CN106834207A - A kind of Shelled Turtle Trionyx Sinensis gonad cell is separated and extracorporeal culturing method - Google Patents
A kind of Shelled Turtle Trionyx Sinensis gonad cell is separated and extracorporeal culturing method Download PDFInfo
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- CN106834207A CN106834207A CN201611152595.8A CN201611152595A CN106834207A CN 106834207 A CN106834207 A CN 106834207A CN 201611152595 A CN201611152595 A CN 201611152595A CN 106834207 A CN106834207 A CN 106834207A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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Abstract
Separated the invention discloses a kind of Shelled Turtle Trionyx Sinensis gonad cell and extracorporeal culturing method, comprised the following steps：(1) the female soft-shelled turtle of sex immature is chosen, in vitro ovary tissue is rinsed after crushing, is placed in container by the sterilized rear in vitro ovary tissue of taking-up with phosphate buffer；(2) using clostridiopetidase A and pancreatin to flushing after in vitro ovary tissue successively carry out digestion process, be then centrifuged for collect cell；(3) cell of collection is placed in treated cell culture container and adds complete medium to be cultivated；(4) after cell propagation is paved with cell culture container bottom, with the culture of pancreatin had digestive transfer culture or freezen protective, that is, Shelled Turtle Trionyx Sinensis gonad cell is obtained.The method step is novel, succinct convenience, repeatability are strong, can obtain the Shelled Turtle Trionyx Sinensis ovary cell line of high-survival rate.
Description
Technical field
Separated the invention belongs to gonad cell and Vitro Culture Techniques field, and in particular to a kind of Shelled Turtle Trionyx Sinensis gonad cell point
From and extracorporeal culturing method.
Background technology
So far, studies in China person has attempted the cell culture of the various body tissues of terrapin animal, including embryo is primary
The histiocytic culture such as cell culture, the brain of tortoise and liver, spleen, kidney and heart.Wherein except the fiber of heart tissue
Cell culture obtains the cell line of stabilization, and only 10 generations occurred as soon as " spontaneous nuclear transformation " for other histiocytic basic cultures of culture
Phenomenon or decline, and then cell cannot continue Secondary Culture.Even having obtained the cardiac fibers cell line of stable cell lines, phase
Equally it is slow-growing for other animal cell lines, has stronger dependence to temperature, generation time etc..Cannot obtain
The testudinate animal cell line of well-grown stabilization is stemmed primarily from does not develop the cell for being suitable to terrapin cell growth training
Support base and breeding condition.
Testudinate animal is used as traditional invigorant, because excessively being opened with high medicinal, nutrition and ornamental value
Hair is utilized, so that its wildlife species resource is increasingly deficient or even endangered.Many testudinate animals, such as Mauremys mutica and soft-shelled turtle
It is extremely limited Deng its wild natural resources, it has also become protection species in imminent danger.Although by making great efforts for many years and experience accumulation, existing
The artificial breeding aspect of various terrapin rareness species achieves breakthrough.But the kind quality guarantee to these species both at home and abroad
Deposit, the research of the aspect such as cell engineering breeding technique is also extremely limited, thus seriously hinder these species enhancement releasing and
The development of the research work such as resource reparation.
The content of the invention
Separated it is an object of the invention to provide a kind of Shelled Turtle Trionyx Sinensis gonad cell and extracorporeal culturing method, the method step is new
Clever, succinct convenience, repeatability are strong, can obtain the Shelled Turtle Trionyx Sinensis ovary cell line of high-survival rate.
Above-mentioned purpose of the invention is achieved by the following technical solution：A kind of Shelled Turtle Trionyx Sinensis gonad cell is separated and body
Outer cultural method, comprises the following steps：
(1) the female soft-shelled turtle of sex immature, the sterilized rear in vitro ovary tissue of taking-up, by vitro ovary tissue phosphate are chosen
After wash buffer is broken, it is placed in container；
(2) using clostridiopetidase A and pancreatin to flushing after in vitro ovary tissue successively carry out digestion process, be then centrifuged for receive
Collection cell；
(3) cell of collection is placed in treated cell culture container and adds complete medium to be cultivated；
(4) after cell propagation is paved with cell culture container bottom, with the culture of pancreatin had digestive transfer culture or freezen protective, that is, obtain
Obtain Shelled Turtle Trionyx Sinensis gonad cell.
