then slow with phosphate
Fliud flushing (phosphate buffer saline, PBS) washes away blood stains, and clean folliculus ovarii is put into the 200mL equipped with PBS
In beaker, it is transferred in cell culture chamber rapidly.
Ovary tissue is taken out, grade ovarian follicle is taken out, the connective tissue and rete vasculosum of outer layer are peeled off, equipped with PBS's
Blood stains are cleaned in 200mL beakers to be put into the culture dish equipped with PBS.Avascular area along ovarian follicle surface handle opposite scratches 1-2cm
Osculum removes yolk and granular cell, then inverts theca layer, cleaned repeatedly in PBS, until yolk and granular cell
It is washed to the greatest extent.Trypsase is added, tryptic digestive juice is dissolved in 1 liter of PBS by 2.5g trypsase and 0.2g EDTA and obtains,
The oscillation incubation 15min in 37 DEG C of water-baths is added fetal calf serum and terminates digestion, trypsase is cleaned with PBS.
Morphology is shredded, be added enzymic digestion liquid, enzymic digestion liquid by mass percentage by 0.3% clostridiopetidase A I types,
0.1%DNase, 4%BSA are dissolved in serum-free DMEM and are formulated, and are transferred to oscillation incubation 30min in 37 DEG C of water-baths.Using ice-cold
(4 DEG C) PBS dilute enzymic digestion liquid, transfer the cell containing film layer enzymic digestion liquid cross 200 mesh stainless steel cell mesh screens, PBS is used in combination
Rinse mesh screen.Cell liquid is transferred to 15ml centrifuge tubes, 10min is centrifuged with 800g after sealing, carefully discards supernatant liquid, is used
Contain improvement Du Shi Eagle's mediums (the dulbecco's modified eagle of 10% serum by percent by volume
Medium, DMEM) cell is resuspended, it dispenses according to demand, is put into incubator and cultivates.Blackboard eraser will be cultivated with 75% alcohol swab
After wiping totally, it is placed in 37 DEG C and contains 5%CO2Incubator constant temperature incubation, 6h change liquid, discard the supernatant containing haemocyte, are placed in culture
Case continues to cultivate.
Cellular morphology is observed：
After cell inoculation, is observed in detail with microscope daily, records cellular morphology and growing state.
Duck folliculus ovarii film layer cellular morphology is observed using inverted microscope, thecal cells are in Epithelial, and ovarian follicle
Pericyte is in threadiness.After the incubation of 1d, film layer cell is closely adherent.The proliferation period of film layer cell continues three days,
Period proliferation is very rapid, and entire culture dish is paved with substantially in 3d caudacorias confluent monolayer cells.Cell integrally enters the stage of stable development later, until
After 5d, film layer cell total body density starts to become smaller, and in 6~7d, its density then significantly decays.
Embodiment 4
Prepare PBS buffer solution：The NaCl of 8g, the KCl of 0.2g are sequentially added in 500mL deionized waters, 1.56g's
Na2HPO4·2H2The KH of O, 0.2g2PO4, pH value is adjusted to 7.2~7.4 with the NaOH of 1mol/L after dissolving, is determined with deionized water
Hold to 1L, sterilize 30min under the conditions of 121 DEG C of 100kpa, spare.Choose the 150 age in days laying periods black duck duck duck neck of health
Portion's sacrificed by exsanguination takes out ovary and ovarian follicle with abdominal cut skin after 75% alcohol swab wiped clean, then uses phosphate buffer
(phosphate buffer saline, PBS) washes away blood stains, and clean folliculus ovarii is put into the 300mL beakers equipped with PBS
In, it is transferred in cell culture chamber rapidly.
Ovary tissue is taken out, grade ovarian follicle is taken out, the connective tissue and rete vasculosum of outer layer are peeled off, equipped with PBS's
Blood stains are cleaned in 300mL beakers to be put into the culture dish equipped with PBS.Avascular area along ovarian follicle surface handle opposite scratches 1-2cm
Osculum removes yolk and granular cell, then inverts theca layer, cleaned repeatedly in PBS, until yolk and granular cell
It is washed to the greatest extent.Trypsase is added, tryptic digestive juice is dissolved in 1 liter of PBS by 2.5g trypsase and 0.2g EDTA and obtains,
The oscillation incubation 10min in 37 DEG C of water-baths is added fetal calf serum and terminates digestion, trypsase is cleaned with PBS.
