CN109371111A - The identification method of cell type in a kind of buffalo stem spermatogonium like cell purification process - Google Patents
The identification method of cell type in a kind of buffalo stem spermatogonium like cell purification process Download PDFInfo
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Abstract
The invention discloses a kind of identification methods of cell type in buffalo stem spermatogonium like cell purification process, the following steps are included: (1) isolates and purifies buffalo testis single cell suspension using the adherent separating method of difference, and collect different type cell obtained in purification process；(2) total serum IgE for the different type cell being collected into total serum IgE kit extraction step (1) then carries out reverse transcription using reverse transcription reagent box and generates cDNA；(3) cDNA for the different type cell for using step (2) to obtain again does quantitative PCR as sample, and respectively with the primer of specific gene, then draws corresponding map.The present invention uses molecular specificity detection method, can quickly and accurately identify the type of cell during difference is adherent.
Description
Technical field
The present invention relates to a kind of technical fields of cellular identification more particularly to a kind of buffalo stem spermatogonium like cell to purify
The identification method of cell type in the process.
Background technique
Buffalo is mainly distributed south China area, is mainly used in labour power, meat and milk production.Buffalo milk nutrition is especially rich
Richness, albumen, electrolytes and minerals etc. are above common milk, according to statistics, nutritive value phase contained by 1 kilogram of buffalo milk
When in 1.85 kilograms of black-and-white flower milk.But the low breeding potential of buffalo and the low output of milk restrict the development of buffalo milk industry, therefore urgently
To be improved by gene technology to buffalo kind.
Currently, the isolation and purification method of stem spermatogonium has magnetic activated cell seperation, selected by flow cytometry apoptosis method, difference patch
Wall separating method, discontinuous density gradient centrifugal process and the adherent combination discontinuous density gradient centrifugal process of difference.Existing research
The stem spermatogonium of buffalo is isolated and purified frequently with difference adherent separating method during buffalo breed improvement, difference is adherent
Separating method is the adherent speed different designs according to stem spermatogonium and other body cells in different extracellular matrixes.It is smart former dry
Cell is easy to adhere on laminin, and other cells are easy to adhere on gelatin, so making testicular cell respectively bright
It cultivates, stem spermatogonium and other cells can be separated on glue and laminin.However, being separated to buffalo stem spermatogonium
Cell type obtained in purification process is not identified, and judges the cell class that final purification obtains using conventional method
Whether type is that stem spermatogonium required for testing is relatively difficult, easily leads to judgement inaccuracy.
Summary of the invention
It is an object of the invention to: in view of the above problems, it is former to provide a kind of accurate, sensitive, convenient buffalo essence
The identification method of cell type in stem cell-like cell purification process.
In order to achieve the above-mentioned object of the invention, The technical solution adopted by the invention is as follows:
The identification method of cell type in a kind of buffalo stem spermatogonium like cell purification process, including including following step
It is rapid:
(1) buffalo testis single cell suspension is isolated and purified using difference adherent separating method, and collects purification process
Obtained in different type cell；
(2) total serum IgE for the different type cell being collected into total serum IgE kit extraction step (1) then uses reverse transcription
Kit carries out reverse transcription and generates cDNA；
(3) cDNA for the different type cell for using step (2) to obtain again is as sample, and drawing with specific gene respectively
Object does quantitative PCR, then draws corresponding map.
Preferably, the specific steps that the difference adherent method separating method isolates and purifies buffalo testis single cell suspension
Are as follows:
(1) the buffalo testis single cell suspension of acquisition is seeded in the culture dish for being covered with gelatin, is cultivated using stem cell
Liquid is incubated overnight, and then will suspend and adherent cell loosely is transferred in the culture dish for being covered with rat tail collagen protein in advance, train
4h is supported, being collected simultaneously the cell being attached on gelatin is G-cell；
(2) training for cell not adherent in the culture dish for be covered with rat tail collagen protein will be seeded in being transferred to laminin
It supports in ware, cultivates 40min, being collected simultaneously the cell being attached in rat tail collagen protein is C-cell；
(3) the cell culture 40min that will be seeded in the culture dish for being covered with laminin abandons floating cells, collects
Attached cell is L-cell.
Preferably, the main component of the stem cell medium are as follows: IMDM basic culture solution, KSR, Fetuin, NEAA,
Chemically Defined Lipidmixture 1, B-27, GDNF, GFR α 1, bFGF, Lif and β-ME.