In the separation of above-mentioned Shelled Turtle Trionyx Sinensis gonad cell and extracorporeal culturing method：
Preferential 1~2 age in the winter female soft-shelled turtle for choosing sex immature in step (1).
Preferably used during sterilization treatment in step (1) volumn concentration for 60~90% alcohol, more preferably 70%.
The concentration of clostridiopetidase A is preferably 0.5~2mg/mL, more preferably 1mg/mL described in step (2), and the clostridiopetidase A is excellent
Choose from sigma companies, digestion time is preferably 20~40min, more preferably 30min；The weight/mass percentage composition of the pancreatin is excellent
Elect 0.01~0.5%, more preferably 0.025% as, the pancreatin is preferably purchased from Invitrogen companies, digestion time is preferably 20
~40min, more preferably 20min.
Cow's serum terminating reaction is preferably used after digestion process is complete in step (2), then cell is collected by centrifugation.
The composition of following consumption is preferably included in step (3) in 1L complete mediums：DMEM in high glucose (basal medium) 13
~14g, 4~5g of pH Hepes, 0.01~0.03mM of nonessential amino acid, 1~2nM of sodium selenite, Sodium Pyruvate 1
~2mM, 200~300 μM of 2 mercapto ethanol, 50~80 μM of antibiotic penicillin/streptomycin, hyclone 100
~200mL, growth factor 4~6 nanogram.
Present inventor studies by lot of experiments and finds, it is thin that the complete medium in the present invention is relatively adapted to culture soft-shelled turtle
Born of the same parents.
Cell culture container described in step (3) is preferably with the gelatin coating that weight/mass percentage composition is 0.1~0.5%
Reason.
The invention has the advantages that：
(1) Shelled Turtle Trionyx Sinensis gonad cell of the present invention is separated and extracorporeal culturing method, and step is novel, succinct convenience, repeatability are strong,
The Shelled Turtle Trionyx Sinensis ovary cell line of high-survival rate can be obtained；
(2) foundation of testudinate animal cell culture technology and cell line that the present invention is carried out, is suitable to the training of testudinate zooblast
Support and the stable culture medium and condition of culture for passing on；
(3) culture of the Shelled Turtle Trionyx Sinensis gonad cell that the present invention is carried out is not only that the research of terrapin Sex Determination Mechanism is provided not
The in vitro study system that can or lack, what is more important provides good in vitro study for research testudinate animals' reproduction physiology etc.
Platform, the research that new technology is bred further to carry out testudinate animal provides Research foundation and platform.
Brief description of the drawings
Fig. 1 is the separation of Shelled Turtle Trionyx Sinensis gonad cell and culture flow chart in embodiment 1-3；
Fig. 2 is the Shelled Turtle Trionyx Sinensis gonad cell of subculture in vitro separately culture in embodiment 1-3；
Fig. 3 is the chromosome analysis of cultured cell in vitro in embodiment 1-3；
Fig. 4 is the gene expression checking of cultured cell in vitro in embodiment 1-3.
Specific embodiment
Specific embodiment of the invention is specifically further illustrated with reference to example.
Embodiment 1
As shown in figure 1, the Shelled Turtle Trionyx Sinensis gonad cell that the present embodiment is provided is separated and extracorporeal culturing method, including following step
Suddenly：
(1) 1 age in winter female soft-shelled turtle of sex immature is chosen, etherization bloodletting, the sprinkling of 70% (volumn concentration) alcohol are gone out
Bacterium, dissect and take out ovary tissue, ovary tissue phosphate buffer is rinsed and shredded, load test tube；
(2) with clostridiopetidase A and pancreatin successively digestion Shelled Turtle Trionyx Sinensis ovary tissue, it is then centrifuged for collecting cell；
(3) cell that will be collected by centrifugation is put into treated Tissue Culture Flask and adds complete medium to be cultivated；
(4) after cell propagation is paved with bottom of bottle, with the culture of pancreatin had digestive transfer culture or freezen protective, that is, Shelled Turtle Trionyx Sinensis ovary is obtained
Cell.
In above-mentioned steps：
Clostridiopetidase A and pancreatin in step (2) digest, and detailed process is：First with the (digestion of sigma companies 1mg/mL clostridiopetidase As
The volume of liquid there was not tissue) room temperature digestion tissue block 40 minutes, it is subsequently adding the pancreatin (final concentration of Invitrogen companies
0.025%, weight/mass percentage composition, digestive juice did not had tissue) room temperature digest 30 minutes, then with 5%, (volume basis contain
Amount) cow's serum terminating reaction.