Morphology is shredded, be added enzymic digestion liquid, enzymic digestion liquid by mass percentage by 0.3% clostridiopetidase A I types,
0.1%DNase, 4%BSA are dissolved in serum-free DMEM and are formulated, and are transferred to oscillation incubation 30min in 37 DEG C of water-baths.Using ice-cold
(4 DEG C) PBS dilute enzymic digestion liquid, transfer the cell containing film layer enzymic digestion liquid cross 200 mesh stainless steel cell mesh screens, PBS is used in combination
Rinse mesh screen.Cell liquid is transferred to 15ml centrifuge tubes, 10min is centrifuged with 800g after sealing, carefully discards supernatant liquid, is used
Contain improvement Du Shi Eagle's mediums (the dulbecco's modified eagle of 10% serum by percent by volume
Medium, DMEM) cell is resuspended, it dispenses according to demand, is put into incubator and cultivates.Blackboard eraser will be cultivated with 75% alcohol swab
After wiping totally, it is placed in 37 DEG C and contains 5%CO2Incubator constant temperature incubation, 8h change liquid, discard the supernatant containing haemocyte, are placed in culture
Case continues to cultivate.
Cellular morphology is observed：
After cell inoculation, is observed in detail with microscope daily, records cellular morphology and growing state.
Duck folliculus ovarii film layer cellular morphology is observed using inverted microscope, thecal cells are in Epithelial, and ovarian follicle
Pericyte is in threadiness.After the incubation of 1d, film layer cell is closely adherent.The proliferation period of film layer cell continues three days,
Period proliferation is very rapid, and entire culture dish is paved with substantially in 3d caudacorias confluent monolayer cells.Cell integrally enters the stage of stable development later, until
After 5d, film layer cell total body density starts to become smaller, and in 6~7d, its density then significantly decays.Although there has been shown and described that
The embodiment of the present invention, for the ordinary skill in the art, it is possible to understand that do not depart from the principle of the present invention and
A variety of change, modification, replacement and modification can be carried out to these embodiments, the scope of the present invention is by appended power in the case of spirit
Profit requires and its equivalent limits.
Claims (6)
1. a kind of isolation and culture method of duck folliculus ovarii film layer cell, which is characterized in that include the following steps：
1) the laying period Liancheng white duck duck neck sacrificed by exsanguination for choosing health, with abdominal cut after 75% alcohol swab wiped clean
Skin takes out ovary and ovarian follicle, PBS buffer solution wash away blood stains, and clean folliculus ovarii is put into the beaker equipped with PBS buffer solution
In, it is transferred in cell culture chamber rapidly；
2) ovary tissue is taken out, grade ovarian follicle is taken out, peels the connective tissue and rete vasculosum of outer layer off, is cleaned with PBS buffer solution
Blood stains are put into the culture dish equipped with PBS buffer solution；
3) avascular area along ovarian follicle surface handle opposite scratches 1~2cm osculums, removes theca layer after yolk and granular cell
Reversion, is cleaned repeatedly in PBS buffer solution, until yolk and granular cell are washed to the greatest extent；
4) tryptic digestive juice is added, in 37 DEG C of water-baths after oscillation incubation 10min, fetal calf serum is added and terminates digestion, uses
PBS buffer solution cleans tryptic digestive juice；
5) morphology is shredded, enzymic digestion liquid is added, is transferred to oscillation incubation 20min in 37 DEG C of water-baths；
6) PBS buffer solution is used to dilute enzymic digestion liquid, the enzymic digestion liquid of transfer cell containing film layer is crossed stainless steel cell mesh screen, is used in combination
PBS buffer solution rinses mesh screen；
7) cell liquid is transferred to centrifuge tube, 10min is centrifuged with 800g, discards supernatant liquid, it will be thin using improvement DMEM culture solutions
Born of the same parents dispense after being resuspended, and are put into incubator and cultivate；
8) culture plate wiped clean is placed in 37 DEG C and contains 5%CO with 75% alcohol swab2Incubator constant temperature incubation discards after 6h and contains
The supernatant of haemocyte is placed in incubator and continues to cultivate.
2. a kind of isolation and culture method of duck folliculus ovarii film layer cell according to claim 1, which is characterized in that institute
The PBS buffer solution stated is：The NaCl of 8g, the Na of the KCl of 0.2g, 1.56g are sequentially added in 500mL deionized waters2HPO4·
2H2The KH of O, 0.2g2PO4, pH value is adjusted to 7.2~7.4 with the NaOH of 1mol/L after dissolving, and 1L is settled to deionized water,
Sterilize 30min under the conditions of 121 DEG C of 100kpa.
3. a kind of isolation and culture method of duck folliculus ovarii film layer cell according to claim 1, which is characterized in that institute
The tryptic digestive juice stated is dissolved in 1 liter of PBS by 2.5g trypsase and 0.2g EDTA and obtains.
4. a kind of isolation and culture method of duck folliculus ovarii film layer cell according to claim 1, which is characterized in that institute
The enzymic digestion liquid stated is formulated by mass percentage by 0.3% clostridiopetidase A I types, 0.1%DNase, 4%BSA.
5. a kind of isolation and culture method of duck folliculus ovarii film layer cell according to claim 1, which is characterized in that institute
The improvement DMEM culture solutions stated are：Contain the DMEM of 10% fetal calf serum by percent by volume.
6. a kind of isolation and culture method of duck folliculus ovarii film layer cell according to claim 1, which is characterized in that institute
It is 200 mesh stainless steel filter screens to state stainless steel filter screen.
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