Preferably, the specific proportion of the stem cell medium are as follows: with IMDM be basic culture solution, the volume ratio of KSR is
The volume ratio of 10%, Fetuin are final concentration of 10 μ L/mL, the Chemically Defined Lipid of 10%, NEAA
The final concentration of 20ng/mL, GFR α 1 of final concentration of 20 the μ L/mL, GDNF of final concentration of 10 the μ L/mL, B-27 of mixture 1
Final concentration of 100ng/mL, the final concentration of 10U/mL of the final concentration of 10ng/mL of bFGF, Lif, β-ME's is final concentration of
0.1mM。
Preferably, the culture is to be cultivated in 37 DEG C of incubator.
Preferably, the buffalo testis single cell suspension the preparation method comprises the following steps:
(1) pretreatment of raw material: tunica albuginea is cut off in super-clean bench after water intaking bull testis disinfection, cleans up and is placed on culture dish
In be cut into small pieces, transfer in EP pipe and shred；
(2) it digests: the testis tissue after shredding in step (1) is transferred in culture dish, level-one digestive juice is added and is placed in
Digestion is digested to testis tissue substantially in after single fine tubule in incubator, is transferred to centrifuge tube centrifugation, and discard supernatant liquid
Obtain lower layer's turbid；
(3) level-one is resuspended: precipitating is resuspended in the lower layer turbid PBS that step (2) is obtained, and centrifugation removal supernatant repeats
Obtain the re-suspension liquid of bottom afterwards twice；
(4) cell separates: the re-suspension liquid of step (3) being transferred to addition second level digestive juice digestion 2-5min in culture dish and is incited somebody to action
Stem spermatogonium releases inside fine tubule, is placed in microscopically observation and blows out individual cells with pipettor, uses
IMDM containing serum is collected cell into centrifuge tube using pipettor after being neutralized, and supernatant is removed after centrifugation and obtains primary
Cell suspending liquid；
(5) second level is resuspended: after the primary cell suspension of step (4) is resuspended using stem cell medium, using
Buffalo testis single cell suspension is obtained after cell net filtration.
Preferably, the level-one digestive juice is the clostridiopetidase A and mass ratio 0.001- of mass ratio 1-3% in step (2)
0.005% DNase I enzyme, the digestion time are 5-15min, and the incubator is carbon dioxide incubator, incubator
Condition of culture is the gas concentration lwevel of 37 DEG C, 5%.
Preferably, the second level digestive juice is the trypsin-EDTA and 0.003- of 0.1-0.4% in step (4)
0.007% DNase I enzyme, the serum are the mixing of any one in fetal calf serum FBS and serum substitute KSR or two kinds
Object, the additive amount of the serum are the 10% of IMDM.
Preferably, in step (5), the specific proportion of the stem cell medium are as follows: with IMDM be basic culture solution, KSR
Volume ratio be 10%, Fetuin volume ratio be 10%, NEAA final concentration of 10 μ L/mL, Chemically Defined
The final concentration of 20ng/mL of final concentration of 20 the μ L/mL, GDNF of final concentration of 10 the μ L/mL, B-27 of Lipid mixture 1,
The final concentration of 10U/mL of the final concentration of 10ng/mL of the final concentration of 100ng/mL of GFR α 1, bFGF, Lif, the end of β-ME are dense
Degree is 0.1mM.
Preferably, the specific gene is OCT4, Nanos2, DDX4, β 1-integrin and GDNF.
In conclusion by adopting the above-described technical solution, the beneficial effects of the present invention are:
(1) in buffalo stem spermatogonium like cell purification process of the invention cell type identification method, using molecule
Specific detection, can rapidly identify the type of cell during difference is adherent, and be accurately judged to finally be enriched to thin
Born of the same parents are largely the stem spermatogonium like cell with stemness, and what is got rid of is body cell substantially.
(2) be used in the adherent assorting room of difference of the invention serum-free stem cell medium, relative to tradition
Increase serum Somatic Cell Culture liquid, stem spermatogonium like cell loss amount is few, and more conducively stem spermatogonium like cell stemness
Maintenance.