In step (3), blake bottle is processed using 0.01% (weight/mass percentage composition) gelatin coating.
In step (3), the composition (1L) of complete medium：14 grams of DMEM in high glucose (Invitrogen, basal medium),
4.76 grams of Hepes (Sigma, pH), 0.01mM nonessential amino acid (Invitrogen), 2nM sodium selenites
(Sigma), 2mM Sodium Pyruvates (Invitrogen), 200 μM of 2 mercapto ethanols (Sigma), 50 μM of antibiotic
(penicillin/streptomycin), 150mL hyclones (Fetal Bovine Serum；PAA,cat.no.A15-
108), 5 nanogram growth factors (PetroTech Inc, cat.no.100-18B).
The Shelled Turtle Trionyx Sinensis gonad cell of acquisition is carried out into the analysis of cellular morphology observation, chromosome number and gene expression detection,
The wherein Shelled Turtle Trionyx Sinensis gonad cell of subculture in vitro separately culture as shown in Fig. 2 as can be seen from Figure 2 cellular morphology rule is homogeneous, carefully
Born of the same parents' adherent growth is in good condition, and the chromosome analysis of cultured cell in vitro is as shown in figure 3, as can be seen from Figure 3 cell dyeing
Body number is consistent with report, illustrate in cell cultivation process without generation " inverting phenomenon (and chromosome deficiency and cause cell to wither
Die) ".
Gene expression analysis as shown in figure 4, the expression analysis of germ cells gene show that cell characteristics do not change, also
Keep (Fig. 4) consistent with the reproduction cell characteristic in ovary tissue.
The present embodiment Shelled Turtle Trionyx Sinensis gonad cell is separated and extracorporeal culturing method includes unique cell isolation method and culture
Method, can obtain Shelled Turtle Trionyx Sinensis ovary cell line that is repeated strong and can obtaining stabilization.
Embodiment 2
As shown in figure 1, the Shelled Turtle Trionyx Sinensis gonad cell that the present embodiment is provided is separated and extracorporeal culturing method, including following step
Suddenly：
(1) choose sex immature 1 age in winter female soft-shelled turtle, etherization bloodletting, 80% (volumn concentration) alcohol sprinkling sterilizing,
Ovary tissue is dissected and taken out, ovary tissue phosphate buffer is rinsed and shredded, load test tube；
(2) with clostridiopetidase A and pancreatin successively digestion Shelled Turtle Trionyx Sinensis ovary tissue, it is then centrifuged for collecting cell；
(3) cell that will be collected by centrifugation is put into treated Tissue Culture Flask and adds complete medium to be cultivated；
(4) after cell propagation is paved with bottom of bottle, with the culture of pancreatin had digestive transfer culture or freezen protective, that is, Shelled Turtle Trionyx Sinensis ovary is obtained
Cell.
In above-mentioned steps：
Clostridiopetidase A and pancreatin in step (2) digest, and detailed process is：First with the (digestion of sigma companies 2mg/mL clostridiopetidase As
The volume of liquid there was not tissue) room temperature digestion tissue block 20 minutes, it is subsequently adding 0.05% pancreatin of Invitrogen companies
(digestive juice did not had tissue) room temperature digests 20 minutes, then with 5% (volumn concentration) cow's serum terminating reaction.
In step (3), blake bottle is processed using 0.03% (weight/mass percentage composition) gelatin coating.
In step (3), the composition (1L) of complete medium：13.37 grams of DMEM in high glucose (Invitrogen, basis culture
Base), 4.76 grams of Hepes (Sigma, pH), 0.02mM nonessential amino acid (Invitrogen), 1nM sodium selenites
(Sigma), 1mM Sodium Pyruvates (Invitrogen), 100 μM of 2 mercapto ethanols (Sigma), 50 μM of antibiotic
(penicillin/streptomycin), 200mL hyclones (Fetal Bovine Serum；PAA,cat.no.A15-
108), 6 nanogram growth factors (PetroTech Inc, cat.no.100-18B).