Detailed description of the invention
Fig. 1 be buffalo testicular cell before purification after SSC-like cells ratio chart, wherein 1 (A) is before difference is adherent
DDX4 positive cell, 1 (B) is the DDX4 positive cell after difference is adherent, and 1 (C) is the PGP9.5 before difference is adherent positive thin
Born of the same parents, 1 (D) are the PGP9.5 positive cells after difference is adherent；
Fig. 2 is summarizing for the adherent front and back DDX4 positive cell of difference and PGP9.5 positive cell proportion；
Fig. 3 is the proof diagram of each section cell type molecular level in purification process；Wherein 3 (A) are OCT4 gene three
Distribution situation in class cell；3 (B) are distribution situation of the Nanos2 gene in three classes cell；3 (C) are DDX4 gene three
Distribution situation in class cell；3 (D) are distribution situation of the β 1-integrin gene in three classes cell；3 (E) are gdnf gene
Distribution situation in three classes cell.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention more comprehensible, preferred embodiment is enumerated below, to this hair
Bright further description.However, it is necessary to illustrate, many details listed in specification are used for the purpose of making reader to this
The one or more aspects of invention have a thorough explanation, also may be implemented even without these specific details of the invention
These aspects.
It is involved in the present invention to English name be explained as follows:
SSC-like cell: stem spermatogonium like cell；IMDM:Iscove's Modified Dulbecco's
The improvement version culture solution of Medium, that is, DMEM；KSR:KnockOut Serum Replacement, serum substitute；Fetuin:
Myosin；NEAA: nonessential amino acid；Chemically Defined Lipid mixture 1: lipid mixture；
GDNF:Glialcellline-Derived Neurotrophic Factor, glial cell line-derived neurotrophic factor；GFRα
1:GDNF family receptor alpha-1, glial cell line-derived neurotrophic factor family receptors α -1；BFGF:basic
Fibroblast growth factor, basic fibroblast growth factor -2；Lif:leukemia inhibitory
Factor, LIF ELISA；β-ME:2-mercapto-Ethanol, β-coloured glaze base ethyl alcohol；DNase I:
Deoxyribonuclease, deoxyribonuclease I；FBS: fetal calf serum.
The total serum IgE kit that the present invention uses is Total RNA Kit I (OMEGA, R6834-02) kit.Reverse transcription
Kit is PrimeScriptTM RT reagent Kit with gDNAEraser (TAKARA, Code NO.RR047A).
Embodiment
1. the preparation of buffalo testis single cell suspension
(1) the buffalo testis that the 3-7 monthly age is taken from slaughterhouse, using 75% alcohol disinfecting 10min, PBS is washed three times, super
Tunica albuginea is cut off in net platform, is washed three times using containing dual anti-PBS, testis tissue is put into clean culture dish and is cut into small pieces,
It is then transferred into sterile EP pipe and is shredded；
(2) tissue after shredding is transferred in clean culture dish, and adds clostridiopetidase A (20mg/ml) and DNase I
Enzyme (1mg/ml) digestive juice is placed 37 DEG C, is digested in 5% carbon dioxide incubator, until most tissue blocks are digested to list
A fine tubule, this process are maintained at 10min or so, shift tissue into the centrifuge tube of 50ml, 2000rpm horizontal centrifugal
5min abandons supernatant；
(3) it is resuspended and is precipitated with PBS, 2000rpm horizontal centrifugal 5min abandons supernatant, is repeated 2 times；
(4) sediment is transferred in the big ware of 100mm using fine tubule, add 0.25% trypsin-EDTA and
DNase I enzyme (1mg/ml) digests 3min, places microscopically observation and is simultaneously blown out individual cells with pipettor, using containing
The IMDM of 10%FBS is neutralized, and collects cell into the centrifuge tube of 50ml, and 2000rpm horizontal centrifugal 5min abandons supernatant；
(5) cell is resuspended using culture solution, respectively with 70 μm and 40 μm of cell net filtration, it is slender obtains buffalo testis
Born of the same parents' suspension.
2. the adherent separating method of difference is isolated and purified
(1) the buffalo testis single cell suspension of acquisition is seeded in the culture dish for being covered with gelatin, in 37 DEG C of incubators, is made
It is incubated overnight with stem cell medium, then will suspend and adherent cell loosely is transferred to and is covered with rat tail collagen protein in advance
It in culture dish, places in 37 DEG C of incubators and cultivates 4h, being collected simultaneously the cell being attached on gelatin is G-cell；
(2) training for cell not adherent in the culture dish for be covered with rat tail collagen protein will be seeded in being transferred to laminin
It supports in ware, places in 37 DEG C of incubators and cultivate 40min, being collected simultaneously the cell being attached in rat tail collagen protein is C-cell；
(3) cell being seeded in the culture dish for being covered with laminin is placed in 37 DEG C of incubators and cultivates 40min, lost
Abandon floating cells, adherent cell collecting L-cell.