The Shelled Turtle Trionyx Sinensis gonad cell of acquisition is carried out into the analysis of cellular morphology observation, chromosome number and gene expression detection,
Wherein subculture in vitro separately culture Shelled Turtle Trionyx Sinensis gonad cell as shown in Fig. 2 cultured cell in vitro chromosome analysis as shown in figure 3,
The expression analysis of germ cells gene, illustrate that cell characteristics do not change, and also keep and the reproduction cell spy in ovary tissue
Property consistent (Fig. 4).
The present embodiment Shelled Turtle Trionyx Sinensis gonad cell is separated and extracorporeal culturing method includes unique cell isolation method and culture
Method, can obtain Shelled Turtle Trionyx Sinensis ovary cell line that is repeated strong and can obtaining stabilization.
Embodiment 3
As shown in figure 1, the Shelled Turtle Trionyx Sinensis gonad cell that the present embodiment is provided is separated and extracorporeal culturing method, including following step
Suddenly：
(1) choose sex immature 2 age in winter female soft-shelled turtle, etherization bloodletting, 90% (volumn concentration) alcohol sprinkling sterilizing,
Ovary tissue is dissected and taken out, ovary tissue phosphate buffer is rinsed and shredded, load test tube；
(2) with clostridiopetidase A and pancreatin successively digestion Shelled Turtle Trionyx Sinensis ovary tissue, it is then centrifuged for collecting cell；
(3) cell that will be collected by centrifugation is put into treated Tissue Culture Flask and adds complete medium to be cultivated；
(4) after cell propagation is paved with bottom of bottle, with the culture of pancreatin had digestive transfer culture or freezen protective, that is, Shelled Turtle Trionyx Sinensis ovary is obtained
Cell.
In above-mentioned steps：
Clostridiopetidase A and pancreatin in step (2) digest, and detailed process is：First (disappeared with sigma companies 1.5mg/mL clostridiopetidase As
The volume for changing liquid there was not tissue) room temperature digests tissue block 30 minutes, and (end is dense to be subsequently adding the pancreatin of Invitrogen companies
Degree 0.0125%, weight/mass percentage composition), (digestive juice did not had tissue) room temperature digests 40 minutes, then with 5% (volume hundred
Divide content) cow's serum terminating reaction.
In step (3), blake bottle is processed using 0.05% (weight/mass percentage composition) gelatin coating.
In step (3), the composition (1L) of complete medium：13.37 grams of DMEM in high glucose (Invitrogen, basis culture
Base), 4.76 grams of Hepes (Sigma, pH), 0.03mM nonessential amino acid (Invitrogen), 1.2nM selenous acid
Sodium (Sigma), 1.2mM Sodium Pyruvates (Invitrogen), 110 μM of 2 mercapto ethanols (Sigma), 60 μM of antibiotic
(penicillin/streptomycin), 100mL hyclones, 6 nanogram growth factors (PetroTech Inc,
cat.no.100-18B)。
The Shelled Turtle Trionyx Sinensis gonad cell of acquisition is carried out into the analysis of cellular morphology observation, chromosome number and gene expression detection,
Wherein subculture in vitro separately culture Shelled Turtle Trionyx Sinensis gonad cell as shown in Fig. 2 cultured cell in vitro chromosome analysis as shown in figure 3,
The expression analysis of germ cells gene, illustrate that cell characteristics do not change, and also keep and the reproduction cell spy in ovary tissue
Property consistent (Fig. 4).
The present embodiment Shelled Turtle Trionyx Sinensis gonad cell is separated and extracorporeal culturing method includes unique cell isolation method and culture
Method, can obtain Shelled Turtle Trionyx Sinensis ovary cell line that is repeated strong and can obtaining stabilization.
The above is only non-limiting implementation method of the invention, for the person of ordinary skill of the art, not
On the premise of departing from the invention design and not making creative work, various modifications and improvements can be made, these are all
Belong to protection scope of the present invention.
Claims (7)
1. a kind of Shelled Turtle Trionyx Sinensis gonad cell is separated and extracorporeal culturing method, it is characterized in that comprising the following steps：
(1) the female soft-shelled turtle of sex immature, the sterilized rear in vitro ovary tissue of taking-up, by vitro ovary tissue phosphate-buffered are chosen
Liquid is rinsed after crushing, and is placed in container；
(2) using clostridiopetidase A and pancreatin to flushing after in vitro ovary tissue successively carry out digestion process, be then centrifuged for collecting thin
Born of the same parents；
(3) cell of collection is placed in treated cell culture container and adds complete medium to be cultivated；
(4) after cell propagation is paved with cell culture container bottom, with the culture of pancreatin had digestive transfer culture or freezen protective, that is, in obtaining
Magnificent soft-shelled turtle gonad cell.