As Fig. 1 be buffalo testicular cell before purification after SSC-like cells ratio chart, wherein 1 (A) is that difference is adherent
Preceding DDX4 positive cell, 1 (B) are the DDX4 positive cells after difference is adherent, and 1 (C) is that the PGP9.5 before difference is adherent is positive
Cell, 1 (D) are the PGP9.5 positive cells after difference is adherent；
Fig. 2 is summarizing for the adherent front and back DDX4 positive cell of difference and PGP9.5 positive cell proportion.
Refer to the poststaining that testis tissue is digested to individual cells before difference is adherent, the positive of DDX4 and PGP9.5 are thin
Born of the same parents.Refer to after difference is adherent with after difference adherent method purifying cells, is being dyed to obtain a positive cell with both antibody
Number.DDX4 positive cell ratio before the difference known to Fig. 1-2 is adherent is 18.1%, the DDX4 positive cell ratio after difference is adherent
Example is 58.2%；PGP9.5 positive cell ratio before difference is adherent is 11.3%, the PGP9.5 positive cell after difference is adherent
Ratio is 49.8%, thus the adherent rear positive cell number of difference increases, and difference adherent method can play the purpose of purifying cells.
3. the identification of cell type during isolating and purifying
(1) total serum IgE for extracting G-cell, C-cell and L-cell for being collected into respectively with total serum IgE kit, then uses
Reverse transcription reagent box carries out reverse transcription and generates cDNA；
(2) corresponding reagent is added, and respectively with base respectively using the cDNA of obtained different type cell as sample again
Because of the primer of OCT4, Nanos2, DDX4, β 1-integrin and GDNF, quantitative PCR is done, then according to obtained each gene table
The recurring number reached is calculated and draws corresponding map again.It is as shown in Figure 3 to draw gained map.
The extraction process of 3.1 total serum IgEs are as follows:
A, G-cell, C-cell and L-cell cell are collected respectively and is centrifuged, and suitable TRK is added.
B, it is centrifuged 2 minutes under maximum (top) speed using RNA Homogenizer Spin pillar, collects the liquid in centrifuge tube
Body.
C, 70% isometric alcohol and mixing are added into the liquid of collection, but is not centrifuged.
D, it is put into new centrifuge tube using HiBind RNA Mini pillar.
E, the sample in the step 3 of 700ul is added in pillar, 10000g is centrifuged 1 minute, abandons waste liquid.It repeats to walk
Rapid e, until sample has all filtered.
F, continue that 500ul is added into pillar, RNA washbuffer I, 10000g are centrifuged 30 seconds, abandon waste liquid.
G, continue that 500ul is added into pillar, RNA washbuffer II, 10000g are centrifuged 1 minute, abandon waste liquid.Weight
Multiple step g is washed once again.
H, centrifugal column is dried in 2 minutes with maximum (top) speed centrifugation completely.
I, HiBind RNA Mini pillar is put into the centrifuge tube of new 1.5ml, the DECP water of 30ul is added, with most
Big revolving speed is centrifuged 2 minutes, abandons pillar, and in centrifuge tube is the total serum IgE being collected into.
The process of 3.2 reverse transcriptions generation cDNA are as follows:
A, removal genomic DNA reaction
By ingredient shown in table 1 in preparing reaction mixture on ice, in order to guarantee reaction solution preparation accuracy, carry out
When items reaction, reaction mixture first should be prepared by the amount of stoichiometric number+2, be then dispensed into each reaction tube again, is eventually adding total
RNA sample (42 DEG C, 2min).
Table 1
| Reagent | Usage amount |
| 5×gDNA Eraser Buffer | 2.0ul |
| gDNA Eraser | 1.0ul |
| Total RNA | *1 |
| RNase Free dH2O | Up to 10ul |
* in 1:20ul reverse transcription reaction system, the Total RNA of 1ug is at most can be used in SYBR Green qPCR method, is visited
The Total RNA of 2ug at most can be used in needle qPCR analytic approach.
B, reverse transcription reaction
By ingredient shown in table 2 in preparing reaction mixture on ice, in order to guarantee reaction solution preparation accuracy, carry out
When items reaction, reaction mixture first should be prepared by the amount of stoichiometric number+2, then dispense 10ul again into each reaction tube, it is soft mixed
Carried out immediately after even reverse transcription reaction (37 DEG C, 15min；85 DEG C, 5s).