2. Shelled Turtle Trionyx Sinensis gonad cell according to claim 1 is separated and extracorporeal culturing method, it is characterized in that：In step (1)
Choose 1~2 age in winter female soft-shelled turtle of sex immature.
3. Shelled Turtle Trionyx Sinensis gonad cell according to claim 1 is separated and extracorporeal culturing method, it is characterized in that：In step (1)
Used during sterilization treatment volumn concentration for 60~90% alcohol.
4. Shelled Turtle Trionyx Sinensis gonad cell according to claim 1 is separated and extracorporeal culturing method, it is characterized in that：In step (2)
The concentration of the clostridiopetidase A is 0.5~2mg/mL, and the clostridiopetidase A is purchased from sigma companies, and digestion time is 20~40min；Institute
It is 0.01~0.5% to state the weight/mass percentage composition of pancreatin, and the pancreatin is purchased from Invitrogen companies, digestion time is 20~
40min。
5. Shelled Turtle Trionyx Sinensis gonad cell according to claim 1 is separated and extracorporeal culturing method, it is characterized in that：In step (2)
Cow's serum terminating reaction is used after digestion process is complete, then cell is collected by centrifugation.
6. Shelled Turtle Trionyx Sinensis gonad cell according to claim 1 is separated and extracorporeal culturing method, it is characterized in that：In step (3)
1L complete mediums include the composition of following consumption：13~14g of DMEM in high glucose, 4~5g of pH Hepes, it is non-must
Need 0.01~0.05mM of amino acid, 1~2nM of sodium selenite, 1~2mM of Sodium Pyruvate, 200~300 μM of 2 mercapto ethanol, antibiosis
50~80 μM of plain penicillin/streptomycin, 100~200mL of hyclone, growth factor 4~6 nanogram.
7. Shelled Turtle Trionyx Sinensis gonad cell according to claim 1 is separated and extracorporeal culturing method, it is characterized in that：In step (3)
The cell culture container weight/mass percentage composition is 0.1~0.5% gelatin coating treatment.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109370979A (en) * | 2018-11-28 | 2019-02-22 | 中国水产科学研究院淡水渔业研究中心 | A kind of separation of Procambius clarkii maturation egg cell and its primary culture method |
| CN115181718A (en) * | 2022-06-14 | 2022-10-14 | 中国水产科学研究院珠江水产研究所 | Isolation culture method of ovary cells of mauremys mutica |
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| CN103756952A (en) * | 2014-01-09 | 2014-04-30 | 中国水产科学研究院黄海水产研究所 | Establishment and application method of ovary cell line of cynoglossus semilaevis |
| CN106085950A (en) * | 2016-07-04 | 2016-11-09 | 山大生殖研发中心有限公司 | Separate the single celled method of variety classes in ovary |
| KR101680269B1 (en) * | 2003-12-30 | 2016-11-29 | 주식회사 에이치바이온 | Embryonic stem cell line and method for preparing the same |
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| KR101680269B1 (en) * | 2003-12-30 | 2016-11-29 | 주식회사 에이치바이온 | Embryonic stem cell line and method for preparing the same |
| CN102250830A (en) * | 2011-07-05 | 2011-11-23 | 西北农林科技大学 | In-vitro separating and culturing method for germline stem cell |
| CN103756952A (en) * | 2014-01-09 | 2014-04-30 | 中国水产科学研究院黄海水产研究所 | Establishment and application method of ovary cell line of cynoglossus semilaevis |
| CN106085950A (en) * | 2016-07-04 | 2016-11-09 | 山大生殖研发中心有限公司 | Separate the single celled method of variety classes in ovary |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109370979A (en) * | 2018-11-28 | 2019-02-22 | 中国水产科学研究院淡水渔业研究中心 | A kind of separation of Procambius clarkii maturation egg cell and its primary culture method |
| CN115181718A (en) * | 2022-06-14 | 2022-10-14 | 中国水产科学研究院珠江水产研究所 | Isolation culture method of ovary cells of mauremys mutica |
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