Table 2
PCR sequence used in 3.3 present invention detection transcript profile gene expressions is shown in Table 3.
Table 3
Referring to the proof diagram (identifying) that attached drawing 3 is each section cell type molecular level during isolating and purifying, wherein 3
It (A) is distribution situation of the OCT4 gene in three classes cell；3 (B) are distribution situation of the Nanos2 gene in three classes cell；3
It (C) is distribution situation of the DDX4 gene in three classes cell；3 (D) are distribution feelings of the β 1-integrin gene in three classes cell
Condition；3 (E) are distribution situation of the gdnf gene in three classes cell.Stem spermatogonium belongs to germline stem cell, and existing stem cell is special
Property, and have reproduction cell characteristic；OCT4 and Nanos2 is the label of multipotential cell, can be used as buffalo stem spermatogonium herein
Label, the label of reproduction cell when DDX4, β 1-integrin and GDNF belong to the label of sertoli cell.The ordinate of Fig. 3 is
The expression quantity of gene in G-cell, C-cell and L-cell cell, three histograms in figure show most some L-
Cell in cell with very strong versatility there is stem spermatogonium characteristic also to have reproduction cell characteristic again simultaneously, comprehensive next
Say to be exactly stem spermatogonium like cell required for final purifying of the invention；β 1-integrin and GDNF are the marks of sertoli cell
Note, is exactly body cell, relatively high in the expression quantity of G-cell and C-cell cell, illustrates that most body cell is all got rid of
?.
Thus by identification method of the invention it is found that this part of cell of G-cell is mainly body cell；C-cell this
Part cell is mainly the cell of not adherent complete body cell and differentiation on gelatin；This part of cell of L-cell is mainly
Stem spermatogonium like cell.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the principle of the present invention, it can also make several improvements and retouch, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. the identification method of cell type in a kind of buffalo stem spermatogonium like cell purification process, it is characterised in that: including with
Lower step:
(1) buffalo testis single cell suspension is isolated and purified using difference adherent separating method, and collects in purification process and obtains
The different type cell arrived；
(2) total serum IgE for the different type cell being collected into total serum IgE kit extraction step (1) then uses Reverse Transcription
Box carries out reverse transcription and generates cDNA；
(3) cDNA for the different type cell for using step (2) to obtain again is done as sample, and respectively with the primer of specific gene
Quantitative PCR, then draw corresponding map.
2. the identification method of cell type in buffalo stem spermatogonium like cell purification process according to claim 1,
It is characterized in that: the specific steps that the difference adherent method separating method isolates and purifies buffalo testis single cell suspension are as follows:
(1) the buffalo testis single cell suspension of acquisition is seeded in the culture dish for being covered with gelatin, uses stem cell medium mistake
Then night culture will suspend and adherent cell loosely is transferred in the culture dish for being covered with rat tail collagen protein in advance, cultivates 4h,
Being collected simultaneously the cell being attached on gelatin is G-cell；
(2) culture dish for cell not adherent in the culture dish for be covered with rat tail collagen protein will be seeded in being transferred to laminin
In, 40min is cultivated, being collected simultaneously the cell being attached in rat tail collagen protein is C-cell；
(3) the cell culture 40min that will be seeded in the culture dish for being covered with laminin abandons floating cells, collects adherent
Cell is L-cell.
3. the identification method of cell type in buffalo stem spermatogonium like cell purification process according to claim 2,
Be characterized in that: the main component of the stem cell medium are as follows: IMDM basic culture solution, KSR, Fetuin, NEAA,
Chemically Defined Lipid mixture 1, B-27, GDNF, GFR α 1, bFGF, Lif and β-ME.
4. the identification method of cell type in buffalo stem spermatogonium like cell purification process according to claim 2,
It is characterized in that: the specific proportion of the stem cell medium are as follows: it with IMDM is basic culture solution, the volume ratio of KSR is 10%,
The volume ratio of Fetuin is final concentration of 10 μ L/mL, Chemically Defined the Lipid mixture 1 of 10%, NEAA
Final concentration of 10 μ L/mL, B-27 final concentration of 20 μ L/mL, GDNF final concentration of 20ng/mL, GFR α's 1 is final concentration of
The final concentration of 0.1mM of the final concentration of 10U/mL of the final concentration of 10ng/mL of 100ng/mL, bFGF, Lif, β-ME.
5. the identification method of cell type in buffalo stem spermatogonium like cell purification process according to claim 2,
Be characterized in that: the culture is to be cultivated in 37 DEG C of incubator.
6. the identification method of cell type in buffalo stem spermatogonium like cell purification process according to claim 1,
Be characterized in that: the buffalo testis single cell suspension the preparation method comprises the following steps:
(1) pretreatment of raw material: tunica albuginea is cut off in super-clean bench after water intaking bull testis disinfection, cleans up to be placed in culture dish and cut
At fritter, transfers in EP pipe and shred；
(2) digest: the testis tissue after step (1) is shredded is transferred in culture dish, and level-one digestive juice is added and is placed in incubator
Middle digestion is digested to testis tissue substantially in after single fine tubule, is transferred to centrifuge tube centrifugation, and discard supernatant liquid and obtain down
Layer turbid；
(3) level-one is resuspended: precipitating is resuspended in the lower layer turbid PBS that step (2) is obtained, and centrifugation removal supernatant is repeated twice
The re-suspension liquid of bottom is obtained afterwards；
(4) cell separate: by the re-suspension liquid of step (3) be transferred in culture dish be added second level digestive juice digestion 2-5min will be fine
Stem spermatogonium releases inside tubule, is placed in microscopically observation and blows out individual cells with pipettor, using containing blood
Clear IMDM is collected cell into centrifuge tube using pipettor after being neutralized, and supernatant is removed after centrifugation and obtains primary cell
Suspension；
(5) second level is resuspended: after the primary cell suspension in step (4) is resuspended using stem cell medium, using thin
Buffalo testis single cell suspension is obtained after born of the same parents' net filtration.
7. the identification method of cell type in buffalo stem spermatogonium like cell purification process according to claim 6,
It is characterized in that: in step (2), clostridiopetidase A and mass ratio 0.001-0.005% that the level-one digestive juice is mass ratio 1-3%
DNase I enzyme, the digestion time are 5-15min, and the incubator is carbon dioxide incubator, and the condition of culture of incubator is
37 DEG C, 5% gas concentration lwevel.
8. the identification method of cell type in buffalo stem spermatogonium like cell purification process according to claim 6,
Be characterized in that: in step (4), the second level digestive juice is the trypsin-EDTA's and 0.003-0.007% of 0.1-0.4%
DNase I enzyme, the serum are the mixture of any one in fetal calf serum FBS and serum substitute KSR or two kinds, the blood
Clear additive amount is the 10% of IMDM.
9. the identification method of cell type in buffalo stem spermatogonium like cell purification process according to claim 6,
It is characterized in that: in step (5), the specific proportion of the stem cell medium are as follows: with IMDM be basic culture solution, the volume of KSR
Final concentration of 10 μ L/mL, the Chemically Defined Lipid for being 10%, NEAA than the volume ratio for 10%, Fetuin
The final concentration of 20ng/mL, GFR α 1 of final concentration of 20 the μ L/mL, GDNF of final concentration of 10 the μ L/mL, B-27 of mixture 1
Final concentration of 100ng/mL, the final concentration of 10U/mL of the final concentration of 10ng/mL of bFGF, Lif, β-ME's is final concentration of
0.1mM。
10. the identification method of cell type in buffalo stem spermatogonium like cell purification process according to claim 1,
Be characterized in that: the specific gene is OCT4, Nanos2, DDX4, β 1-integrin and GDNF.
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Citations (3)
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| CN103820383A (en) * | 2014-03-31 | 2014-05-28 | 广西大学 | Method of inducing spermatids with Ba-ma mini pig spermatogonia stem cells |
| CN104004708A (en) * | 2014-06-16 | 2014-08-27 | 内蒙古大学 | Method for separation and purification, long-term passage cultivation, cryopreservation and recovery of sheep spermatogonia stem cells |
| CN108795850A (en) * | 2018-06-26 | 2018-11-13 | 西北农林科技大学 | A kind of Spermatogonial Stem Cells are without feeder layer long-period culture method |
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| CN103820383A (en) * | 2014-03-31 | 2014-05-28 | 广西大学 | Method of inducing spermatids with Ba-ma mini pig spermatogonia stem cells |
| CN104004708A (en) * | 2014-06-16 | 2014-08-27 | 内蒙古大学 | Method for separation and purification, long-term passage cultivation, cryopreservation and recovery of sheep spermatogonia stem cells |
| CN108795850A (en) * | 2018-06-26 | 2018-11-13 | 西北农林科技大学 | A kind of Spermatogonial Stem Cells are without feeder layer long-period culture method |
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Application publication date: 20190222 |